SWIFS Toxicology Laboratory Procedure Manual v2.3 (03.13.2009) 267 Pages

Dallas County Southwestern Institute of Forensic Sciences
TOXICOLOGY LABORATORY PROCEDURE MANUAL, Version 2.3
Authorized by:
Elizabeth Todd, Ph.D., Chief Forensic Chemistry Chris Heartsill, Supervisor Toxicology Laboratory Karen Young, Quality Manager March 13, 2009
Effective date:
This is a non-controlled copy of a controlled document
Dallas County Institute of Forensic Sciences Toxicology Procedure Manual Summary of Changes from Previous Manual Version Previous Version: Toxicology Procedure Manual, version 1.0 Current Version: Toxicology Procedure Manual, version 2.x 1. Purpose - The section on Case Numbering was moved to Evidence Handling Procedure. The sections on Testing Validation and Critical Reagents were moved to Quality Management. 2. Scope of Toxicology Services - Section formally added to the procedure manual. 3. The Evidence Handling and Results Reporting section was reorganized and broken into more useable sections: Evidence Management, Evidence Handling Procedures, Evidence Hold Release and Destruction, Laboratory Referrals (updated with current vendor specifications), Reports and Release of Information. 4. Three new sections were added to provide more instruction on evidence management: Management of Histology Specimens, Tox Lims Operating Instructions, and DWI Database Operating Instructions. 5. The Quality Management section now includes a description of the process for case review, peer/technical review, determination of expiration dates for reagents and standards, and housekeeping 6. A section of abbreviations is added. 7. The Introduction to Procedures and Testing Protocols has been added to provide guidance for selection of applicable procedures by case type. 8. The section on Testimony was moved from the Section Administrative Manual to the Toxicology Laboratory Manual and updated. 9. Acetaminophen no technical change; version changed indicating electronic format 10. Acid Neutral Drug Screen Update equipment used, correct spelling of Liquichek standard, update amount injected on GC/MS, add methocarbamol as an analyte, and update concentration of calibrators used. 11. Alcohols and Acetone - no technical change; version changed indicating electronic format 12. Alkaline Drug Screen no technical change to procedure, Shimadzu GC/MS added, training notes updated, version changed indicating electronic format, 13. Alkaline Retention Index - no technical change; version changed indicating electronic format 14. Arsenic - no technical change; version changed indicating electronic format 15. Benzodiazepines - no technical change; additional explanation added regarding controls; version changed indicating electronic format 16. Carboxyhemoglobin Zeroing Solution updated to Triton X-100 17. Cocaine and Metabolites/Hamilton Updated amount of ethyl acetate for reconstituting and updated training notes. 18. Cyanide - no technical change; version changed indicating electronic format 19. Electrolytes - no technical change; version changed indicating electronic format 20. ELISA - no technical change; assay specific information originally located in the Training Notes section was moved into the procedure; version changed indicating electronic format 21. Ethchlorvynol - no technical change; version changed indicating electronic format 22. Ethosuximide - no technical change; version changed indicating electronic format
Dallas County Institute of Forensic Sciences Toxicology Laboratory 1 Changes from Previous Version Version 2.0
23. Ethylene Glycol - no technical change; version changed indicating electronic format 24. GHB Update concentration of control sample, give more detail in procedure, and update training notes 25. Lead - no technical change; version changed indicating electronic format 26. Lithium - no technical change; version changed indicating electronic format 27. Mercury - no technical change; version changed indicating electronic format 28. Opiates GC/MS Update concentration of working internal standard mixture; update pipettes used; reporting guidelines are clarified; reporting criteria updated to reflect C. Heartsill 8/29/07 memo 29. Salicylates procedure is updated to reflect that a recorder is no longer used and pipettes are updated. 30. THC in Blood - no technical change; version changed indicating electronic format 31. THC in Urine - no technical change; version changed indicating electronic format 32. Valproic Acid - no technical change; version changed indicating electronic format 33. Volatiles by GC - no technical change; version changed indicating electronic format 34. Volatiles by GC/MS - no technical change; version changed indicating electronic format 35. Volatiles by Multiple Headspace - no technical change; version changed indicating electronic format
Dallas County Institute of Forensic Sciences Toxicology Laboratory
Changes from Previous Version Version 2.0
This is a non-controlled copy of a controlled document.
Toxicology Procedure Manual Version 2.x Revisions and Corrections Effective Date 2/28/08 2/28/08 Description Authorized by ELT
2/28/08 2/28/08 2/23/09
2/23/09 2/23/09 2/23/09
2/23/09
2/23/09 2/23/09
2/23/09 3/13/09 3/13/09 3/13/09
Chap 15 Reports and Release of Information, addition of Section 1.C.1 to clarify the components of an M case file report. (v 2.1) Chap 17 Quality Management, addition of Section, addition of ELT Section IV.C.3.b to clarify documentation of administrative review for D cases. (v 2.1) Alcohols and Acetone, addition of reporting to the section: Range ELT of Detection and Reporting Criteria. (v 2.1) Opiates by GC/MS, clarification of reporting criteria for morphine. (v ELT 2.1) Cocaine and Metabolites: Change from Hamilton automated ELT extraction system to a vacuum manifold system (VacElute). (v 2.1, effective date 1/20/09) GHB: Change from Hamilton automated extraction system to a ELT vacuum manifold system (VacElute). (v 2.1, effective date 1/20/09) ELISA: Change from Tecan Miniprep to the Tecan EVO instrument. ELT (v 2.1) Chap 17 Quality Management: Section IV Case Review was updated ELT to reflect that the primary analyst signs the case report; other minor changes were made to improve clarity of the case review and technical review process. (v 2.2) Alcohols and Acetone: The section on stocks, standards, controls, and ELT calibrators was revised to clarify that the high and low calibration standards used for DWI analyses contains only ethanol and is not a mixed alcohol/acetone standard. In the section Qualitative Identification, the criteria for reporting of isopropanol from transplant samples was clarified. Other minor changes were made to improve clarity and formatting. (v 2.2) Lead: The expiration date for the 10% Triton X-100 was corrected. (v ELT 2.1) Lithium: A typographical error under Calculations was corrected, ELT lead was corrected to lithium. Minor changes were made to improve clarity. (v 2.1) Valproic Acid: The Stocks, Standards, Controls, and Calibrators ELT section was rearranged to improve clarity. (v 2.1) New procedure added: Fentanyl by GC/MS ELT Histology Specimen Management: Update holding period for blocks ELT from 5 to 10 years to remain consistent with NAME standards. Histology Block Storage Policy: Update holding period for blocks ELT from 5 to 10 years to remain consistent with NAME standards.
Dallas County Institute of Forensic Sciences Toxicology Procedure Manual
Corrections and Revisions Version 2.3

Dallas County Southwestern Institute of Forensic Sciences Toxicology Laboratory STATEMENT OF PURPOSE The Dallas County Forensic Laboratory is a division of the Dallas County Southwestern Institute of Forensic Sciences. It is an independent laboratory established through joint agreement of the Dallas County Commissioners Court and UT-Southwestern Medical School. The Laboratory is not a part of any police agency, the Dallas County District Attorneys Office, nor the Dallas County Sheriffs Office. The Laboratory operates on a fee-for-services basis, and its services are available to anyone paying the fees prescribed by the Dallas County Commissioners Court within the policies and procedures established by the Commissioners Court and the Director of the Institute. The purpose of the Toxicology Laboratory of the Dallas County Forensic Laboratory is as follows: 1. To analyze submitted specimens for a wide variety of drugs, metabolites, poisons, and other substances to aid Medical Examiners in determining cause and manner of death, the criminal justice system in the investigation of driving under the influence cases and sexual assault cases, and other submitters as needed within the scope and expertise of the Laboratory; and 2. To testify in court proceedings regarding testing results, methods of analysis, and the general effects of substances identified by this Laboratory.
The purpose of this manual is to outline the activities conducted by this Laboratory and to provide an overview of sample analysis strategy. Because of the unique nature of many of the samples submitted to this Laboratory, exact analytical procedures cannot be prescribed for every case. Variance from procedures should be noted in the case file.
Statement of Purpose Version 2.0
SCOPE OF TOXICOLOGY SERVICES The toxicology services offered by the Dallas County Institute of Forensic Sciences Toxicology Laboratory are designed to identify and quantitate a wide variety of substances and metabolites including abused drugs, clinical drugs, alcohols, acetone, solvents, carbon monoxide, cyanide, metals, ethylene glycol and many other substances. The General Screen provides comprehensive screening of biological fluids for several hundred drugs and metabolites. Upon request, other testing is provided. General Screen Alcohols and Acetone Cannabinoid Screen by ELISA with reflex of positives to GC/MS assay Drug Screen Alkaline Drug Screen Acid/Neutral Drug Screen Opiate Screen by ELISA with reflex of positives to GC/MS assay Cocaine Metabolites Screen by ELISA with reflex of positives to GC/MS assay Alcohols and Acetone ethanol methanol isopropanol acetone Drug Screen Alkaline Drugs Screen detects >200 drugs and metabolites including but not limited to cocaine and cocaethylene amphetamine, methamphetamine, MDMA, MDA, etc. some opiates: codeine, hydrocodone, oxycodone, meperidine, etc. propoxyphene and norpropoxyphene benzodiazepines tricyclic antidepressants other antidepressants including bupropion selective serotonin reuptake inhibitors including citalopram, fluoxetine, sertraline some cardiac drugs Drug Screen Acid/Neutral Drugs Screen detects approximately 30 drugs and some pesticides acetaminophen barbiturates anticonvulsants carisoprodol and meprobamate salicylate
Scope of Services Version 2.0
Opiate Narcotics Confirmation by GC/MS codeine hydrocodone hydromorphone morphine 6-monoacetylmorphine Cocaine and Metabolites by GC/MS cocaine cocaethylene ecgonine methyl ester benzoylecgonine Marihuana and Metabolites by GC/MS tetrahydrocannabinol carboxytetrahydrocannabinol Other Analyses electrolytes carboxyhemoglobin metals: lead, arsenic, mercury, lithium cyanide volatiles: freons, butane, propane, toluene, etc. ethylene glycol LSD screen (quantitation to referral lab) gamma-hydroxybutyrate (GHB) Reference Lab Upon request specimens may be sent to referral laboratories for other testing. Drug Testing o Examples of drugs sent to referral laboratories for analysis include gabapentin, sildenafil (Viagara), zaleplon (Sonata), eszopiclone (Lunesta), warfarin (Coumadin), oxymorphone, buprenorphine (Subutex), butorphanol (Stadol), digoxin, and steroids. Medical Testing o The Toxicology Laboratory routinely sends out various specimens for medical testing in support of the Office of the Medical examiner including standard clinical chemistry testing; metabolic testing; infectious disease testing; tryptase; antibody testing for allergens including wasp, bee, and fire ant venoms; and histology.
Scope of Services Version 2.0
TOXICOLOGY EVIDENCE MANAGEMENT I. Responsibilities A. It is the responsibility of all Toxicology staff to operate in a manner that 1. preserves the quality of evidence submitted to the Laboratory 2. ensures security and provides accountability of evidence 3. protects evidence from contamination, cross transfer, and loss B. Proper evidence management is the responsibility of all toxicology staff. 1. The Evidence Registrar typically manages receipt, storage, and release of evidence for M and H cases. 2. The Toxicology Chemist III typically manages receipt, storage, and release of evidence for D cases. 3. Evidence Registration duties may be performed by all Toxicology staff including the Laboratory Aide. 4. The term Evidence Registrar applies to the management of evidence by any Toxicology staff member. II. Evidence Receipt A. Evidence is usually received in person, by mail, from a lockbox, or by express package service. B. The individual receiving evidence performs the following as applicable: 1. documents the chain of custody 2. obtains proper submitter/billing information including complete address information for non-routine submitters 3. describes the item(s) received 4. determines services requested 5. assesses the status of the evidence seals 6. ensures that the evidence package is properly sealed for storage or is immediately inventoried and logged in III. Chain of Custody A. All specimen transfers are documented at the time of evidence transfer on paper or in TOX-LIMS. 1. Chain of custody transfers are documented between the submitter and Institute staff, between Institute staff, and between storage locations and Institute staff. 2. Initials or electronic identifier are acceptable in place of the signature for Institute staff, and it is not necessary to note IFS affiliation. 3. Chain of custody must legibly document the following: a) Name, signature, and affiliation of individual(s) releasing evidence or the site of release b) Name, signature, and affiliation of individual(s) receiving evidence or the site of receipt c) Date and time of evidence transfer d) General description of evidence transferred e) Status of specimen seal (except during analysis) f) Case name and/or Institute case number; agency case number as applicable
Dallas County Institute of Forensic Sciences Toxicology Laboratory 1 Evidence Management Version 2.0
g) Method of shipment and tracking number if applicable(Fed Ex, courier, etc.) IV. Evidence Seals A. Proper seal means 1. The seal is tamper evident and entry into the specimen container results in obvious damage/alteration to the container or its seal, and 2. The seal is initialed by the person making the seal, the submitter, or by Institute staff receiving the evidence. B. Evidence received into the Laboratory must be stored under proper security seal until it is ready for inventory, log-in, and analysis. 1. Evidence which is not submitted with a proper security seal by the submitting agency and which will not be logged-in and/or analyzed immediately must be sealed by Toxicology personnel receiving the evidence. a) This may be accomplished by (1) initialing an un-initialed, tamper-evident agency seal, (2) placing a new, tamper evident seal on the evidence and initialing the seal, or (3) by grouping individual tubes into an outer container which is properly sealed and initialed 2. Within one month of the completion of analysis, all evidence is properly sealed and stored. a) Specimen containers may be individually sealed with a security seal or grouped in an outer container which has a proper seal. C. In addition to the security seal, evidence containers must be physically sealed and stored in such a manner that the contents cannot readily escape or otherwise be subject to cross contamination. 1. Evidence must remain physically sealed at all times during the storage process. V. Specimen Storage A. Specimens should be stored in a manner which preserves their security, condition, and integrity. 1. All evidence storage locations are secure and restricted to authorized Toxicology staff. 2. Tissues are typically stored in the Toxicology freezer until they are released or disposed. 3. Liquid biological specimens are typically stored in the Toxicology refrigerator, DWI refrigerator, or in long term refrigerated storage until they are released or disposed. 4. Other evidence is stored in the best available manner so as to preserve its condition to the extent reasonably possible. VI. Specimen Inventory, Log-in, and Use A. Each Toxicology case is assigned a unique case number. B. The contents of each case are inventoried. C. A unique lab identifier is assigned to each specimen. D. Evidence is logged into Tox LIMS. E. Cases are entered into the Toxicology Case Logs as applicable.
Dallas County Institute of Forensic Sciences Toxicology Laboratory 2 Evidence Management Version 2.0
F. Physically-sealed specimen containers are placed in a secure refrigerator or freezer accessible only by authorized Toxicology personnel pending analysis. G. Specimens are removed from the secure refrigerator/freezer by authorized personnel for sampling throughout the analytical process. a) All specimen transfers are documented using established chain of custody procedures. H. Specimens may be sent to referral laboratories as applicable including: 1. Histology 2. Clinical lab services 3. Toxicology lab services VII. Reporting Results and Release of Information A. A report is generated detailing the results of analysis. B. A copy of the report is provided to the submitter. C. A record of entities receiving copies of the report is maintained in the case file. VIII. Specimen Retention, Release, and Disposal A. Within one month of the completion of analysis, samples are properly sealed for storage. B. Specimens are typically maintained for up to one year as noted in the Evidence Retention, Release, and Hold policy and returned to the submitter or disposed. C. Disposition of evidence is documented in the case file.
Evidence Management Version 2.0
PROCEDURE FOR TOXICOLOGY EVIDENCE HANDLING I. Discrepancies A. Significant discrepancies in specimen inventory and/or identification should be reported immediately to a supervisor and/or the submitting agency. 1. An example of a discrepancy is when case number and case name or initials on evidence do not match paperwork. 2. Discrepancies should be noted in the case file. II. Recommended Conventions for Describing Evidence A. Relevant information on the evidence label should be included in the specimen inventory. 1. Name and date/time of collection noted on the tube should be included on the inventory for ante-mortem samples where available. B. Descriptions quoted verbatim from the evidence container should be placed in quotation marks. C. Information added as clarification by the Evidence Registrar should be placed in parenthesis. D. If the container label has a typed name matching the name on the submission paperwork, write name. E. If the container has a date and time, write the date and time in quotation marks. F. If the container label is hand written, write handwritten next to the relevant information. G. If the name on the container does not match the name on the submission paperwork write the name, date, and/or time on the container in quotation marks. If present on the label, include hospital number or other identifying number in quotation marks. H. If several specimens have the same label information, the label may be documented on the first piece of evidence and subsequent items may be described as same as [item#]. III. Case Numbering A. M-Cases are submitted by the Dallas County Medical Examiners Office. 1. Case designations are of the following format YYMNNNN, NNNN-YY, and/or NNNNYY where N = number, Y= the last two digits of the year, and M = Medical Examiner. 2. These numbering schemes exist due to conventions in the Medical Examiners Office and the billing system. The preferred numbering is YYMNNNN. B. H-Cases include specimen submitted by medical examiners outside the Institute, police departments, and other agencies. C. D-Cases include legal alcohols and drug screens which are submitted by law enforcement agencies or other criminal justice agencies usually related to driving under the influence cases IV. ME Specimen Handling A. ME Submissions 1. The Office of the Medical Examiner (ME) typically submits specimens to the Toxicology Laboratory using the secure Medical Examiner/Toxicology Refrigerator (METR).
Evidence Handling Procedure Version 2.0
a) ME staff place specimens in the METR and paperwork in the designated location on the METR. 2. ME submissions may also be made in person. 3. ME specimens are submitted sealed. 4. ME submissions typically consist of a) biological specimens collected at autopsy for toxicology testing b) histology specimens to be sent to the histology vendor by the Toxicology Laboratory c) MCAD cards to be sent to the metabolic testing vendor by the Toxicology Laboratory. d) other case related evidence B. Inventory of ME Specimens 1. Medical Examiner specimens are taken from the METR when the Evidence Registrar is ready to log in and inventory specimens. a) If for some reason, specimens are taken from METR and cannot be inventoried immediately, the Evidence Registrar must receive the specimens and store them in the Toxicology refrigerator, freezer, or other secure storage. (1) The Evidence Registrar must follow items 2 and 3 below with the exception of 3a5. (2) If evidence is not properly sealed, it must be properly sealed prior to storage and pending inventory. 2. Each case submitted by the ME should have submission paperwork. a) The Evidence Registrar matches each case with its corresponding submission sheet. b) If paperwork or specimens are missing for a case, the Evidence Registrar returns the case to the refrigerator with a note that paperwork is missing. 3. The Evidence Registrar completes the chain of custody at the bottom of the ME submission paperwork. a) Enter (1) Received from (individual, METR, etc.) (2) Received by (initials of Toxicology staff receiving) (3) Date of receipt (date evidence was received or taken from METR) (4) Time of receipt (time evidence was received or taken from METR) (5) Inventoried by (initials of Toxicology staff inventorying evidence) (6) Items Recd (the number of bags received or a description of other items received) (7) Sealed (note if the bag was received with a tamper evident seal in place) (8) Initialed (note if the seal is initialed) 4. Each piece of evidence is assigned a unique identification typically by the medical examiner:
a) a case number b) a unique container letter designation 5. The Evidence Registrar compares the specimens received with the specimens marked by the medical examiner on the specimen submission sheet. a) If the ME has checked that a specimen was sent, but it was not received with the case, mark NR date/initial on the submission sheet and continue logging in the rest of the case. 6. If a piece of evidence is not assigned a unique container identification by the medical examiner, the Evidence Registrar assigns that designation except as noted below. a) When it is not feasible to mark the evidence, the evidence will be placed in a bag or other container, and the proximal container will be labeled. (1) Evidence will be stored in the properly labeled container. b) In some cases, multiple hospital tubes are received in one bag. (1) Assign the outer container a letter designation. (2) Choose 2-3 tubes to assign that letter and a sequential number. (a) Choose the hospital tubes with the earliest date/time of collection containing an adequate amount of specimen for testing. (Example: bag label NNNN-YYK, tube labels NNNN-YYK1, NNNN-YYK2, etc) (3) Not all hospital tubes will be individually identified. 7. Specimens for each case are logged in and inventoried using Tox-Lims. a) Refer to the Tox-Lims Procedure. C. Description of evidence in Tox-Lims and on the submission paperwork 1. Container labels should be compared to information on the submission paperwork. a) If there is variance between information on the submission sheet and information on the evidence container regarding type of specimen, use the description on the evidence container. 2. Assumptions cannot be made about the type of specimens or the identity of a specimen. a) The medical examiner should be contacted to resolve questions. b) Do not log-in questionable specimens until the problems are resolved. c) If questions cannot be clearly resolved in a timely manner, seek supervisor assistance. d) Ultimately, if the type of specimen is not known, describe it generally (yellow liquid, clear liquid, tissue, etc.) 3. Each piece of evidence should be described briefly including a) specimen type (blood, urine, etc.) b) number of tubes c) type of tube (red top, gray top, etc) d) volume estimation to the nearest milliliter (3 mL, 10 mL, <1mL, etc.) e) source of collection (femoral, heart, etc.) as applicable. 4. Lengthy descriptions are not necessary; however, noting something out of the ordinary (bloody vitreous, dark urine, etc.) is helpful.
D. E.
F.
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5. Description of antemortem or hospital specimens should include name, date, volume, and time information recorded on the tube as applicable. The Evidence Registrar prints a copy of the Evidence Receipt form from Tox-Lims, initials the form, and places it in the case file. The Evidence Registrar stores ME specimens for analysis. 1. Liquid specimens and specimens in glass containers are typically stored in the toxicology refrigerator. a) ME cases are placed in racks in numerical case order and alphabetical order within the row. 2. Tissues should be contained in plastic cups and properly sealed one to a bag. a) More than one cup can be placed into a Kapak bag. b) Tissues are typically stored in the freezer in case order. c) All bags stored in the freezer must be properly heat sealed because other types of seals release at low temperatures. The Evidence Registrar prepares a case folder. 1. The case folder should contain the following: a) ME submission paperwork containing completed chain of custody information and specimen receipt information b) Evidence receipt sheet from Tox-Lims (initialed) c) Administrative Billing sheet with the name and case number filled out. 2. Expedite cases are marked in red and placed in the expedite area. 3. Homicide cases are marked in blue and placed in the homicide case area. 4. For Stat cases, the Evidence Registrar should inform the analyst in charge of that assay of the stat or notify a supervisor. The Evidence Registrar enters cases into the ME Toxicology Case Log 1. The ME Toxicology Case Log is generated using the following program: tox_clp4.xls. 2. Conventions and abbreviations: a) Black ink is used for routine cases. b) Blue ink is used for homicide cases c) Red ink is used for expedite cases or stat cases d) NS no specimens received (yellow highlight the case number) e) NR no case received (yellow highlight the case number) f) NT no tests requested (yellow highlight the case number) 3. Enter the following information into the ME Toxicology Case Log: a) ME the initials of the ME responsible for the case b) RECD - date case was received by Toxicology (usually from the METR) c) RELEASED date completed in the lab (entered at the time of case completion) d) If Alcohols and Acetone analysis is requested by the ME on the ME Toxicology Submission sheet, enter the following into the ALC column if the specimens were submitted (1) B - postmortem blood (2) V - vitreous (3) A - antemortem blood
e) If General Screen is selected by the ME on the ME Toxicology Submission sheet, enter the following (1) In the ALK column enter B for postmortem blood (2) In the A/N column, enter B for postmortem blood (3) In the ELISA column, enter B for postmortem blood f) If electrolytes are selected by the ME on the ME Toxicology Submission sheet and if vitreous was submitted, enter V in the VHP column. g) If carbon monoxide is selected by the ME on the ME Toxicology Submission sheet, enter B (for postmortem blood) in the CO column. h) MISC List in any other tests that are marked or any special instructions noted on the ME Toxicology Submission sheet. H. Supplemental Requests for Analysis 1. Supplemental requests for analysis received after the initial submission request must be documented in the case file including the name of the requestor, date, additional analyses requested, and initials of the individual receiving the request on behalf of the Toxicology Lab. a) The supplemental request should be written on the original request form if the case is not complete. b) If the case is complete, the supplemental request should be written on a new Administrative Billing Summary form, record of conversation, etc. c) The ME Tox Case Log should be updated as applicable. I. Completed ME Cases 1. When a case has been completed, the responsible analyst will place it in the completed bin. 2. The Evidence Registrar will enter the completion date in the RELEASED column of the ME Toxicology Case Log and yellow highlight the case number. 3. Print a chain of custody from Tox-Lims and initial. 4. The Evidence Registrar will properly reseal evidence where applicable and log it out of Tox-Lims. (see Tox-Lims procedure) a) A copy of the Tox-Lims Chain of Custody is initialed and placed into the case file. 5. Cases are released for typing. 6. Properly sealed evidence may be released to long-term storage as needed. 7. Once a case is logged out of Tox-Lims, chain of custody may be tracked manually and kept in the case file. V. H Case Evidence Handling A. H identifies cases which are not driving while intoxicated cases (D cases) or regular Dallas County ME cases (M cases). Submitters are usually police agencies (sexual assault toxicology kits), other crime laboratories, other Medical Examiners, etc. 1. Most toxicology sexual assault kits are initially received by the Physical Evidence Section (PES) and transferred to the Toxicology Lab. B. Standard H-Cases (excluding sexual assault toxicology kits from PES):
1. Evidence is usually submitted by mail, delivery service, or in-person. a) Submission by mail or delivery service (1) Initial receipt into the Laboratory may be documented on a Supplemental Chain of Custody form or in the received by portion of the Toxicology Evidence Receipt form. (2) A record of shipper tracking numbers should be retained. (3) Once evidence is properly received into the lab, it may be stored under proper seal in the Toxicology refrigerator and inventoried at a later date using the Toxicology Evidence Receipt form. b) In-person submission (1) The Toxicology Evidence Receipt form is used for in-person submissions. 2. Toxicology Evidence Receipt Form: Specimen Receipt, Description, Inventory, and Identification a) All evidence received should be accompanied by the following legible information: (1) Submitting agency name (2) Contact name (3) Complete address and phone number (required for non-routine submitters) (4) Billing information if different from submitter (required for non-routine submitters) (5) Method of shipment and tracking information if applicable (6) Date and time received (7) Name of individual receiving the evidence (8) Condition of seal (9) Case name (10) Agency case number (11) Description of the item received (a) This is usually a description of the main outer package received, for example: 1 brown paper bag (12) Analyses requested b) Item(s) Received (1) Enter a description of the inner contents of the main package received. (2) The description should include discussion of packaging and seal conditions of inner items, notation of identifying information on the items submitted (for example case name, case number, date, etc.), and a general description of what was received (3) For example, 1 sealed brown bag with paperwork containing 1 sealed plastic baggie with 2 grey top tubes Smith, Joe 12/25/07 1549 c) Case Number Assignment
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4. 5.
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(1) Each case will be assigned a unique lab number which will be written on or affixed to the Toxicology Evidence Receipt (07H####). (2) Each piece of evidence will be assigned the case number plus a unique letter identification which will be placed on the evidence container (07H####A, etc.) (3) When it is not feasible to mark the evidence, the evidence will be placed in a bag or other container, and the proximal container will be labeled. (4) Evidence will be stored in a properly labeled container. Specimens are logged into Tox-Lims. a) An Evidence Receipt sheet is printed, initialed, and placed into the case file. Cases are entered into the H-case spiral notebook which records the date, case name, submitter, items submitted, tests requested, and agency numbers. Cases are entered into the H-case Toxicology Log a) AGENCY the submitting agency b) RECD - date case was received in the Toxicology Lab c) RELEASED date completed in the lab (entered at the time of case completion) d) Enter the specimen designation into the applicable test column. (1) For example, enter B under ALC to indicate that blood is to be analyzed for Alcohols and Acetone. (2) Seek supervisory assistance if submission requests and/or evidence are unclear or non-standard. e) The following protocol is used for routine submissions of sexual assault cases: (1) Enter B under ALC for a blood Alcohols and Acetone (2) Enter U under ALK for a urine alkaline screen (a) If the urine is tested and found to be positive for drugs, the blood is tested and quantitated as applicable. (3) Enter B under ELISA for an ELISA screen of blood (4) Enter U under SA ELISA for a urine screen sexual assault ELISA screen (5) Enter U under GHB for a urine screen for GHB (a) If the urine is tested and found to be positive for GHB, the blood is tested and quantitated as applicable. (6) MISC list any other tests or special instructions noted on the requests Completed Standard H-Cases a) When a case has been completed, the responsible analyst will place it in the completed bin. b) The Evidence Registrar will enter the completion date in the RELEASED column of the H-Case Toxicology Case Log and yellow highlight the case number. c) Print a copy of the chain of custody from Tox-Lims and intial.
d) The Evidence Registrar will properly reseal evidence where applicable and log it out of Tox-Lims. (refer to Tox-Lims procedure) (1) A copy of the Tox-Lims Chain of Custody is initialed and placed into the case file. e) Cases are released for typing. f) Properly sealed evidence may be released to the submitter or otherwise handled per instruction of the submitter. g) Once a case is logged out of Tox-Lims, chain of custody may be tracked manually and kept in the case file. C. Sexual Assault Toxicology Kits from PES 1. The Evidence Registrar contacts PES Registration to determine if sexual assault Toxicology kits are available for pick up (usually Wednesday and Friday). 2. If kits are available, the Evidence Registrar collects kits from PES. a) A Physical Evidence Section Chain of Custody form is completed for each kit received. (1) A copy of the Physical Evidence Section Chain is given to PES Registration to hold while Toxicology has the kit. b) The Tox Collection Kit Pick Up Log which summarizes the kits received on a particular day is completed, signed, and dated by the Toxicology Evidence Registrar. (1) The Tox Collection Kit Pick Up Log is retained in Toxicology. 3. The Evidence Registrar takes kits to the Toxicology Lab for processing and storage. a) Kits received from Irving, Mesquite, Duncanville, and DeSoto (1) The Evidence Registrar opens kits received from these agencies to determine if the physician has requested toxicology testing. (a) If testing is requested, the case is logged in and handled as a Standard H Case (see above procedure). (b) If testing is not requested, the kit is properly resealed and stored in the Toxicology Refrigerator. b) Kits from other submitters (1) The Evidence Registrar ensures that the kit is properly sealed and it is stored in the Toxicology Refrigerator. (2) Requests for testing these kits will be made by the submitting agency or through PES. (a) When testing is requested, the case will be handled as a Standard H-Case (see above procedure.) 4. Release of Physical Evidence Toxicology Kits a) Kits are properly sealed and returned to PES Registration (1) After 45 days of storage with no testing, or (2) Upon case completion for cases that were analyzed. b) Kits are returned with the original Physical Evidence Section Chain of Custody and any other case specific paperwork received from PES. (1) Chain of Custody is completed.
(2) Copies are made of the completed chain of custody for Toxicology; original chain of custody is retained by PES. c) Chain of custody is filed in Toxicology (1) If kits that were analyzed, chain of custody is filed in the Toxicology case file. (2) If kits were not analyzed (just stored) the chain of custody is placed in the Returned SA Kits to PES notebook. VI. D Case Evidence Handling A. D identifies cases which involve driving while intoxicated offenses. 1. Submitters are usually police agencies or other crime laboratories. B. DWI Evidence Submission Types 1. DPD a) Dallas Police Department submits the majority of their samples in person using a portable lockbox including (1) An Offense/Incident Report which serves as chain of custody for the lockbox and is signed by the DPD submitter and IFS receiver; this is retained by the Laboratory for processing. (2) Yellow DIC-23A Specimen Routing Reports if available (3) Blood Alcohol Submission forms and specimen tubes. b) An inventory of specimens received is generated using the DWI computer program dBase4 DWI Database. The inventory is printed onto the Offense Incident Report. (1) A copy of the Offense Incident Report is placed in each case file. (2) The original is returned in person to the DPD submitter at a future delivery. 2. Lockbox Lockboxes are located in the Parkland ER and the lobby of the Institute. a) Police agencies may submit samples directly into the Parkland lockbox or the locked refrigerator in the Institute lobby. b) Specimens are typically accompanied by the Blood Analysis Request sheet or the Toxicology Evidence Receipt form. 3. Mail or delivery service a) Initial receipt into the Laboratory may be documented on a Supplemental Chain of Custody form or the Toxicology Evidence Receipt form. b) A record of shipper tracking numbers should be retained. c) Once evidence is properly received into the lab, it may be stored under proper seal in the Toxicology refrigerator and inventoried at a later date using the Toxicology Evidence Receipt form. 4. In-person a) When evidence is delivered in person, a Toxicology Evidence Receipt form is used to receive evidence. C. All evidence received should be accompanied by the following legible information which may be entered on the Toxicology Evidence Receipt form: 1. Submitting agency name
2. Contact name 3. Complete address and phone number (required for non-routine submitters) 4. Billing information if different from submitter (required for non-routine submitters) 5. Method of shipment and tracking information if applicable 6. Date and time received 7. Name of individual receiving evidence 8. Condition of seal 9. Case name 10. Agency case number if provided 11. Description of the item received a) For items received by mail, this is usually a description of the main outer package received, for example, 1 brown paper bag 12. Analyses requested D. Specimen Inventory, Description, and Identification will be documented on one of two forms 1. Blood Analysis Request Form: a) When a sample is submitted by lockbox, a Blood Analysis Request form is usually submitted with the sample or filled out by the person logging in the case. b) Information on the evidence container is compared to the submission paperwork; a red check mark is made on submission paperwork to document verification of case information. (1) Discrepancies in information will be specifically noted on submission paperwork. c) Where possible, the submitting agency case number or arrest number is recorded on the Blood Analysis Request form. d) Evidence from the same person or with the same agency case number submitted on the same date will be given a unique laboratory case number (07D####). (1) The case number is written or affixed to the Blood Analysis Request form. (a) A description of the specimens is recorded on the barcode label (1-grey, 2-grey, 1 red, 1-cup, etc.). (b) This label is initialed by the person logging in the evidence. (2) Each container of evidence within the case is assigned and labeled with the case number plus a unique container letter (07D####A). 2. Toxicology Evidence Receipt Form a) All other evidence should be received using a Toxicology Evidence Receipt form; evidence description, inventory, and identification are added to this form. b) Item(s) Received (1) This typically provides a description of the inner contents of the main package received.
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E. F. G.
H.
(2) The description should include discussion of packaging and seal conditions of inner items, notation of identifying information on the items submitted (for example case name, case number, date, etc.), and a general description of what was received. (3) For example: 1 sealed brown bag with paperwork containing 1 sealed plastic baggie with 2 grey top tubes Smith, Joe 12/25/07 1549 c) Case Number Assignment (1) Each case is assigned a unique lab number which will be written on or affixed to the Toxicology Evidence Receipt form (07D####). (a) A description of the specimens is recorded on the barcode label (1-grey, 2-grey, 1 red, 1-cup, etc.). (b) This label is initialed by the person logging in the evidence. (2) Each piece of evidence will be assigned the case number plus a unique letter identification which will be placed on the evidence container (07D####A, etc.) (3) When it is not feasible to mark the evidence, the evidence will be placed in a bag or other container, and the proximal container will be labeled. (4) Evidence will be stored in a properly labeled container. 3. General a) Abnormalities such as blood clots or broken tubes are noted on the submission paperwork. b) Any discrepancies in specimen inventory and/or identification should be reported to a supervisor and/or the submitting agency and noted in the case file. Specimens are logged into Tox-Lims; refer to Tox-Lims procedure. Case information is entered into the dBase4 DWI Database if it was not entered previously. Work flow for routine D cases 1. D cases with blood will be tested for alcohols and acetone. a) D cases in which the blood contains 0.09% ethanol or greater are considered complete and ready to report. b) D cases in which the blood contains 0.08% ethanol or lower will be tested for drugs. 2. Intoxication Manslaughter and Intoxication Assault blood cases are tested for alcohols and acetone and routine drug screen. 3. Urines are tested for drugs only. 4. Specific instructions by the submitter will be followed as applicable. D Case Completion 1. The Tox III that is responsible for the case will enter the results in the dBase4 DWI Database.
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a) A report and billing sheet are generated from the dBase4 DWI Database. b) Corrections to reports can be made in Word (c:\dbase\DB2\REPORTS\07DXXXX). c) A copy of the report is sent to the requesting agency. d) Copy of cases to DPS (1) The analyst will review all D-cases in which an alcohol analysis was performed to determine if the report should be sent to the DPS as part of the Administrative License Revocation (ALR) program. (a) If the report meets ALR criteria, an ALR affidavit will be printed on the report. (b) A copy of the signed report will be notarized and sent to DPS along with the Yellow DIC-23A Specimen Routing Report if it is available. (i) No copy is kept of the notarized affidavit or the DIC-23A form. I. Evidence Release of D Cases 1. Evidence is properly sealed within 30 days of completion. 2. Evidence is returned to the submitting agency or otherwise held and/or disposed as directed by the requesting agency or per court order, subpoena, or other legal direction. a) Evidence is typically returned to the agency after completion by mail, delivery service, or in-person transfer. 3. Samples may be disposed with permission from the submitting agency.
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TOX-LIMS OPERATING INSTRUCTIONS I. Purpose A. The purpose of this procedure is to provide instructions for Toxicology Laboratory staff to log in specimen using the computerized case tracking software, Tox-Lims. II. System Overview A. Tox-Lims is an Access based program which tracks specimen through the lab as they are received, used for analysis, and released. B. Tox-Lims documents the 1. Chain of custody of toxicology specimens within the Laboratory 2. Inventory and description of evidence C. Use of Tox-Lims is limited to authorized personnel. 1. Staff must be authorized as a system user by a supervisor. 2. Use of Tox-Lims requires a user name and confidential password. D. Tox-Lims utilizes a barcode label containing a unique case number to identify case evidence. E. The program is used to track all categories of specimens submitted to the Toxicology Lab 1. Dallas County Medical Examiner (M) cases 2. H-cases 3. D-cases F. Tox-Lims users must log in and log out of the system for each session. 1. Many of the program features use the identification of the person logged in to automatically populate certain fields. a) Therefore, a user must log in so Tox-Lims can accurately reflect who is using the database. b) A user must log out so other users can log in for a new session. G. Manual inventory and chain of custody may be used in place of Tox-Lims. III. Process Overview A. Initial evidence receipt and chain of custody is typically tracked using hard copy forms. B. Initial Processing for M-Cases and H-Cases. 1. Evidence inventory is performed using Tox-Lims. 2. The user logs into Tox-Lims using their user name and password. 3. Specimen are initially inventoried and logged in using Initial Log In of Specimen. 4. An Evidence Receipt is printed, initialed, and placed into the case file. C. Initial Processing for D-Cases 1. Evidence inventory is performed manually on the transmittal sheet. 2. Evidence is logged in to Tox-Lims to allow sample tracking. a) The user logs into Tox-Lims using their user name and password. b) Specimen Logging is used to initially enter D-cases into Tox-Lims. D. ToxLims is used throughout the analytical process to document specimen chain of custody for all toxicology specimens. 1. The user logs into Tox-Lims using their user name and password.
Tox-Lims Operating Instructions Version 2.0
E.
F.
G. H.
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2. Specimen Logging is used to track cases as they are checked in and out of the toxicology refrigerator/freezer or other location. a) It is assumed that the storage location is the Toxicology refrigerator/freezer. If another storage location is used, the individual taking or releasing custody of the specimen must note the storage location in the Comments field. Once analysis is complete, chain of custody documentation is printed from Tox-Lims. 1. The user logs into Tox-Lims using their user name and password. 2. Reports is used to print a case chain of custody which is initialed and placed in the case file. Within one month of the completion of analysis, samples are properly sealed and logged out of Tox-Lims. 1. The user logs into Tox-Lims using their user name and password. 2. LogOut Specimen to Long Term Storage is used to complete the chain of custody in Tox-Lims. 3. The case chain of custody is printed, initialed, and placed into the case file. 4. Specimens are properly sealed and may be released or stored. Chain of custody of specimens from this point forward is typically performed using hard copy forms. If a specimen in storage is identified for reanalysis, it may be logged back into ToxLims for identification and tracking. 1. Use the Specimen Logging function and follow instructions for D-Cases to track the sample in Tox-Lims. (It is not necessary to reinventory the specimens since this information is already in the case file.) 2. Upon completion of the additional analysis, specimens are logged out using the Specimen Logging function of Tox-Lims as with the D-cases. 3. A chain of custody form is printed, initialed, and placed into the case file. System Outage 1. If at any time Tox-Lims is down or cannot be accessed, a manual chain of custody must be kept and placed in the case file. a) A Temporary Possession of Specimens form (TPS) may be used to manually track evidence.
IV. Accessing Tox-Lims A. The log-on screen can be opened by double clicking on the Specimen Logging icon found on the desktop of all users.
B. Users access Tox-Lims by entering their user name and password. C. The main menu screen allows access to all of the functions of the software. D. The name of the individual using the software should appear in the upper left hand corner of the page. 1. If the message Not Logged In appears in the upper left hand corner of the page, the user must exit and attempt to log in again or contact a supervisor.
V. Log-in and Inventory of M-Cases and H-Cases

Dallas County Institute of Forensic Sciences Toxicology Laboratory 3 Tox-Lims Operating Instructions Version 2.0
A. The Initial Log In of Specimens screen is used to log in Medical Examiner (M) cases and H-cases. B. It allows the user to record the receipt of the specimen, inventory and describe the specimen, and print an evidence receipt sheet. C. From the Tox-Lims main screen, select Initial Log In of Specimens.
D. The Submissions screen opens ready to input a new submission (New Submission button) or add to a batch of previously submitted evidence (click in the line of the submission you want to add to).
E. To enter a new submission, select New Submission.
F. The New Submissions page opens.
G. Enter Received From information. 1. You may select an item from the drop-down list or type a custom name in the Received From field. H. Type the name of the individual that delivered the specimen in the Delivered By field. I. Select a time and date from the calendar that will appear when you click in the Received Date/Time field. J. In the Received By box, select the initials of the individual that received the specimen from the drop-down box or type them if they are not available. K. Click the OK button when all fields are populated. L. A new screen will appear, showing your entries. M. Review the data entered for correctness. 1. To correct an error in data entry, click on the Change Submission Info button. a) Do not try to correct an error directly from the populated boxes on this page. 2. The Change Submission Info button opens the Submissions page. 3. Select the appropriate submission information and correct as necessary.
4. Once corrections are complete, click OK to move back the below screen.
N. Click on the Case & Item box at the right of the Print button to activate it. O. Scan the barcode for the first tube or specimen to be logged in. 1. You may also type the case number and hit the Enter key. P. The specimen description box will appear. 1. Specimens should be inventoried in a manner that fully describes specimen appearance, identity, volume if applicable, and condition for the case record. 2. Assumptions should never be made about the type of specimens or the identity of a specimen. a) The submitter should be contacted to resolve questions. b) If questions cannot be clearly resolved, a supervisor should be contacted.
Q. Enter Specimen, Blood Type if applicable, Container, Amount if applicable, and description if applicable. 1. Data may be entered using a) the barcode scanner and selecting responses from the barcodes printed on the barcode sheet located near the log-in computer or b) using the dropdown boxes for each entry. c) If using the scanner, cursor will automatically move from one field to the next upon scanning data; if you select options using the dropdown list, you must tab from one field to the next. R. When data is correctly entered for this specimen, select OK. S. The Specimen Log In screen reopens, and the Case and Item box is active. T. Scan the barcode for the next piece of evidence in this case, the Specimens screen opens, and continue with step Q above. 1. You may also type the case number and hit the Enter key. U. Repeat this process until all of the specimen in a case are logged in. V. When all specimen for a case are logged in, click on the Print button to print the Evidence Receipt sheet. 1. Initial the Evidence Receipt sheet and place it in the case file.
W. Clicking the Print button clears the previous case information and allows you to begin entering another case using the same submission information. (see step O above.) X. When case entry for this submitter and/or submission is complete, click on the Exit button. 1. This will take you to the main menu and allow you to begin the process again for another submitter or perform other functions. 2. Multiple specimens may be logged under a single receipt date and time as long as it is the same submitter. 3. If different agencies submit samples at the same time, enter the time separated by 5 minutes. a) The software will not allow entries from different submitters at the same time. Y. To add additional specimens or cases to a previous batch (i.e. with receipt date, time, and submitter already in the system) 1. Select Initial Log In of Specimens from the main menu 2. Click on the appropriate line on the Submissions page 3. Click OK 4. If the submission does not appear on the screen, you may scroll down to find it using the scroll bar on the right. Z. To reprint an Evidence Receipt sheet 1. Select Initial Log In of Specimen from the main menu 2. Select the appropriate submission information a) If the submission does not appear on the screen, you may scroll down to find it using the scroll bar on the right. 3. Select OK once the appropriate submission is identified 4. The Specimen Log In screen opens. 5. Select Print (make sure that the Case & Item box is blank). 6. This will prompt a case number request. 7. Type the case number and hit OK. 8. The evidence receipt sheet for the specific case will print. VI. Log-in and Inventory of D-Cases A. D-cases are inventoried manually on the transmittal sheet submitted with the specimen. 1. Instructions for inventory of D-cases may be found in the Procedure for Toxicology Evidence Handling. B. Cases are logged in to Tox-Lims to allow tracking of specimen use and documentation of internal chain of custody. C. D-cases are initially entered into Tox-Lims by selecting Specimen Logging on the main menu of the Tox-Lims entry screen.
D. The Specimen Logging screen opens.
VII.
E. Select Login from the drop down list on the In/Out box. This will prompt the Case & Item box to appear. F. Scan the barcode on the tube. 1. Alternataively, you may enter the case number and specimen identifier and hit Enter. 2. An example of the format used for the case number and specimen identifier is 1234-07B. G. Scan or enter the next specimen and continue for all evidence in the case. 1. The Date & Time, In/Out, and User boxes will be filled in automatically: a) The Date & Time will be the current date and time. b) The User will be whoever is logged in to ToxLims at the time of the entry c) The In/Out is what the user has selected to do. d) You may also enter a comment if appropriate. 2. As a matter of practice, specimens are usually entered one case at a time. However, specimens may be entered into Tox-Lims irrespective of case number; specimens will be put in numerical order when the user closes out of the screen. H. Click Exit when done. Sample Tracking and Documentation of Chain of Custody (M, H, and D Cases) A. All transfers of specimen between locations and people must be tracked to document chain of custody. 1. Internal transfers are usually tracked electronically in Tox-Lims. 2. Transfers may be tracked manually on hard copy documents in the case file. B. Sample logging must occur at the time of specimen transfer from one person or place to another. C. Documentation of Chain of Custody using Tox-Lims: 1. Select Specimen Logging on the initial Tox-Lims entry screen.
2. The Specimen Logging screen opens.
3. Conventions for use of the In/Out dropdown list: a) Login is used to initially enter D cases (see Section IV) or re-enter other cases for reanalysis. b) Logout may be used in place of Log Out Specimen to Long Term Storage feature for certain cases. (1) This feature does not automatically print a chain of custody and is not the preferred method. c) Out is used when an analyst is taking possession of a specimen; for example, taking a specimen out of the refrigerator or freezer. (1) If the Toxicology refrigerator/freezer is not the site of specimen storage, the name of the secure storage location such as the hood must be entered in the Comment section. d) In is used when an analyst is returning a specimen; for example, putting a specimen back into the refrigerator. e) If one analyst is taking a specimen from another analyst, the specimen must first be logged in by the original analyst before the new analyst can log it out. (1) If the original analyst is unavailable, the new analyst can log the specimen in, and a prompt will appear that will allow the new analyst to explain why they are logging a specimen in for another analyst. 4. Select the appropriate entry from the In/Out dropdown list. 5. Scan the barcode of the first specimen container to transfer. a) Alternatively, you may enter the case number and specimen identifier and hit Enter. b) An example of the format used for the case number and specimen identifier is 1234-07B. 6. Scan or enter the next specimen, and continue for all specimens to be transferred. 7. The Date&Time, In/Out, and User fields are populated automatically upon case number entry. a) The Date&Time is the current date and time. b) The In/Out is selected by the user. c) The User is the analyst logged in to ToxLims at the time of the entry.
Dallas County Institute of Forensic Sciences Toxicology Laboratory 9 Tox-Lims Operating Instructions Version 2.0
8. Click Exit when done to return to the main menu. VIII. Logging Out Specimens to Long Term Storage A. Within one month of the conclusion of testing, specimens are properly sealed, logged out of Tox-Lims, and transferred to Long Term Storage. B. Log Out Specimens to Long Term Storage logs the specimen out of Tox-Lims and automatically generates a chain of custody record which is placed in the case file. C. M, H, and D cases are handled using this function. D. Before using this function, all evidence in a case must be accounted for and present as a group. E. To log out specimens from Tox-Lims, select Log Out Specimens to Long Term Storage from the main menu.
F. The Log Out to Long-term Storage screen opens.
G. Scan one of the tubes in the case that you wish to log out. 1. Alternatively type in the case number and tube identifier and hit Enter. H. The screen will populate with all of the specimens that were logged in under that case number.
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I. To log out the specimens in a case, scan each one using the barcode scanner. 1. Alternatively, enter the case number and specimen identifier and hit Enter. J. A check mark will appear in the Log Out box located at the right for each tube as you scan it verifying that all specimens were accounted for.
K. When all specimens for a case have been scanned and all boxes in the Log Out column are checked, a chain of custody report will print and automatically clear the screen for the next case. L. Continue until all applicable cases are logged out to storage. IX. Reports A. Tox-Lims provides several types of automated reports. B. Select Reports from the main menu.
C. Types of reports follow: 1. Print Chain for a Case: enter the case number and a copy of the internal chain of custody will print. 2. Specimen Checked out by an Analyst: enter the analyst username to list all specimen that are currently checked out to an individual analyst. a) Users may view any analyst that has specimen in their custody. 3. Items Logged in on a Specific Date: enter the date to view all cases/specimens that were logged in on a given date.
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X. Edit A. Tox-Lims allows editing of certain specimen specific or submission specific information. B. Select Edit from the main menu.
C. Edit Specimen Info: allows the analyst to edit specimen specific information such as the type of container, the description, the specimen code, etc. This will only update the specific specimen and will not be reflected for all specimen within a case.
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D. Edit Submission Info: allows editing of submission specific information. This is a global edit and will affect all specimens and all cases logged in under the specific submission. This can be used when there was a entry error for a particular submission such as a date or time or initial. Corrections may be made using this edit function prior to the completion of logging in or after the completion of logging in such as during the review process.
XI. Changing a User Password A. Change Password allows the analyst to change his/her confidential password. B. Select Change Password from the main menu and follow the prompts.
XII.
Exiting Tox-Lims A. Select Exit on the main menu to exit the Tox-Lims program. 1. A user must log off at the completion of an individuals session to allow others to log on and use the database.
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Dbase IV and ACCESS DWI DATABASE OPERATING INSTRUCTIONS

Overview: Blood and urine specimen are received from multiple police agencies for analysis of alcohol and drugs in driving while intoxicated (DWI) cases. The Dbase IV program is used as a database to log information for a given case such as name of suspect, submitting agency, type and number of specimen(s), submitting information, agency numbers, and dates. This information is linked to an Access 97 database that is used for billing, report generation, and printing batch results for archival purposes. DBASE IV Open the Dbase IV program by double clicking on the icon at the left. It is located on the desktop of the Toxicology Chemist III computer (DWI computer). This is an independent program and will run without the need for additional software or having Dbase installed on a computer. Upon opening the program will go to a menu screen. It may run a bit slow to open up on newer versions of windows and if it seems stalled, you can just hit enter and it should move to the open menu. The open menu is as follows:
Only items 1,2,3,7, and Q will be used. The other items are not used or are controlled through the Access 97 database that is discussed in more detail later in this document. You may enter the appropriate number or letter in the box next to ENTER YOUR CHOICE. The cursor will be in the box by default. 1. Log-in cases: this is used to enter information about the case such as name, receipt information, sample type, dates, agency information, etc.
Dallas County Institute of Forensic Sciences Toxicology Laboratory 1 DWI Database Operating Instructions Version 2.0
2. Enter alcohol results: is used to enter alcohol results only. It will require the addition of duplicate results. 3. Enter Drug Results: is used to enter drug results when specimens are tested for drugs. 7. Print receipt form: is used to print the inventory of the lockbox on the receipt form submitted by DPD. Q. Quit to DOS: is used to exit the program and return to Windows.
Log-in Cases When you are ready to enter case information and log in the cases, type 1 in the Open Menu selection box (Enter Your Choice). After typing 1 from the Open Menu the following screen will appear:
Type in the case number of the sample to be logged in or edited and hit enter. The following screen will appear:
Begin filling in the appropriate information and use the tab or enter key to move from one field to another. Use the following conventions on entering the data:
DWI Database Operating Instructions Version 2.0
NAME: Type last name first, first name last. OFF. DATE: This is the date of the offense. Type the day of the offense. The database will use the current month and will use the previous month if the day entered is later than the current day. For cases that are received where the offense occurred more than one month prior simply enter the day of the evidence receipt. # OF TUBES: Type the number of tubes received (typically blood/serum tubes). # OF CUPS: Type the number of cups received (typically urine cases). ARREST #: Type the arrest or service number of the submitting agency when available. UCR CODE: Use the space bar to select DWI (driving while intoxicated), DRE (drug recognition evaluation), IA (intoxication assault), or IM (intoxication manslaughter). AGENCY: Select the appropriate agency from the pick list that will appear when the field is selected. If the agency is not in the pick list select unknown from the list. This needs to be the agency in which the specimen will be returned. RCVD:DPD: Type T if the specimen were delivered from DPD and type F if it was any other delivery method. RCVD:EOR: Type T if the specimen were retrieved from the Parkland Lock Box and type F for any other delivery method. RCVD:OTHER: Type the name of the person delivering or the method of delivery such as M. Gibbons # 4419 or mail. RECVD BY: Type the name of the person that received the evidence. RECPT DATE: Type the date the evidence was received. Hit the enter key to write the data to the database. You will move to the case number input screen to input the next case numbers information or you may hit the escape key to move back to the Open Menu when you are done with all of the cases. At this point log the specimen into the Toxicology LIMS database for specimen tracking. Enter Alcohol Results At the completion of the alcohol analysis, type 2 from the Open Menu to enter alcohol results into the database. The following screen will appear:
Enter through these fields. These fields are no longer used. Another set of entry boxes will come up to enter control, standard, and response factor information. Enter through
these fields as well until you come to the field to enter your case number for the case of interest.
Enter the case number for the appropriate case and hit enter. The following screen will appear:
Enter your first and then your second alcohol results. Then type the name of the analyst that ran the alcohol assay. Hit enter. You will be prompted to determine if the case is completed. Type f for not complete and t if the case is complete after the alcohol analysis. You will return to the case number entry page. You may enter a new case or hit escape to return to the Open Menu. Enter Dug Results If a drug analysis was requested or if one was done per the protocol of the Toxicology Laboratory, type 3 on the Open Menu to enter these drug results. You will be prompted to enter the analyst that performed the work (in the case of multiple analysts, use the primary analyst).
Type the name of the analyst and hit enter to move to the next screen:
Enter the case number of the appropriate case. Always type F for the input box lableled A/N and ALK are negative? This is no longer used and a complete entry of all analysis done is needed for correct billing information. The following screen will allow you to input your drug results:
Use the following conventions on entering the data: SPEC: This is the specimen code used in Toxicology (03 = blood, 30 = urine, etc) ANAL: This is the analysis code used in Toxicology (97 = drug screen, 06 = thc screen, etc.). If entering multiple drugs under one analysis such as the drug screen, enter 99 in the anal field for all entries after the first. The first entry will be the real analysis code. GROUP: Group refers to the first two numbers in the four digit code for drugs. The drug code for diphenhydramine is 1121. The group is 11. CMPD: Compound refers to the second two numbers in the four digit code for drugs. The drug code for diphenhydramine is 1121. The compound code is 21. CONC: This refers to the concentration of the drug.
RESULT: Type the result of the individual drug here. This is what will show up on the report and needs to be spelled out with the drug concentrations. Even though the drug code and concentration have been entered, type the name of the drug in full, for example 0.005 mg/L carboxytetrahydrocannabinol. Another line will appear to enter more drug results. If you have no other results to enter, simply enter through the fields and you will return to the case number entry screen. You may enter another case or hit escape to take you back to the Open Menu. Print Receipt Form This menu item is used for receipt of the DPD lockbox and the associated paperwork only. After all of the items have been entered into the computer for a DPD delivery select 7 from the Open Menu. The following screen will appear:
Type the number of the first case of the batch received from DPD on a given delivery. This will move you to the next entry:
Type the number of the last case of the batch received from DPD on a given delivery. This will move you to the next prompt:
At this point insert the receipt sheet from DPD (shown below) into the printer so that the information will print appropriately on the sheet. This configuration may be different on each printer so printing a test page prior to inserting the DPD sheet into the printer is a good idea. A test page can be generated by following this process without putting the receipt sheet into the printer. This will print on a blank sheet of paper which can be compared to the spacing on the receipt sheet.
The printing will fill in the spaces under SWIFS Case#, suspects name, etc. This sheet is copied and a copy put in every case file that is represented in that batch. The original is returned to DPD upon their next delivery. Quit To DOS This saves and quits the database. You will return to your Windows Desktop.
Access 97 Database The access 97 database is linked to the DBASE IV database and is used for printing reports, billing sheets, results sheets, and editing reports. Double click on the Access icon shown on the desktop of the DWI computer.
This will open the database and bring you to the main menu. The main menu is:
When you are ready to print the reports for the cases that were entered in the DBASE IV database click on this button. You will be prompted for information on the following screen:
The report date is the date the report is generated (todays date). The Reviewer will be the analyst or supervisor that will be technically reviewing the case. The notary will be the person that will notarize the report if needed for the Department of Public Safety. After the correct data is entered, hit the Print Reports button and all available reports will print. These are cases that have been designated as complete in the DBASE IV database and have not been previously printed.
To print billing statements click on the correct button on the main menu. All available billing statements will print. These are designated as complete and not billed in the DBASE IV database. The billing statements will print and you will be returned to the main menu.
You may print results of the analysis by date or by case number. To print out reports by date click on the appropriate button. You will be prompted to enter the beginning date and the ending date. All results for every case between the chosen dates will be printed. These can be archived. The format for both case number approach and date approach is the same.
You may print results of the analysis by date or by case number. To print out reports by case number click on the appropriate button. You will be prompted to enter the beginning case number and the ending case number. All results for every case between the chosen cases will be printed. These can be archived. The format for both case number approach and date approach is the same.
If a case report needs to be edited click on the Edit DWI Letters button from the main menu. You will be prompted to enter the case number. You must use the format YYD0000 (07D0214). This will open Microsoft Word and the report you selected. You may make changes, additions, corrections, etc. Using standard Microsoft Word menu items, save the report and print the edited version. Below is the window that prompts you to enter the case number:
The cancel button will take you back to the main menu. The close form button will take you back to the main menu.
The exit button will save the database and exit the database.
11
HISTOLOGY SPECIMEN MANAGEMENT I. Requests: Requests for histology services will usually come from three sources: A. The initial submission of formalin fixed tissue from the Morgue submitted with toxicology specimens to the Toxicology Lab. B. A request from the Medical Examiner to the Senior Secretary for a special stain or recut; this request will usually be accompanied by an H&E slide to match with the paraffin block. C. A request from Records to the Senior Secretary for a recut of a slide set. II. Requests Received by Toxicology Evidence Registration A. Sealed histology specimens and a Histology Request form are delivered to the Toxicology Laboratory by the OME with other toxicology specimens typically via the Medical Examiner Toxicology Refrigerator (METR). B. Toxicology personnel receiving the specimens 1. Match paperwork and specimens a) Incomplete submissions are returned to the Morgue via the METR for further processing. 2. Complete chain of custody including: received from, received by, time, date, items received, and condition of the seal. 3. Place specimens in the outgoing histology box. III. Requests Received by Senior Secretary A. Requests for special stains and recuts are typically made by Records or the Medical Examiner to the Senior Secretary using the Histology Request form. 1. For special stains, H&E slides will usually be submitted along with the request form to allow submission of the proper paraffin block. B. Applicable blocks are pulled, placed into a properly sealed bag, and placed in the outgoing histology box in Toxicology. 1. The H&E slide if submitted is returned to the Medical Examiner. IV. Histology Vendor A. The Lab Aide or other staff transports specimens to the histology vendor for processing and notes the date transported on the Histology Request form. B. The Lab Aide or other staff picks up slides, blocks, and paperwork from the histology vendor notes the date received on the Histology Request form. 1. The Lab Aide reconciles the number of blocks received with the number of slides received and notes this on the Histology Request form. 2. Discrepancies are reported to the Senior Secretary for resolution. a) The Lab Aide will take blocks, slides, and paperwork to the Senior Secretary for resolution. b) Slides are not distributed to the Medical Examiner until all discrepancies are resolved. C. The Senior Secretary is responsible for resolving problems encountered by the histology vendor. 1. The Senior Secretary may verify spelling of names that are not legible with the histology vendor by phone.
Histology Specimen Management Version 2.1
2. Other discrepancies in case name and/or number require that the cassettes and paperwork be returned to the Institute for resolution prior to processing by the vendor. V. Slide Distribution A. Slides are placed into slide folders by case and by medical examiner and distributed. B. Discrepancies are reported to the Senior Secretary for resolution. 1. The Lab Aide will take blocks, slides, and paperwork to the Senior Secretary for resolution. 2. Slides are not distributed to the Medical Examiner until all discrepancies are resolved. C. The original Histology Request form is forwarded to the Senior Secretary for processing payment. VI. Block Storage A. Histology blocks are filed timely in numerical order by case and year by the Lab Aide. B. Blocks are stored for ten years. After this time they are available for disposal. C. Histology blocks may be held beyond ten years in additional one-year increments provided all of the following conditions are met prior to the anniversary date of the autopsy: 1. The Toxicology Laboratory must have a written request from the outside agency/individual requesting that histology blocks are retained for an additional one-year period. This written notice must include the case name; the IFS case number; the name, phone number, and complete mailing address of the agency/individual requesting specimen hold; and a description of the types of specimens to hold. 2. The Laboratory must be in receipt of a check for the amount of the Annual Specimen Storage Fee authorized by the Dallas County Commissioners Court. The check must be made payable to Dallas County and reference the case name and/or case number. 3. It is the responsibility of the agency/individual requesting hold of histology blocks to renew the annual specimen hold prior to the anniversary date of the autopsy. Otherwise, samples will be disposed per Institute policy. The Institute will not provide annual notice of this requirement to renew the sample hold request and provide payment. D. Payment of the specimen hold fee does not infer exclusive control of blocks nor does it guarantee release of histology blocks or production of slides. E. Blocks may be placed on hold by more than one entity. Should specimens be released by the action of one entity, effort will be made to preserve a portion of each block for use by other entities; however, this is not guaranteed. F. Production of histology slides 1. Recutting of blocks and production of slides requires written authorization from the next of kin/executor of the estate (and a copy of their drivers license or other government photo ID) or other legal authority, such as court order or subpoena, directing disposition of the specimens by this Laboratory in addition to approval of the Chief Medical Examiner, submitting agency, and/or proper legal authority.
Dallas County Institute of Forensic Sciences Toxicology Laboratory 2 Histology Specimen Management Version 2.1
2. Release of blocks in their entirety is at the discretion of the Chief Medical Examiner. G. Costs for recutting blocks, production of slides, specimen transportation, and handling are the responsibility of the requestor; the Chief Medical Examiner may require payment in advance. H. Dallas County reserves the right to change this policy at any time. Reasonable effort will be made to notify the outside agency/individual requesting the hold; however, no guarantee is made in this regard. It is the responsibility of the agency/individual requesting specimen hold to keep the Institute advised of address changes, etc. I. All correspondence and payment must be sent to Dallas County Institute of Forensic Sciences, Attn: Section Chief Forensic Chemisty, 5230 Medical Center Drive, Dallas, TX 75235 (214-920-5990). All correspondence and payment must be received prior to the anniversary date of the autopsy VII. Slide Storage A. Slides are returned to the slide holding area in the OME by the Medical Examiners. 1. Neuropathology collects and processes neuropathology specimens. a) The slides are returned to IFS and distributed to the appropriate medical examiner. b) Slides are returned to the slide holding area in the Fellows office and filed with other case slides. B. Slides are filed timely in numerical order by case and year by the Clerk/Typist. C. Slides are held indefinitely. D. Slides may not be released from the Institute without approval of the Chief Medical Examiner. VIII. Histology Vendor Payment A. The Senior Secretary receives the invoice from the histology vendor, reconciles the bill with services received, and RMRs the invoice. IX. Histology Request Form A. The original Histology Request form is filed in the Toxicology case file.
Histology Specimen Management Version 2.1
SOUTHWESTERN
INSTITUTE OF FORENSIC SCIENCES

AT DALLAS
TELEPHONE: 214-920-5990 FAX: 214-920-5812 5230 Medical Center Drive Dallas, Texas 75235
HISTOLOGY BLOCK STORAGE POLICY

1. ROUTINE STORAGE AND DISPOSAL: Histology blocks are routinely held at no charge for ten years from the date of autopsy and then disposed unless specific action is taken by the submitter or other interested individual as noted below. 2. REQUIREMENTS TO HOLD HISTOLOGY BLOCKS BEYOND 10 YEARS: Histology blocks may be held for additional one-year increments provided all of the following conditions are met prior to the anniversary date of the autopsy: a. The Toxicology Laboratory must have a written request from the outside agency/individual requesting that histology blocks are retained for an additional oneyear period. This written notice must include the case name; the IFS case number; the name, phone number, and complete mailing address of the agency/individual requesting specimen hold; and a description of the types of specimens to hold. b. The Laboratory must be in receipt of a check for the amount of the Annual Specimen Storage Fee authorized by the Dallas County Commissioners Court ($60). The check must be made payable to Dallas County and reference the case name and/or case number. c. It is the responsibility of the agency/individual requesting hold of histology blocks to renew the annual specimen hold prior to the anniversary date of the autopsy. Otherwise, samples will be disposed per Institute policy. The Institute will not provide annual notice of this requirement to renew the sample hold request and provide payment. 3. Payment of the specimen hold fee does not infer exclusive control of blocks nor does it guarantee release of histology blocks or production of slides. 4. Blocks may be placed on hold by more than one entity. Should specimens be released by the action of one entity, effort will be made to preserve a portion of each block for use by other entities; however, this is not guaranteed. 5. PRODUCTION OF HISTOLOGY SLIDES: Recutting of blocks and production of slides requires written authorization from the next of kin/executor of the estate (and a copy of their drivers license or other government photo ID) or other legal authority, such as court order or subpoena, directing disposition of the specimens by this Laboratory in addition to approval of the Chief Medical Examiner, submitting agency, and/or proper legal authority. Release of blocks in their entirety is at the discretion of the Chief Medical Examiner. 6. COSTS: Costs for recutting blocks, production of slides, specimen transportation, and handling are the responsibility of the requestor; the Chief Medical Examiner may require payment in advance. Requests for recuts of slides and costs of this service must be made to Records, Dallas County Institute of Forensic Sciences, 5230 Medical Center Drive, Dallas, TX 75230 (214-920-5922). 7. Dallas County reserves the right to change this policy at any time. Reasonable effort will be made to notify the outside agency/individual requesting the hold; however, no guarantee is made in this regard. It is the responsibility of the agency/individual requesting specimen hold to keep the Institute advised of address changes, etc. 8. CORRESPONDENCE: All correspondence and payment must be sent to Dallas County Institute of Forensic Sciences, Attn: Elizabeth Todd, 5230 Medical Center Drive, Dallas, TX 75235 (214-920-5990). All correspondence and payment must be received prior to the anniversary date of the autopsy. Version 2.1
EVIDENCE RETENTION, RELEASE, AND DISPOSAL I. IFS Medical Examiner Evidence A. Biological specimens submitted to the Toxicology Laboratory by IFS Medical Examiner staff are retained for a minimum of one year after collection unless directed otherwise by the Medical Examiner. 1. After that time, specimens are disposed as regulated biological waste unless a proper request to hold specimens is received. B. Non-biological specimens are released to the Medical Examiners Office at the completion of the case. C. Medical Examiners may request that specimens are retained for more than one year for their own use. II. Non-Medical Examiner Evidence A. Specimens are returned to the submitter at the completion of analysis unless arrangements are made otherwise. 1. A submitting agency may provide standing instructions for disposition of evidence at the completion of testing. 2. If standing instructions are not available, the submitting agency will be advised that specimens are available for release in person at the Institute during normal business hours. 3. If a submitter fails to respond to the notice of specimen availability, the specimens will be returned to the agency at the submitters expense. 4. Upon notification by the submitting agency, the Institute may dispose of biological specimens as regulated biological waste. (1) The Institute will usually not dispose of non-biological evidence. B. Agencies may choose to place specimens on hold and must follow standard procedures for holding and releasing toxicology specimens. III. Methods of Specimen Release A. Evidence is usually released in person, by mail, to a lockbox, or by express package service IV. Responsibilities - Responsibilities of the individual releasing evidence include the following, as applicable: A. Documenting the chain of custody B. Documenting the identity of items released C. Verifying that the evidence is properly sealed D. Verifying identity of the individual receiving the evidence if that person is not know to Toxicology staff in a personal transfer E. Ensuring that the package is properly packaged for transport V. Specimen Hold A. General Provisions 1. Upon request and payment of a fee as applicable, Toxicology specimens may be held for more than one year from collection or submission to the Laboratory. 2. Records are maintained regarding the storage, release, disposition, and/or disposal of specimens held by the Toxicology Laboratory. B. Retention of Evidence by Medical Examiners
Dallas County Institute of Forensic Sciences Toxicology Laboratory 1 Evidence Retention, Release, and Disposal Version 2.0
1. Medical Examiners may request that specimens are retained more than one year for their own use by submitting a written request to the Section Chief Forensic Chemistry. C. Retention of Evidence by Entities other than the Medical Examiner 1. The Toxicology Laboratory stores samples for one year from the date of receipt or collection at the Institute at no charge 2. Requests to hold evidence beyond one year may be made by both submitters and non-submitters. 3. Toxicology samples may be held for additional one-year increments provided the following conditions are met prior to the anniversary date of specimen receipt or collection at the Institute: a) The Toxicology Laboratory must have a written request from the outside agency/individual requesting that specimens are retained for an additional year. (1) This written notice should include the case name; the IFS case number; the name, phone number, and complete mailing address of the agency/individual requesting specimen hold; and a description of the types of specimens to hold. b) The Laboratory must be in receipt of a check for the amount of the Annual Specimen Storage Fee authorized by the Dallas County Commissioners Court, and the check should be made payable to Dallas County and reference the case name and/or case number. c) It is the responsibility of the agency/individual requesting specimen hold to renew the annual specimen hold prior to the anniversary date of sample receipt or collection at IFS; otherwise samples will be disposed per Institute policy. (1) The Institute will not provide annual notice of the requirement to renew the sample hold request and provide payment, and 4. Dallas County reserves the right to change this policy at any time. a) Reasonable effort will be made to notify the outside agency/individual requesting the sample hold; however, no guarantee is made in this regard. b) It is the responsibility of the agency/individual requesting specimen hold to keep the Institute advised of address changes, etc. 5. All correspondence and payment to Dallas County should be sent to the Chief of Forensic Chemistry, Dallas County Institute of Forensic Sciences, 5230 Medical Center Drive, Dallas, TX 75235. 6. This policy pertains to retention of specimen only and does not guarantee specimen release nor does payment of the specimen hold fee infer exclusive control of specimens. a) Specimens may be placed on hold by more than one entity. b) Should specimens be released by the action of one entity, every effort will be made to preserve a portion of each specimen type for use by other entities; however, this is not guaranteed c) Release of specimens requires approval of the Chief Medical Examiner, submitting agency, and/or proper legal authority.
Dallas County Institute of Forensic Sciences Toxicology Laboratory 2 Evidence Retention, Release, and Disposal Version 2.0
VI. Release of Specimens to Non-Submitting Agencies A. Typically, specimen release to non-submitting agencies requires written permission of the next of kin (M case) or submitting agency, a court order, or subpoena. 1. The Chief Field Agent will review and approve requests on behalf of the Medical Examiner. B. Costs of specimen handling and transport are the responsibility of the requestor. C. The releasing agent for IFS will document the items released and establish chain of custody for the release. D. It is the policy of IFS not to release the entirety of an IFS Medical Examiner specimen type without prior approval of the medical examiner responsible for the case or the Chief Medical Examiner. VII. Release of Specimens for Outside Testing A. Records will be kept regarding release of specimens for testing outside IFS including the date of specimen release, the type of testing ordered, the individual preparing the specimen for release, and the type and/or tube number of specimen(s) released. B. If the original container is released, a record to this effect will be included in the case file. C. Costs for specimen handling, transportation, and/or testing where applicable are the responsibility of the requestor and are usually collected in advance. VIII. Container Breakage and Specimen/Container Damage A. If a specimen container breaks or is otherwise damaged such that the original tube creates a safety hazard and/or the specimen is contaminated, lost, or rendered unacceptable for analysis, the container and/or contents may be disposed with proper notation made in the case file.
Evidence Retention, Release, and Disposal Version 2.0
SOUTHWESTERN
INSTITUTE OF FORENSIC SCIENCES

AT DALLAS
TELEPHONE: 214-920-5990 FAX: 214-920-5812 5230 Medical Center Drive Dallas, Texas 75235
TOXICOLOGY EVIDENCE STORAGE POLICY

1. ROUTINE STORAGE AND DISPOSAL: Specimen submitted to the Toxicology Laboratory will be held at no charge for one year after specimen collection or receipt at IFS. After this time, specimens will be disposed unless specific action is taken by the submitter or other interested individual as noted below. 2. REQUIREMENTS TO HOLD SPECIMENS BEYOND ONE YEAR: Toxicology samples may be held for additional one year increments provided all of the following conditions are met prior to the anniversary date of specimen receipt or collection at IFS: A. The Toxicology Laboratory must have a written request from the outside agency/individual requesting that specimens are retained for an additional year. This written notice must include the case name; the IFS case number; the name, phone number, and complete mailing address of the agency/individual requesting specimen hold; and a description of the types of specimens to hold. B. The Laboratory must be in receipt of a check or money order for the amount of the Annual Specimen Storage Fee authorized by the Dallas County Commissioners Court ($60). The check must be made payable to Dallas County and reference the case name and/or case number. C. It is the responsibility of the agency/individual requesting specimen hold to renew the annual specimen hold prior to the anniversary date of sample receipt or collection at IFS. Otherwise samples will be disposed per Institute policy. The Institute will not provide annual notice of this requirement to renew the sample hold request and provide payment. 3. Payment of the specimen hold fee does not infer exclusive control of blocks nor does it guarantee release of specimens. 4. Specimens may be placed on hold by more that one entity. Should specimens be released by the action of one entity, effort will be made to preserve a portion of each specimen type for use by other entities; however this is not guaranteed. 5. RELEASE OF SPECIMENS: Release of specimens requires written authorization from the next of kin/executor of the estate (and a copy of their drivers license or other government photo ID) or other legal authority directing disposition of the specimens by this Laboratory in addition to approval of the Chief Medical Examiner, submitting agency, and/or proper legal authority. Costs of specimen handling and shipping and additional testing are the responsibility of the requestor: $32 made payable to Dallas County if shipping fees are paid by the receiving lab or other entity or $64 made payable to Dallas County if Dallas County pays shipping fees. 6. Dallas County reserves the right to change this policy at any time. Reasonable effort will be made to notify the outside agency/individual requesting the sample hold; however, no guarantee is made in this regard. It is the responsibility of the agency/individual requesting specimen hold to keep the Institute advised of address changes, etc. 7. CORRESPONDENCE: All correspondence and payment must be sent to Dallas County Institute of Forensic Sciences, Attn: Elizabeth Todd, 5230 Medical Center Drive, Dallas, TX 75235 (214-920-5990). All correspondence and payment must be received prior to the anniversary date of sample receipt or collection at IFS. 10/07, Version 2.0
LABORATORY REFERRALS I. Referral laboratories are used to perform analyses not routinely provided by the Toxicology Laboratory including clinical diagnostic testing requested by the Office of the Medical Examiner. A. A list of commonly used referral laboratories and their specialty is provided in the attached Referral Lab and Specimen Selection Matrix. II. Responsibilities A. Evidence Registrar - The Evidence Registrar usually coordinates requests for reference laboratory referrals, prepares related paperwork, pulls specimens, and packages and ships specimens prepared by the analyst. B. Toxicology Chemist - The analyst typically has responsibility for reviewing the request, verifying appropriateness of the specimen and the referral laboratory, logging specimen out, aliquoting specimen into appropriate tubes, documenting the specimen sent on the original container and related paperwork, packaging and sealing the specimens for release, logging in specimens, and re-racking original containers. C. The Section Secretary has responsibility for tracking referral lab requests, distributing and filing results, and processing invoices for services. III. Process A. Referral lab requests are typically processed daily. B. A request for referral lab testing may be submitted by 1. OME a) Requests for infectious disease testing or other clinical diagnostic testing are submitted using on an Infectious Disease Exposure Request form, the Medical Examiners Request for Toxicology form, email, or phone. 2. Toxicology staff a) Toxicology staff reviews the service request form, investigative information provided, and testing results to determine when referral laboratory testing is indicated. C. Questions about referral laboratory samples should be referred to a supervisor prior to preparation of specimens. D. Appropriate specimens are checked out using Tox-Lims or manual chain of custody. E. A referral laboratory service requisition form or service request letter is prepared including the case name, case number, date specimen prepared, identity of specimen submitted, test requested, analyst name or initials, and other relevant information. 1. Usually the red top tube (D) is selected for clinical laboratory testing. 2. A gray top tube is usually selected for drug testing. 3. Additional information regarding submission of specimens may be found in the Referral Lab and Specimen Selection Matrix appendix to this procedure. F. The analyst marks the new aliquot container with a unique lab number: either the IFS case number or the referral lab requisition number. 1. If the referral lab requisition number is used, this is also placed on the original specimen tube as a reference and should appear on the Toxicology Laboratory copy of the requisition slip. G. The appropriate specimen is transferred to the new aliquot container.
Dallas County Institute of Forensic Sciences Toxicology Laboratory 1 Reference Laboratory Referral Version 2.0
1. If serum was removed from the original container, the original container is marked serum removed to advise other users that the composition of the tube has been altered. H. The container is placed in a biohazard transport bag and properly sealed. I. If the specimen requisition form contains a chain of custody section, it is completed. J. The original specimen requisition form or letter is inserted into the outside of the biohazard transport bag and sent with the specimen. K. The Toxicology Laboratory retains a copy of the requisition form or letter which is placed in the case file. L. A notation is made of all reference lab sendouts in the Drug Send Out line of the Quality Control form. The name of the test sent out is noted and a requisition form specimen identification sticker is attached as applicable. M. On some occasions, the original specimen container and its contents are sent to the reference lab. 1. A note is made on the original specimen receipt form (Medical Examiners Request for Toxicology) that the original container was released, by whom, and the date. 2. In addition, this information is placed on the Toxicology Labs copy of the requisition sheet or letter. 3. A notation is made in Tox-Lims if applicable. N. Original specimens are logged back in. IV. Sample Handling and Shipping A. Lockbox Specimens to be sent to the clinical laboratory vendor are placed into a lock box for courier pickup. B. Personal delivery Specimens may be delivered to the referral lab in person; for example, insulin specimens are transported to the UT-Southwestern Diabetes Laboratory. C. Overnight package delivery service 1. Specimens should not be sent by overnight package delivery on Friday or before a holiday. 2. The biohazard bag containing the specimen is placed into another fluid resistant bag with a liquid absorbent to contain an unexpected leak in transit. 3. The packaged specimen is placed into a shipping container such as a foam box with cold pack, if appropriate, and placed into a plastic, shipping bag such as a FedEx diagnostic specimen envelope. 4. Shipping documents are prepared and package pickup is arranged. D. Questions regarding proper shipping preparation should be directed to the package carrier such as FedEx. V. Distribution of Referral Laboratory Reports A. The Toxicology Lab copy of the requisition form or letter is transferred to the Section Secretary for entry into the Referral Log. B. The secretary matches service requests with lab results, files the Toxicology Lab copy of the service request and the original outside lab report in the case file, distributes copies of results as appropriate, and processes invoices for payment. 1. Results of referral toxicology testing are reviewed by a supervisor prior to distribution to the submitter (typically the Medical Examiner).
Dallas County Institute of Forensic Sciences Toxicology Laboratory 2 Reference Laboratory Referral Version 2.0
2. Results of clinical testing are distributed to the Medical Examiner without review. C. A copy of all infectious disease (HIV, hepatitis, and RPR/syphilis) reports are also distributed to the Chief Field Agent for review and submittal to the local health authority as appropriate. D. Submitters receive a copy of the referral laboratory report. 1. Results of outside laboratory testing are not transcribed into the Toxicology Report. VI. Procedure: Infectious Disease Submission Criteria for ViroMed Labs A. ViroMed/LabCorp will not analyze specimens received outside the following collection windows: 1. Refrigerated specimen within 5 days of collection a) 81337 Hepatitis B Core Antibody total 2. Refrigerated specimen within 7 days of collection a) 81347 Hepatitis B Surface Antigen b) 81351 Hepatitis C Virus Antibody c) 81204 HIV Antibody d) 81300 RPR (syphilis) e) Panel 76504: HIV Antibody, Hepatitis B Surface Antigen, Hepatitis C Antibody, RPR f) Panel 76502: HIV Antibody, Hepatitis B Surface Antigen, Hepatitis C Antibody 3. Frozen specimen within several months of collection a) All tests listed above B. To respond to this policy the following will be implemented 1. The Evidence Registrar will review infectious disease requests on the day they are received. 2. Requests received which are outside these collection windows (greater than 5 or 7 days) will not be tested. 3. The Evidence Registrar will a) Write Lab unable to test; specimen outside acceptable collection window on the infectious disease request form. b) Give a copy to the applicable Medical Examiner c) Give the original to the Section Secretary. 4. Requests received which are at the collection window will be communicated that day to the Tox Chemist assigned to send-outs. a) Tox Chemist will spin down sample and freeze serum in a plastic tube and then send for testing. 5. The Tox Chemist assigned send-outs will process infectious disease send-outs daily. 6. If a specimen is noted to be at or near the cut-off, it will be spun down and serum frozen in plastic prior to sending to Viro-Med/LabCorp.
Reference Laboratory Referral Version 2.0
Referral Lab and Specimen Selection Matrix Test Standard clinical tests Laboratory Recommended Specimen serum: thyroid panel, LabCorp place specimens in lockbox lipid panel, glucose, for transport antibodies 972-566-7500 blood: hemoglobin fractionation, sickle www.labcorp.com cell prep Comments Refer to bid for test codes. Refer to www.labcorp.com for specimen requirements. Hemoglobin A1C, hemoblobin fractionation, or sickle cell prep: Place blood in purple top tube. Refrigerated specimens must be received within 5 days of collection to be acceptable for testing. Serum should be frozen before shipping if the hold period will be exceeded. 76504: RPR, HIV, HBSAG, HCV 76502:HIV, HBSAG, HCV 81337: HBCore Ab 81347: HBSAG 81351: HCV 81204: HIV 81300: RPR 81182: Western Blot
Infectious disease testing
LabCorp/Viromed 6101 Blue Circle Drive Minnetonka, MN 55343 800-582-0077 www.viromed.com
Serum is recommended; blood may be sent if serum is not available.
Tryptase
Dr. Lawrence Schwartz Division of Rheumatology, Allergy and Immunology Virginia Commonwealth University MCV Campus 1112 East Clay Street McGuire Hall, Room 4110 Richmond, VA 232980263 804-828-6322
1-2 ml blood
Test Drugs/chemicals
Laboratory
National Medical Services 3701 Welsh Road Willow Grove, PA 190900437 800-522-6671 www.nmslab.com Insulin UT-Southwestern Diabetes Laboratory G.5.246 214-648-2868 Steriods Dr. David L. Black Aegis Analytical Laboratories 345 Hill Ave Nashville, TN 37210 615-255-2400 www.aegislabs.com Metabolic testing Pediatrix PO Box 219 90 Emerson Lane Bridgeville, PA 15017 412-220-2300 www.pediatrix.com Venom antibody Johns Hopkins University tests Asthma and Allergy Ctr. DACI Reference Laboratory Room 1A20 5501 Hopkins Bayview Circle Baltimore MD 21224 800-344-3224 www.hopkinsmedicine.org /allergy/daci/index.html
Recommended Specimen Blood from gray top usually sent. LSD usually screened in urine; wrap in foil to protect from light. 2 ml serum
Comments Refer to NMS Directory of Services for test numbers and specimen requirements: www.nmslab.com
urine or blood
blood
Use specimen submission cards and packaging.
serum
Covers all common wasps and bees in developed world: anti-yellow jacket, honey bee, yellow hornet, white faced hornet, Polistes wasp, fire ant, bumble bee (note: yellow jacket has some cross reactivity with yellow hornet and white faced hornet)
Other
Mayo Medical Laboratory 3050 Superior Drive NW Rochester, MN 55901 800-533-1710 www.mayomedicallaborato ries.com
Factor V Leiden and prothrombin
Molecular Diagnostics Lab UT-Southwestern 214-648-5105/4075
blood less than 5 days old, frozen tissue (liver/kidney/lung)
REPORTS AND RELEASE OF INFORMATION I. Results Reporting A. At the completion of testing, the case is reviewed and signed by the designated analyst, a report is generated, the case is administratively and technically reviewed by a supervisor who signs the final report, and a copy of the report is sent to the agency requesting the analysis. 1. A billing sheet is generated that provides for billing of services rendered. 2. If the final report is provided to entities other than the submitting agency, this will be noted in the case file or addressed by standing practice. A) D case results are made available to the Dallas County District Attorney via database. B. The final report must accurately reflect the work performed and be fully supported by data present in the case file. C. The report will include the unique case number, a description of the evidence received, notation of tests performed, listing of substances identified and quantitated as applicable, the date the evidence was received into the laboratory, who delivered it, who received it, the date of completion, and the submitting agency. 1. The final report for M cases is a multi-page document that includes the following documents: Toxicology Report, Medical Examiners Request for Toxicology, and Evidence Receipt. D. If it is determined that a revised or corrected report must be generated, the report will bear the notation Corrected Report. 1. Copies of a corrected report are sent to agencies receiving the previous copy of the report from Toxicology. 2. The corrected report is filed along with the original report in the case folder. 3. A copy of the corrected report is sent to the Quality Manager for review. E. If additional work is requested on a completed case, the report of additional testing will usually be added to information in the original report, the new report will be marked Amended Report, and a copy will be provided to all agencies receiving the original report from Toxicology. 1. If the report provides only the results of the new testing, it will be marked Supplemental Report and distributed as above. II. Release of Information to the Submitting Agency A. Release of Preliminary Results 1. Case results are considered preliminary until a final report is generated. 2. At the discretion of a supervisor, preliminary results may be released to the submitting agency after case review by a supervisor. a) In extenuating circumstances, preliminary results may be released by an analyst after review of the case by a second analyst; the case should be reviewed with a supervisor as soon as possible. 3. Documentation of release of preliminary results should be made in the case file including the information released, to whom, and when. B. Toxicology staff is authorized to provide a case status report to requesting entities. C. Release of Complete Reports
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1. A copy of the completed and signed report is provided to the submitting agency. D. Requests for interpretation of toxicology results should be referred to a supervisor or the Medical Examiner if applicable. III. Release of Information to Non-submitting Agencies A. Typically case results and other case related information will be released only to the submitting agency or entities authorized by the submitting agency. B. Requests to release information to other entities will follow the direction of or be referred to the submitting agency or the Civil Section of the Dallas County District Attorneys Office. IV. Security of Case Files A. The case file consists of the final report, all analytical work performed by the analyst which is reflected in or relied upon for the written report, sample submission documents, chain of custody and sample disposition documents, instrument output, worksheets, records of conversation, photos, peer review sheets, and/or other relevant information. B. During analysis, the case file is maintained by the Toxicology Evidence Registrar for M and H cases and by the chemist performing legal alcohols for D cases. C. Completed case files may be stored on-site in Section file storage or off-site in the Dallas County Records Storage Facility. 1. Files are retained in accordance with Dallas County policy as implemented by the Dallas County Records Manager. 2. Access to case files is restricted to Toxicology staff and Section administrative personnel.
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COURT TESTIMONY, DEPOSITIONS, and other LEGAL PROCEEDINGS 1. General 1.1. The Dallas County Institute of Forensic Sciences is an independent laboratory which provides services on a fee for services basis under direction of the Dallas County Commissioners Court and the Director of the Institute. 1.2. As such, the Institute is not part of any police agency, the Dallas County Sheriffs Office, or the District Attorneys Office. 1.3. Most cases submitted to the Laboratory are forensic in nature and employees may reasonably be expected to provide testimony in legal proceedings regarding facts and opinions related to submitted evidence. 1.4. As outlined in the mission statement of the Institute, it is the goal of Institute staff to perform forensic laboratory analyses with accuracy, integrity, and reliability and to present written and oral results of analyses in a complete and impartial manner. 2. Fact Testimony 2.1. All employees of the Forensic Chemistry Section are subject to call for fact testimony by virtue of their employment with the Institute. 2.2. This type of testimony is used for building a record at trial concerning events occurring prior to trial such as receipt and release of evidence, custody of evidence, typing of reports, etc. 2.3. Employees called as fact witnesses must limit their testimony to personal actions taken (receipt and release of evidence, record generation, lab number assignment, typing, etc.). 2.4. Use of laboratory records by fact witnesses shall be limited to identification of applicable records and actions personally conducted by the employee. 2.5. The fact witness should not respond to questions about legal or scientific aspects of the case; these should be referred to other witnesses. 3. Introducing a Business Record 3.1. Laboratory staff may be called to introduce a laboratory report as a business record. 3.2. This typically occurs when the testifying staff did not perform work on the case under discussion. 3.3. The witness will answer a standard set of questions designed to establish the case report as a business record. 3.4. Then the witness will read the report into the record. 4. Expert Witness Testimony 4.1. Most testimony performed by analysts and supervisors will blend fact testimony with opinion or expert testimony. 4.2. Testifying staff are expected to maintain proficiency in the following areas: 4.2.1. A thorough understanding of applicable Texas and federal laws. 4.2.2. A broad general knowledge of natural science as applicable.
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4.2.3. An appropriate subject matter expertise in the employeees discipline. 4.2.4. Knowledge of the legal requirements for maintaining chain of custody of evidence and for ensuring care, custody, and control of evidence and official records of their analyses. 4.2.5. Mastery over the scientific and technical aspects of the analytical methods used and the legal and scientific certainty of the validity of their results. 4.2.6. The ability to defend their analytical findings under court examination with clear and concise testimony using acquired forensic skills. 4.3. Until such time as an employee is considered acceptable by the Section Chief to give courtroom testimony, he shall not provide expert testimony in those cases in which he has prepared the report but shall refer such cases to the designated analyst or supervisor who will have co-signed the case report. 4.4. Expert witness testimony will be limited to the analysts area of expertise and will be further limited to testimony in cases in which the analyst actually performed work, work was performed under their supervision, or testimony is provided with approval of a supervisor. 4.5. The analyst is responsible for testimony concerning laboratory policy or interpretation of the law only as it pertains to the analytical conclusion reached in the specific case at trial. 4.6. Because expert knowledge is not subject to subpoena, analysts cannot be required to provide testimony in any case in which they did not perform analyses. 4.7. Permission to conduct outside employment requires prior permission of the Section Chief forwarded up to the Institute Director; the policy relating to outside employment is covered in the Dallas County Code. 4.8. An employee providing testimony as part of his County duties, must produce a subpoena unless testimony is provided for the Dallas County District Attorneys Office or meets other standing arrangement. 4.8.1. Pre-arrangement has been made with the Dallas County District Attorneys Office to honor telephone or fax requests in order to avoid the expense and inconvenience of issuing subpoenas for Institute employees. 4.9. Individuals leaving the building to go to Court should check out with the Section Secretary or Evidence Registrar. 4.10. All requests including subpoenas for testimony which is unrelated to the analysis of a specific Institute case must be reviewed prior to trial with a supervisor or Institute Director in the absence of a supervisor. 4.11. The individual receiving a subpoena is responsible for advising the requestor of applicable preparation, testimony, and travel fees to be charged by Dallas County related to the subpoena; written agreement to pay fees should be received from the requestor prior to the date of the proceeding. 4.11.1. Disagreement about payment of fees should be brought to the attention of a supervisor and the Civil Section, Dallas County District Attorneys Office, prior to the proceeding. 4.11.2. The Dallas County Commissioners Court has ordered that payment is required in advance (two hours minimum) for all services provided to private attorneys and any costs in excess of the minimum are payable at the time service is rendered.
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4.11.3. At the discretion of the Director, payment may be required before any service is rendered. 5. Testimony in the Capacity of Supervisor 5.1. The Eighth District of Texas, Court of Appeals in the case of Antoine Delano Caw v. the State of Texas (No: 08-92-00155-CR) has determined that chemists employed by the Dallas County Forensic Laboratory do not meet the definition of law enforcement personnel. Therefore, test results sponsored by a supervisor are admissible as a business records exception to the hearsay rules. 5.2. A supervisor usually enters the report as a business record. He then provides testimony regarding education and training of the analyst and their oversight of the analyst. After review of case file documentation, the supervisor can also testify as an expert witness based upon his own evaluation of data in the case file. 6. Dress 6.1. Professional business attire should be worn to all legal proceedings. 6.2. This usually includes a tie and/or jacket for men and dress, skirt, or pantsuit for women. 6.3. Shorts, jeans, and tee shirts are not appropriate. 6.4. Physical appearance should be neat and professional. 6.5. Dress code for federal may be more stringent; direction is usually provided with the subpoena if applicable. 7. Review of Testimony 7.1. Testimony of staff will be reviewed annually as described in the Quality Management Program. 8. General Guidelines for Testimony 8.1. Tell the truth at all times. 8.2. If you do not know or are unsure of an answer, clearly state this; you may state that you are not trained in this area, refer the question to a supervisor, etc. 8.3. Remain composed and professional; treat all parties with respect. 8.4. Keep on sound scientific ground; stay within your area of expertise. 8.5. Be prepared; review case prior to trial. 8.6. Understand the difference between possibility and probability. 8.7. Do not answer questions you do not understand. Ask for questions to be repeated or rephrased. 8.8. Do not provide answers to unasked questions. 8.9. Explain scientific concepts in clear laymans terms; avoid use of jargon. 8.10. Speak to the jury (jury trial) or judge (trial before the court). 8.11. When problems or confusion arise, speak to the Judge. 8.12. Speak slowly and distinctly. Speak in a tone that can be heard by the jury and court reporter.
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8.13. Discuss any concerns with your supervisor.
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QUALITY MANAGEMENT I. General A. Quality control and quality assurance are integral parts of the forensic activities performed at the Institute. B. Quality processes are described in detail in the Quality Manual and procedure specific quality issues are noted in procedures. C. Results of quality control testing may be found in the case file and/or in the Toxicology Laboratory. II. Toxicology Quality Committee A. The Toxicology Quality Committee is made up of two or more Toxicology Chemists selected by the Toxicology Supervisor. Criteria for serving on the Committee includes but is not limited to a broad understanding of overall laboratory function, organization and communication skills, attention to detail, etc. 1. It is important that at least two individuals serve on this Committee so that no member reviews their own work. B. Committee responsibilities include 1. Review, sign, and archive each quality control run in the Toxicology Laboratory a) Review includes the following as applicable (1) Compare QC results to target and acceptable ranges (2) Review QC for typographical errors (3) Review linearity of quantitation curves (4) Review peak shape and area counts (5) Review QC for consistency with written procedures 2. Maintain awareness of processes and procedures used in the Laboratory and identify departures from written procedures or good laboratory practice 3. Recommend changes to procedures, policies, and processes to ensure and improve Laboratory quality 4. Advise Laboratory staff and/or a supervisor to initiate action when QC is out of tolerance or other quality issues are identified C. The Quality Committee functions independently within the Laboratory with reference to quality process review. 1. When quality issues are identified that cannot be addressed through routine policies and procedures, the Quality Committee member should immediately bring this to the attention of the Toxicology Supervisor, Lab Chief, and/or the Institute Quality Manager. a) Disagreement regarding appropriate action will be referred to the Executive Committee. D. All Toxicology staff is expected to 1. actively participate in quality processes as outlined in the Institute Quality Manual and 2. run appropriate QC as noted in analytical procedures 3. place QC documentation in designated area for review by the Quality Committee in a timely manner
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4. participate actively with the Quality Committee to resolve noted issues and to further quality processes in the Laboratory. III. Expiration Dates A. Expiration dates for reagents, standards, and chemicals are as follows unless noted otherwise in a procedure: 1. Expiration dates are established by the manufacturer of the reagent, standard, or chemical. 2. The expiration date for reagents, chemicals, and standards made in-house is one year from the date the item was made. IV. Case Review A. Primary Analyst - The Primary Analyst is defined as a Toxicology Chemist who has completed Stage I of training and who performed the Alkaline and Acid/Neutral Drug Screen on the case or as designated by the Toxicology Supervisor. B. Case review is conducted as follows for Toxicology cases: 1. Each analyst performing work on a case will review their own work for completeness, accuracy, and consistency with procedures and policy prior to entering results into the case file. 2. The Primary Analyst will perform a review of the entire case contents as noted below, initial each page of the technical case record, and sign the case report indicating that the review was acceptable. 3. A supervisor will perform a technical/peer review of each case prior to release and sign the report indicating the review was acceptable. C. Components of Primary Analyst Toxicology Case Review 1. Administrative Review The Primary Analyst will ensure that the following conditions are met where applicable: a) The case report is free of typographical errors. b) The case number is on each page in the case file. c) Analyst initials are on each non-administrative page of the case file. d) Evidence registrar initials are on applicable administrative paperwork. e) Specimen tube designator is on all analytical work. f) Appropriate units are used and dilutions are properly noted and factored into calculations. (Note: pay special attention to tissues and non-routine situations) g) Case file contains appropriately completed: (1) Evidence Receipt and Chain of Custody (2) Administrative Billing Summary (3) Quality control sheets (4) Tissue Homogenate Worksheet as applicable h) All test requests have been run, sent out or accounted for. i) The meaning of all notes is clear. j) Notes are legible and initialed or signed. k) The Primary Analyst should review Substance Suspected, Relevant Findings, or other submitter case information to ensure that this information has been addressed during analysis as applicable. 2. Technical Review The Primary Analyst will ensure that the following conditions are met where applicable:
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a) The case report accurately reflects the work conducted and is consistent with Institute and Laboratory policy and procedures. b) Positive screening results have been appropriately confirmed and substances identified on multiple assays are consistently present: (1) ELISA: opiates, cocaine, THC, etc. are confirmed by specific assay (2) A/N: presence of valproic acid, salicylate, ibuprofen (sendout), theophylline (send out) are confirmed by specific assay or sent out as applicable (3) Alkaline: presence of cocaine, hydrocodone, codeine, carisoprodol, meprobamate, alprazolam, temazepam, etc. are confirmed by specific assays (4) A/N: positives are quantitated, (5) A/N: carbamazepine is verified on Alkaline Screen c) Proper specimens are analyzed. d) Urine or vitreous opiates were run appropriately following a positive morphine in blood. e) Alcohol was analyzed using two different blood tubes and vitreous run in two different batches as appropriate. f) Femoral blood was used appropriately, i.e. alkaline drugs were given priority. g) Electrolytes were given priority over alcohols when limited vitreous was submitted. h) Sendouts (1) Substances identified but not quantitated were sent out as applicable, for example, fentanyl, high ibuprofen, theophylline, ephedrine, etc. (2) Testing not performed by IFS has been sent to appropriate reference lab. i) Analytical (1) Alkaline and A/N Drug Screen (a) Consistent peak patterns appear on both columns, coelution has been reviewed, and drugs are properly reported. (b) A complete mass spectrum is included for all reported analytes (c) Quantitation is within linear range and dilutions have been made as appropriate. (d) Automatic instrument quantitation matches the calculated and reported results as applicable. (e) Drugs reported in GC/MS library search have been evaluated in the FID chromatogram. (f) Drugs identified in the alkaline or A/N GC have been evaluated on GC/MS.
(g) Search has been made for co-eluting components on GC and GC/MS, for example, zolpidem, quinine, others, etc. (2) Alcohol and Acetone Assay (a) Alcohol duplicates are within tolerance allowed in procedure. (b) Alcohol results between specimens meet tolerance; if not, all tubes have been analyzed and reported. (3) SIM Methods (special assays) (a) Curve fit is acceptable (above 0.995 on linear fit) (b) Reported results are within linear range or reported as > or < a value. (c) Qualifiers are within acceptable range. (d) Dilutions have been accounted for in calculations. (4) Electrolytes and CO assays (a) Duplicates are with acceptable agreement. (b) Unsuitable samples in which reports are not generated are documented appropriately. (5) General Analytical Review (a) Results identified in two or more assays are in general agreement. (b) Metabolites and parent drugs are present in reasonable proportion. (c) Inconsistent or unexpected findings have been evaluated. (d) Quantitations reflect dilutions; pay special attention to tissues. (e) Duplicate analyses are within expected tolerance. (f) Appropriate confirmation has been done (i) drugs of abuse including phencyclidine, methamphetamine, amphetamine, cocaine are confirmed on a second specimen or tube as applicable. (6) ELISA or another technique (SIM) done is performed on appropriate analytes. 3. Completion of Primary Analyst review a) Once the case review is successfully completed, the Primary Analyst will sign the case report. D. Technical/Peer Review 1. Technical/peer review is conducted by a supervisor including Toxicology Chemist III, Toxicology Supervisor, and/or Section Chief or Deputy Chief. 2. A Primary Analyst may not technical/peer review their own case. 3. Toxicology cases receive 100% technical/peer review prior to report release. a) Components of the technical/peer review include (1) Repeat of the Primary Analyst review detailed above. (2) Technical/Peer review elements noted in the Quality Manual.
V. Testing Validation A. Validation and verification of new testing methods is summarized in the Quality Manual. B. The Supervisor, Deputy Chief, and/or Section Chief will typically design a specific testing protocol to validate the accuracy of a new method or to verify the accuracy of a method transferred from one instrument to another. 1. Transfer of a Method to New Instrumentation a) Proper calibration and operation of new instrumentation are verified and documented using the applicable instrument calibration process. b) Analytical results are compared between the new and old instrumentation. (1) Where possible, side by side operation of old and new instrumentation is compared using the same samples or extracts. c) Standards, internal standards, and quality control samples should meet their respective criteria described in the analytical procedure. d) Test specimens should also be run and should meet acceptability criteria established for quality control samples or similar proficiency test results. e) Replicate analytical runs should be made on a single day and also compared between multiple days; criteria should meet those established for standards, internal standards, and quality controls; test specimens should meet acceptability criteria established for quality control samples or similar proficiency test results. f) As applicable, analysts are trained in operation of new instrumentation. 2. Development of a New Analytical Method a) A new analytical method may be proposed based upon literature, other established procedures, or knowledge and experience of the materials to be analyzed. b) The target sensitivity and linear range is established based upon expected sample conditions and instrument performance. c) Standard curves are run to assess the applicability of the proposed analytical technique in comparison to the targeted analytical needs. d) A formal procedure is written. e) The method is evaluated for interference from expected matrices or cooccurring substances. f) The method is evaluated for stability by repeating analysis of single prepared samples and duplicate preparation of samples both within day and between day. g) Variability of standards, internal standards, quality control samples, and specimens are evaluated with respect to literature values and/or similar established procedures. h) Criteria for acceptable standard curves, internal standards, and/or quality control samples are established. The method is revised and revalidated as necessary. Applicable analysts are trained, and results are reviewed for stability within analyst.
i) Method results may be evaluated against external standards or specimen results from reference laboratories; criteria for acceptability will be similar to that established for quality control samples VI. Critical Reagents A. Critical reagents listed in this procedure manual include: 1. Standards 2. Internal Standards 3. Quality Control Samples VII. Housekeeping A. Laboratory cleanliness is the responsibility of the chemists, registrars, and lab aids that work in the toxicology lab. B. There are two types of cleaning that will take place: scheduled and unscheduled cleaning. C. Each area that is to be cleaned on a scheduled basis has been assigned a number (see Toxicology Cleaning Map at the end of this procedure). D. All paper towels used to clean and all bench top absorbent paper are considered a biohazard and must be disposed of properly. E. All areas are to be cleaned with a 10% bleach solution that is prepared fresh with each use or approved disinfectant. F. Appropriate safety measures should always be followed when cleaning. G. Lab coats, lab gloves, and other appropriate safety gear should always be worn. H. Situations involving broken glass should be handled according to procedures outlined in the EHS Manual. I. Unscheduled Cleaning 1. Unscheduled cleaning is performed when the need arises and is independent of the scheduled cleaning. a) Examples of unscheduled cleaning include biological spill, chemical spill, exposure of an area to a biological, or directly after performing work such as extractions. 2. Unscheduled cleaning can be as simple as clearing an area of used glassware and putting away specimens (after extracting) or as complex as a complete removal of absorbent bench paper, disinfecting benches, and replacing absorbent paper after a biological spill. 3. Unscheduled cleaning is performed on the affected area or areas such as bench tops, floors, instruments, or computers as needed. J. Scheduled Cleaning 1. Scheduled cleaning is to be done on a routine basis and recorded in the scheduled Toxicology Cleaning Checklist except as noted below. 2. It is the responsibility of all chemists, registrars, and lab aids working in toxicology to adhere to, execute, and document the scheduled cleaning. 3. Refer to the Toxicology Cleaning Map at the end of this section for more detail: a) Areas 1- 6 are sample prep areas generally used by chemists. These are located in the main toxicology lab.
b) Area 7 is the specimen receiving area and includes specimen receiving, both specimen logging computer areas, and the refrigerator/freezer. (1) Although the refrigerator/freezer is the responsibility of the evidence registrar, each chemist must do their utmost to maintain a clean refrigerator/freezer. This includes returning specimen in an accurate and timely manner to the proper location, maintaining control of reagent storage, and keeping their respective box clean. c) Area 8 includes two hoods in the main toxicology lab. These are the water bath hood and the waste hood. d) Area 9 is the tissue preparation hood located in the back of the specimen logging area. 4. Daily Cleaning a) Each analyst must clean their work area when work is completed; this routine cleaning is not recorded on Cleaning Checklist. (1) This includes putting away specimen, picking up glassware, replacing reagent bottles to their proper location, and emptying waste containers. b) Each biohazard trash box must be evaluated at the end of the day to determine if a replacement is necessary; this is not recorded on the Cleaning Checklist. (1) These boxes should not exceed 20 pounds and if there is a question about the weight they should be replaced. c) The evidence receiving area is the responsibility of the evidence registrar. Specimen should be put away at the end of each day and the area disinfected. 5. Weekly Cleaning (last day of workweek) a) Straighten areas around all instrumentation and disinfect as appropriate. (1) The Tecan, Hamilton, Nova, and Co-oximeter will be wiped down in the areas that come in contact with specimen. b) Change the absorbent paper on each bench top and disinfect the bench top with freshly prepared bleach solution. c) Change the absorbent paper in the evidence receiving area and disinfect the bench top with freshly prepared bleach solution. d) Change the bench top biohazard disposal bags. e) Wipe down centrifuges. f) Dump the waste from the CO-oximeter. g) Check reagent levels; restock as necessary. h) Check supply drawers; restock as necessary. 6. Monthly Checklist (last week of month) a) Deep clean centrifuges. Remove the rotor and clean inside and out. b) Deep clean areas 8 and 9. c) Discard old sample extracts around instrumentation. d) Vacuum around instruments.
Toxicology Laboratory Area Map
ABBREVIATIONS USED IN THE TOXICOLOGY LABORATORY 1. Standard scientific abbreviations may be used. 2. In addition the following abbreviations may be used: Abbreviation + A/N or AN AA Ace Aceta Alc Alk Assoc B or Bld BARB BE BR Cal C/A CAP CE CO COC COOH-THC Dec DNR DOD DOT EME ETOH exp Fluz GC GFAA gry Hep Histo IA IM Inc Inst IS Iso LC Meaning positive Acid Neutral Drug Screen atomic absorption acetone Acetaminophen alcohol Alkaline Drug Screen Associates Blood Barbiturates benzoylecgonine brain Calibrator cholestane/alphaprodine ratio College of American Pathologists cocaethylene carbon monoxide cocaine Carboxy-tetrahydrocannabinol Decrease do not report date of death Department of Transportation ecgonine methyl ester ethanol expired/expiration Flunitrazepam gas chromatograph graphite furnace AA grey Hepatitis Histology intoxication assault intoxication manslaughter Increase Instrument(s) Internal Standard isopropanol liquid chromatograph
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6-mam, 6-mono 6-monoacetylmorphine
LC Li Maint ME MeCl MeOH Meth METR MS Mus NC Neg NR Opi PC Pos QNS qs quant RF SA sat Sero SIM THC Tox U UCT VHP Vol
Low Control liver Maintenance Medical Examiner methylene chloride methanol Methamphetamine ME/Tox refrigerator/lockbox mass spectrometer muscle Negative Control negative not received opiate Positive Control positive quantity not sufficient fill to quantitation Response Factor sexual assault saturated Serology Selected Ion Monitoring Tetrahydrocannabinol Toxicology Laboratory urine United Chemical Technologies Vitreous Humor Electrolytes Volume
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INTRODUCTION TO PROCEDURES AND TESTING PROTOCOLS I. The following provides a brief description of the routine tests run in the toxicology laboratory and a summary of the application of this testing to Medical Examiner cases, Hcases, and DWI cases. II. Toxicology Procedures A. Alcohols and Acetone Screen 1. Analytical technique: headspace GC/FID 2. Analytes: a) Routine: ethanol, acetone, isopropanol, and methanol (1) The detection of acetone in a sample will prompt an Electrolytes analysis even if it has not been requested. (a) Acetone is associated with high glucose in uncontrolled diabetes. b) Other volatile compounds are also detected such as anasthetic gasses, freons, toluene, and others. (1) The detection of these will be reviewed and may prompt a volatile screen. B. Opiates Screen 1. Analytical technique: ELISA 2. Analyte: morphine is the ELISA target analyte 3. A positive response prompts the Opiates by GC/MS assay. C. Cocaine Metabolite Screen 1. Analytical technique: ELISA 2. Analyte: benzoylecgonine is the ELISA target analyte 3. A positive response prompts the Cocaine Metabolites by GC/MS assay. D. Cannabinoid Screen 1. Analytical technique: ELISA 2. Analyte: Carboxy-THC is the ELISA target analyte 3. A positive response prompts the Cannabinoids by GC/MS assay. E. Drug Screen 1. Components: The general drug screen consists of the Alkaline Drug Screen, the Acid/Neutral Drug Screen, and the Opiate Screen (discussed above). 2. Analytical technique: GC/FID with GC/MS confirmation 3. Analytes: several hundred drugs and metabolites may be identified and quantitated 4. Drugs detected in this screen may also prompt other testing such as: a) Acetaminophen Acetaminophen by GC/FID b) Valproic Acid Valproic Acid by GC/MS c) Ethosuximide - Ethosuximide d) Fentanyl sendout for quantitation e) Cocaine Cocaine Metabolites by GC/MS f) Salicylates Salicylate by fluorescence spectrophotometry g) Temazepam Benzodiazepines by GC/MS h) Alprazolam Benzodiazepines by GC/MS
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i) Theophylline sendout for quantitation Opiates by GC/MS 1. Analytical technique: GC/MS SIM 2. Analytes: codeine, hydrocodone, hydromorphone, morphine, and 6monoacetylmorphine Cocaine and Metabolites by GC/MS 1. Analytical technique: GC/MS SIM 2. Analytes: cocaine, benzoylecgonine, ecgonine methyl ester, and cocaethylene Cannabinoids by GC/MS 1. Analytical technique: GC/MS SIM 2. Analytes: tetrahydrocannabinol and carboxy-tetrahydrocannabinol (carboxyTHC) GHB by GC/MS 1. Analytical technique: GC/MS SIM 2. Analyte: gamma-hydroxybutyrate. 3. GHB is routinely tested in sexual assault H cases and analyzed upon request for other cases. Benzodiazepines by GC/MS 1. Analytical technique: GC/MS SIM 2. Analytes: temazepam, clonazepam, oxazepam, triazolam, lorazepam, alprazolam, alpha-hydroxy alprazolam, flunitrazepam, 7-amino flunitrazepam, and 7-amino clonazepam Ethylene Glycol 1. Analytical technique: GC/MS 2. Analyte: ethylene glycol Volatiles Screens 1. Analytical technique: GC/MS with a cooled inlet, multiple headspace GC, or headspace GC. 2. Analytes: many volatiles including freons, inhalents, solvents, and anesthetic gasses Metals Screen 1. Analytical technique: AA 2. Analytes: arsenic, lead, and mercury Lithium Screen 1. Analytical technique: AA 2. Analyte: lithium Electrolytes Screen 1. Analytical technique: electrolyte analyzer 2. Analytes: sodium, chloride, potassium, glucose, and urea nitrogen. Carboxyhemoblobin (carbon monoxide exposure) 1. Analytical technique: blood gas analyzer/co-oximeter 2. Analytes: different forms of hemoglobin including carboxyhemoglobin LSD Screen 1. Analytical technique: ELISA 2. Analyte: LSD is the ELISA target analyte 3. Positive screening results are sent out for analysis.
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R. Cyanide 1. Analytical technique: conway diffusion 2. Analyte: cyanide S. Valproic Acid 1. Analytical technique: GC/MS 2. Analyte: valproic acid T. Ethosuximide 1. Analytical technique: GC/MS 2. Analyte: ethosuximide U. Ethchlorvynol 1. Analytical technique: GC with confirmation by GC/MS 2. Analyte: ethchlorvynol V. Acetaminophen 1. Analytical technique: GC with confirmation by GC/MS 2. Analyte: acetaminophen W. Salicylates 1. Analytical technique: fluorescence spectrophotometry 2. Analyte: salicylate X. ELISA Screen 1. ELISA may be used to screen for a variety of other drugs such as barbiturates and 7-amino flunitrazepam. 2. Confirmation will follow by other in-house or referral lab testing. III. Routine Testing Approach by Case Type A. Medical Examiner Cases: 1. Specimen a) Routine specimens received for Medical Examiner toxicology include blood, urine, vitreous, bile, muscle (particularly in decomposed cases), and antemortem (hospital) blood or serum if available; other specimens may be submitted for testing. b) The preferred site for collection of blood for toxicological analysis is the femoral vein. c) Specimens collected by transplant may be submitted in addition or in lieu of ME specimens. 2. Testing protocol a) General Screen is the routine testing protocol and includes: (1) Alcohols and Acetone Screen (2) Drug Screen: Alkaline Screen and Acid/Neutral Screen (3) ELISA drug screening: cocaine metabolite, carboxy-THC and opiates b) Prioritization of specimens (1) Absent other direction from the ME, specimen use will be prioritized as follows: (a) Limited femoral blood in a gray top tube should be prioritized for analysis of alkaline, cocaine, and/or alcohol.
(b) Limited vitreous should be prioritized for electrolyte analysis if requested. c) Other tests offered by the Toxicology Lab are available upon request or as follow-up to initial testing. d) Referral laboratories: (1) Specimens may be sent to another laboratory for testing of substances requested by the ME which are not offered by this Laboratory. B. H-Cases: 1. H-cases include law enforcement cases (sexual assault), hospital cases, other medical examiner cases, or any other submission that is not generated by the Dallas County Medical Examiners Office and not relating to a driving offense. 2. Specimen a) The normal specimens received for H-cases include blood, urine, vitreous, bile, and antemortem (hospital) blood or serum. b) However, many types of non-routine types of evidence may also be received including syringes, food substances, beverages, or any other type of evidence that is suitable to test for drugs. 3. Testing protocol a) Submitters may indicate the type of testing required from the panel of tests offered by the Laboratory; samples may be sent to a referral laboratory if requested by the submitter. b) Sexual assault testing protocol The following protocol is used for routine sexual assault drug screens with reflex to other analyses as indicated: (1) Urine Alkaline Screen: positive results are evaluated in blood using the appropriate assay (2) Urine ELISA Sexual Assault Panel for barbiturates and 7amino flunitrazepam: positive results are evaluated in blood using the appropriate assay (3) Urine GHB Screen: positive results are then evaluated in blood using the GHB by GC/MS assay (4) Blood Alcohols and Acetone Screen (5) Blood ELISA Screen for cocaine metabolite, cannabinoids, and opiates. C. DWI Cases 1. D-cases are submitted for alcohol and drug testing in driving related offenses. 2. Specimen: The normal specimens received for DWI cases include blood, urine, and hospital blood or serum. 3. Testing a) Routine blood DWI cases within Dallas County (1) Alcohols and Acetone (a) If the result is 0.09% or higher the testing stops. (b) If the result is 0.08% or lower it will prompt a general screen (Alkaline and Acid/Neutral Drug Screens and
Dallas County Institute of Forensic Sciences Toxicology Laboratory 4 Introduction to Procedures Version 2.0
ELISA for opiates, cocaine metabolite, and carboxyTHC. b) Intoxication Manslaughter/Assault within Dallas County (1) Alcohols and Acetone screen and a general screen regardless of the alcohol result. c) Specific testing is performed as requested by the submitter and within the scope of services offered by this Laboratory; samples may be sent to a referral laboratory if requested by the submitter.
ACETAMINOPHEN QUANTITATION BY GAS CHROMATOGRAPHY Principle of Assay: Acetaminophen is identified using the standard acid/neutral drug screen with GC/MS confirmation. It is quantitated by the procedure described here. This method describes the quantitation of acetaminophen by gas chromatography. Acetaminophen is extracted from blood at a weak acid pH (potassium dihydrogen phosphate) using ethyl acetate. After centrifugation the supernatant is analyzed by GC. Equipment: Hewlett-Packard model 6890 Gas Chromatograph, autosampler, and ChemStation: Two capillary columns, inserted into same injection port for simultaneous analysis of a Sample on two columns such as: DB-1 (cross-linked methyl silicone gum phase) 25m x 0.32mm x 0.52um film thickness DB-5 (cross-linked 5% phenyl silicone gum phase) 25m x 0.32mm x 0.52um film thickness Eppendorf pipettes 50, 100, 200, 1000 Microfuge tubes Autosampler vials with fixed inserts, 32 mm x 11 mm, and Teflon lined seals Vortex mixer Pasteur pipettes Hamilton syringe, 10 uL VWR/SMI Positive displacement glass capillary tip pipettor 250 uL Reagents: Potassium dihydrogen phosphate Ethyl acetate, reagent grade Acetaminophen Stock Standard (1 mg/mL) Blank blood Barbital, free acid Instuctions for Reagent Preparations: Potassium dihydrogen phosphate buffer (KH2PO4), saturated: Add 270 grams potassium dihydrogen phosphate to 900 mL water in a beaker containing a stirring bar. Warm on a hot plate while stirring until the potassium dihydrogen phosphate goes into solution. Cool before use.
Acetaminophen by GC Version 2.0
Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Ethyl acetate is a volatile solvent. Avoid contact with skin, eyes, and mucous membranes; do not breathe vapors. Stocks, Standards, Controls, Calibrators: 0.2 mg/mL Barbital Working Internal Standard: Transfer 50 mg barbital free acid to 250 mL volumetric flask and dilute to volume with deionized water. Make fresh every 6 months. 1.0 mg/mL Acetaminophen Working Standard: Transfer 10 mg acetaminophen to a 10 mL volumetric flask. Bring to volume with deionized water. Store in refrigerator. Before using, allow standard to adjust to room temperature. External QC Sample: Prepared by manufacturer such as BioRad. Liquichek (TDM Level 2) Note: When not in use, standards, controls, calibrators, and stocks should remain refrigerated. Sample Requirements and Preservation: Types of samples for analysis: whole blood (gray top tube if available), serum/plasma, urine. Liquids are usually refrigerated during storage. Analytical Procedure: Preparation of Calibrators: To a microfuge tube add the following and vortex for 5 seconds: 10 mg/L calibrator: Add 10 uL working acetaminophen standard to 1 mL blank blood. 50 mg/L calibrator: Add 50 uL working acetaminophen standard to 950 uL blank blood. 100 mg/L calibrator. Add 100 uL working acetaminophen standard to 900 uL blank blood. 300 mg/L calibrator. Add 300 uL working acetaminophen standard to 700 uL blank blood. (Negative Control: Extract 250 uL blank blood without standard added.)
Preparation of Samples: To each microfuge tube add 1. 200 uL KH2PO4 2. 100 uL 0.2 mg/mL Barbital Working Internal Standard 3. Using a positive displacement glass capillary pipettor, add 250 uL of sample, blank blood (negative control), calibrator, or QC sample 4. Vortex for 5-10 sec 5. Add 200 uL Ethyl acetate; Vortex 5-10 sec 6. Centrifuge 3 minutes 7. Transfer supernatant to autosampler vial with fixed insert. 8. Analyze, using ANQuant Method Instrument Operating Procedure: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebook. Instrument Operating Procedure: Refer to the Instrument Methods Notebook. Calculations: The concentration of acetaminophen is calculated using an Excel spreadsheet, Acetaminophen Calculation procedure found on the QC/AA computer. Concentration is plotted on the x-axis and ratio of area drug/area internal standard is plotted on the y-axis. Enter the area ratio (acetaminophen area/I.S. area) for calibrators, controls and samples. In multipoint calibration, a series of standards are run and the best curve that fits these data is determined. The best straight line correlating the calibration data is the least squares line. The correlation coefficient is an indicator of how well the line fits these data points. Using only that portion of the curve between the lowest and the highest calibrators demonstrates the validity of the curve at the analyte concentration. Samples with concentrations above the highest calibrator should be diluted and rerun. Quality Control: The lower limit of quantitation of the assay is 10 mg/L, and the assay is linear up to 300 mg/L. An external control (BioRad) is included in each run. Results should be within range noted by manufacturer or within 20% of target.
References: Acid/Neutral Screening Procedure from Bexar County Forensic Science Center, San Antonio, Texas.
ACETAMINOPHEN BY GC TRAINING NOTES 1. Dilutions: For sample concentrations greater than 300 mg/L dilute the sample with DI water then use 250 uL of the sample dilution in the extraction procedure. To calculate the concentration of the drug in the original sample, multiply by the dilution factor. Always use a positive displacement glass capillary tip pipettor. If sample, control, or calibrator does not separate from organic phase after centrifugation, either re-vortex followed by re-centrifugation or place sample in refrigerator for 10 min, then re-centrifuge. Run a blank consisting of ethyl acetate prior to analysis.
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ACID/NEUTRAL DRUG SCREEN BY GC/MS Qualitative and Quantitative Analysis Principle of Assay: The acid/neutral drug screen is used for the comprehensive screening of approximately twenty-five drugs which are acidic or neutral in chemical nature. Acidic drugs often contain an hydroxyl (-OH) or carboxylic acid (-COOH) functional group. Following the screening assay, quantitation of acid and neutral drugs is performed on a second aliquot of specimen using this or another assay as noted below. Acid and neutral drugs and the internal standard, barbital, are extracted from biological specimens at a weak acid pH (potassium dihydrogen phosphate) using toluene-ether as the extracting solvent. After evaporating the extracting solvent, the residue is partitioned between acetonitrile and hexane. As a result of this partitioning, fat and other lipophilic matrix components stay in the hexane, and the drugs of interest are concentrated in the acetonitrile. The extract is screened by GC/MS. Positive screening results are followed by quantitation using a variety of assays: Quantitation by Acid/Neutral Drug Screen: butalbital, meprobamate, carisoprodol, phenobarbital, phenytoin, primidone, pentobarbital, and secobarbital Quantitation by analyte specific methods: acetaminophen, valproic acid, ethchlorvynol, trichloroethanol, ethosuximide, methocarbamol, and salicylic acid Reported as detected and/or sent out for quantitation: ibuprofen, chlorpropamide, theophylline, naproxen, topiramate, and phenylbutazone Other barbiturates and analytes: This procedure is appropriate for quantitation of a wide range of barbiturates and other acid and neutral drugs using appropriate calibration curves and controls.
Equipment: Agilent 5973 gas chromatograph/mass spectrometer, autosampler, and computer with capillary column such as: DB-1, 30m x 0.25 mm x 0.25 um film thickness Screw top culture tubes, 16mm x 125 mm, screw cap with Teflon liner Screw top conical tubes, 5 mL screw cap with Teflon liner Eppendorf pipet: 100 uL, 50 uL Repipet dispenser/diluter: 0.5, 1.0, 5.0 mL volumes Rotator Centrifuge Autosampler vials, 32 mm x 11 mm, Teflon lined seals and micro-volume glass inserts Vortex mixer Hamilton syringes
Dallas County Institute of Forensic Sciences Toxicology Laboratory 1 Acid/Neutral Drug Screen Version 2.0
Pasteur pipettes Serological pipettes, 5ml Reagents: Toluene, reagent grade Anhydrous diethylether, reagent grade Acetonitrile, reagent grade Hexane, reagent grade Potassium dihydrogen phosphate Barbital, free acid Instructions for Reagent Preparation: Toluene-ether (1:1): In a hood, combine equal volumes of toluene and diethylether. Potassium dihydrogen phosphate, saturated: Add 270 grams potassium dihydrogen phosphate to 900 mL water in a beaker containing a stirring bar. Warm on a hot plate while stirring until the potassium dihydrogen phosphate goes into solution. Cool before use. Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Ether is very volatile and very flammable. Do not use near a source of electrical spark or open flame. Caution: vapors may pool and travel down a lab bench or floor to an ignition source. Toluene-ether should be made in a hood. Avoid repeated contact with the skin, eyes, and mucous membranes. Other solvents are also flammable and should be used in a hood or well ventilated area. Avoid repeated contact with the skin and contact with eyes or mucous membranes. Stocks, Standards, Controls, Calibrators: 0.2 mg/mL Barbital Internal Standard: Transfer 50 mg barbital free acid to 250 mL volumetric flask and dilute to volume with deionized water. Make fresh every 6 months.
Acid/Neutral Drug Screen Version 2.0
1.0 mg/ml Drug Stock Standard: Transfer 10 mg free drug to a 10 mL volumetric flask and dilute to volume with methanol. If the drug is in the form of a salt or contains waters of hydration, the 10 mg must be adjusted accordingly to add 10 mg of the free base. The calculation to convert an amount of free acid to the salt is as follows: mg of salt = mg of free drug x molecular weight of salt / molecular weight free drug A/N Working Standard Mix 0.2 mg/ml (200mg/L): Using a volumetric pipette, transfer 2.0 mL each of a 1.0 mg/ml stock solution of up to 5 standards to a 15 mL screw cap culture tube. Add methanol if needed to bring the total volume to 10 mL. During normal use, this solution is used-up before stability becomes a problem. A/N Working Mix 1: butalbital, meprobamate, carisoprodol, phenobarbital, and phenytoin A/N Working Mix 2: pentobarbital, secobarbital, and primidone A/N QC Mixture 0.1 mg/ml (100mg/L): Using a volumetric pipette, transfer 1.0 mL each of a 1.0 mg/ml stock solution of selected standards to a 10 mL volumetric flask and dilute to volume with methanol. During normal use, this solution is used-up before stability becomes a problem. A/N QC Mix 1: butalbital, meprobamate, carisoprodol, phenobarbital, and phentoin A/N QC Mix 2: pentobarbital, secobarbital, primidone External Control: Biorad Liquichek Level 2 or equivalent containing acidic and neutral drugs in the expected range. Reconstitute or dilute and store per manufacturers directions; reagent expires as noted by manufacturer or one month after opening. Negative control: Extract 1 mL of blank blood containing only the internal standard. All solutions are kept in the refrigerator. Allow to come to room temperature before using. Sample Requirements: Acceptable specimens: blood, serum/plasma, urine, bile, gastric, vitreous, tissue homogenates, pharmaceutical preparations Sample Collection and Preservation: It is recommended but not required that blood specimens are preserved with sodium fluoride (gray top); however, most acid and neutral drugs are not as susceptible to in vitro enzymatic degradation as alkaline drugs. Femoral blood is preferable; however, acid and neutral drugs are typically less susceptible to postmortem redistribution than alkaline drugs. Liquid specimens are usually refrigerated during storage; solid specimens in plastic cups are usually frozen during storage. Sample Preparation:
Tissues:
Homogenize 5 grams tissue in 15.0 mL deionized water. Use 1 mL of the homogenate for analysis Gastric: Measure the total volume of gastric submitted before sampling; record in the case file. If gastric is a homogenous, pipettable liquid, it may be used as is in the analysis. If gastric is non-homogenous and/or thick, homogenize as tissue. For initial analysis, dilute gastric 1 to 100 with water. A significant quantity of drugs will overload the column. If dilution is negative, an undiluted or lesser diluted sample may be run. Other samples: Use 1.0 mL of sample in the analysis. High samples: Gastric, blood, and other specimens may require dilution. In this case, extract less than 1 mL and make up the volume with deionized water Non-standard dilutions and homogenates must be so noted in the case file. QUALITATIVE PROCEDURE: Analytical Procedure: 1. Set up one 15 mL screw cap tube for the internal QC and each sample. 2. Using a serological pipette, add 1.0 mL saturated potassium dihydrogen phosphate to each screw top culture tube. 3. For the Internal QC sample, add 100 uL A/N QC Mixture with an Eppendorf pipette. Vortex. 4. To each tube, add 100 uL of the 0.2 mg/mL barbital internal standard solution using an Eppendorf pipet. Vortex. 5. Using a wide bore glass pipette, add 1.0 mL blank blood to the Internal QC sample. Vortex. 6. Using a wide bore glass pipette, add 1.0 mL sample to each sample tube. Vortex. 7. Add 5.0 mL toluene/diethylether to each tube using repipet dispenser. 8. Rotate for 10 minutes at medium speed. 9. Centrifuge for 3 minutes. 10. Using a Pasteur pipette, transfer the toluene/diethylether layer (top) to a 5 mL conical tube. 11. In a hood, dry to residue using air and hot water bath (approximately 55 oC). Remove from water bath as soon as the specimen reaches dryness.* 12. To each tube, add 100 uL acetonitrile using pipet and 500 uL hexane using repipet dispenser. 13. Vortex for 1 minute. 14. Transfer acetonitrile layer (bottom) and some of hexane layer (top) to an autosampler vial with insert. Centrifuge on low for 1 minute. 15. Inject 1.0 uL on the GC/MS using AN method. 16. A/N drugs identified by this procedure are placed on the appropriate quantitation list for additional analysis, sent out for quantitation, or noted as detected in the report. *Note: If an oily residue remains after the extract is taken to dryness in step 10, perform the following procedure:
Add 200 uL acetonitrile and 1.0 mL hexane. Vortex for one minute; centrifuge. Discard the top (hexane) layer. Take the bottom (acetonitrile) layer to dryness. Continue with step 11. If this procedure is used, it must be noted in the case file. Qualitative Identification: Substance identity is determined by GC/MS identification and GC retention time. Positive screening results are typically followed by quantitation as noted in the Principle of Assay section of this method. QUANTITATIVE PROCEDURE: This procedure provides quantitation and confirmation of butalbital, meprobamate, carisoprodol, phenobarbital, phenytoin, primidone, pentobarbital, and secobarbital. It is also appropriate for quantitation of a variety of other barbiturates and other acid/neutral drugs using appropriate calibration curves and controls. Analytical Procedure: 1. Set up one 15 mL screw cap tube for each calibrator, internal QC, external QC, negative control, and sample. 2. Using a serological pipette, add 1.0 mL saturated potassium dihydrogen phosphate to each screw top culture tube. 3. Instructions for Preparation of Calibration Standards: Calibrator (1 mg/L): Using a Hamilton syringe, transfer 5 ul working mix standard (0.2 mg/ml) to a 15 ml screw top culture tube containing 1.0 ml saturated potassium dihydrogen phosphate. Vortex. Calibrator (10 mg/L): Using a 50 ul Eppendorf pipet, transfer 50 ul working mix standard (0.2 mg/ml) to a 15 ml screw top culture tube containing 1.0 ml saturated potassium dihydrogen phosphate. Vortex. Calibrator (20 mg/L): Using a 100 ul Eppendorf pipet, transfer 100 ul working mix standard (0.2 mg/ml) to a 15 ml screw top culture tube containing 1.0 ml saturated potassium dihydrogen phosphate. Vortex. Calibrator (40 mg/L): Using a 100 ul Eppendorf pipet, transfer 200 ul working mix standard (0.2 mg/ml) to a 15 ml screw top culture tube containing 1.0 ml saturated potassium dihydrogen phosphate. Vortex. 4. For the QC sample, add 100 uL A/N QC Mixture with an Eppendorf pipette to a 15 ml screw top culture tube containing 1.0 ml saturated potassium dihydrogen phosphate. Vortex.
Dallas County Institute of Forensic Sciences Toxicology Laboratory 5 Acid/Neutral Drug Screen Version 2.0
5. To each tube, add 100 uL of the 0.2 mg/mL barbital internal standard solution using an Eppendorf pipet. Vortex. 6. Using a wide bore glass pipette, add 1.0 mL blank blood to each Calibrator, the Negative Control, and the Internal QC sample. Vortex. 7. Using a wide bore glass pipette, add 1.0 mL sample to each sample tube. Vortex. 8. For the external control, add 1 mL of reconstituted sample (such as the Biorad) with a serological pipette. Vortex. 9. Add 5.0 mL toluene/diethylether to each tube using repipet dispenser. 10. Rotate for 10 minutes at medium speed. 11. Centrifuge for 3 minutes. 12. Using a Pasteur pipette, transfer the toluene/diethylether layer (top) to a 5 mL conical tube. 13. Take to residue in a hood using air and hot water bath (approximately 55 oC). Remove from water bath as soon as the specimen reaches dryness.* 14. To each tube, add 100 uL acetonitrile using pipet and 500 uL hexane using repipet dispenser. 15. Vortex for 1 minute. 16. Transfer acetonitrile layer (bottom) and some of hexane layer (top) to an autosampler vial with insert. Centrifuge on low for 1 minute. 17. Inject 1.0 uL on the GC/MS using AN method. *Note: If an oily residue remains after the extract is taken to dryness in step 10, perform the following procedure: Add 200 uL acetonitrile and 1.0 mL hexane. Vortex for one minute; centrifuge. Discard the top (hexane) layer. Take the bottom (acetonitrile) layer to dryness. Continue with step 11. If this procedure is used, it must be noted in the case file. Quantitation: This GC/MS method (ANSIM or ANSIM2) can be used to quantitate most acid/neutral drugs by generating a four-point calibration curve for each drug. GC/MS results are reported in mg/L. Tissues: The calculated result represents the concentration of drug in the homogenate. To calculate the concentration of drug in the tissue, multiply by the dilution factor. If the standard tissue homogenate is used (1 part tissue + 3 parts water = 1:4 dilution), multiply the homogenate concentration by 4 to obtain the concentration of drug in the tissue. Results are expressed in mg/Kg. Other Dilutions: The calculated result represents the concentration of drug in the diluted sample. To calculate the concentration of drug in the original sample, multiply by the dilution factor. For example if 0.5 mL of gastric + 0.5 mL of water was analyzed instead of 1 mL gastric, multiply the calculated concentration by 2 to determine the concentration of drug in the undiluted specimen,. If the original specimen was a solid and weighed, units are mg/Kg. If the original specimen was a pipettable liquid, units are mg/L.
Gastric: Gastric is reported as mg/ total volume of gastric. A notation must be made in the case file regarding how all dilutions were made. Quality Control: A quality control and external control sample will be included with each batch of samples for quantitation. QC sample results should be +/- 20% of the target concentration. QC results outside this range must be reviewed by a supervisor who will determine whether results may be reported or repeated. QC results are maintained in the Acid Neutral Drug Screen QC file for the qualitative assay or AN Quant file for qualitative results. These files are located in the Toxicology Laboratory. External controls such as Biorad controls: refer to the manufacturers acceptable ranges. Instrument Operating Procedures: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebooks. Instrument Methods: Refer to the Instrument Methods Notebooks. References: E. H. Foerster, J. Dempsey, and J.C. Garriott. A gas chromatographic screening procedure for acid and neutral drugs in blood. J. Anal. Toxicol. 3: 87-91 (1979)
ACID/NEUTRAL DRUG SCREEN - TRAINING NOTES 1. Several drugs are detected by this method but quantitated by other methods including salicylic acid, valproic acid, acetaminophen, ethchlorvynol, trichloroethanol, methocarbamol, and ethosuximide. See the individual drug specific method for quantitation of these drugs. The following drugs are reported as detected and may be sent to a referral lab if needed: ibuprofen, chlorpropamide, theophylline, naproxen, topiramate, and phenylbutazone. This procedure is appropriate for quantitation of a wide range of barbiturates and other acid and neutral drugs using appropriate calibration curves and controls. 2. Chromatographic Interferences: Phenobarbital and the carbamazepine metabolite co-elute by GC. Identification is made by GC/MS. Salicylic acid is detected above approximately 100 mg/L. Acetaminophen in high concentrations will co-elute with butalbital. Acetaminophen in high concentration will sometimes be identified by library match as paracetamol. Ibuprofen co-elutes with a common decomposition compound. 3. Diethylether is extremely volatile and extremely flammable. Use caution to prevent solvent or vapors from reaching ignition source. 4. Valproic acid, ethchlorvynol, trichloroethanol, and ethosuximide are volatile. Remove dried toluene/ether extract promptly from water bath to prevent loss of these volatile drugs. 5. Typically drugs identified and quantitated by this procedure are not as susceptible to in vitro enzymatic degradation or postmortem redistribution as basic drugs. 6. Some specimens particularly in decomposed cases have an oily residue left after evaporation of toluene/ether. Use the alternate method described in the procedure to handle this type of sample. Use of the alternate method should be noted in the case file. 7. Once the analytical process is begun, it should be followed to conclusion in a timely manner. The extraction process should not be left mid-stream for extended periods of time because sample degradation may occur with some chemicals at specific stages of the extraction process. 8. The wash bottles for the autosamplers should be filled with hexane washed acetonitrile to ensure solvent compatibility with the sample. 9. Do not report concentrations < 1 mg/L. 10. Caffeine, carbamazepine, and lidocaine are detected in the A/N screen but quantitated using the Alkaline Drug Screen. 11. Alkaline drugs of high concentration may be detected by the A/N screen. The GC/MS spectrum should also be printed for these alkaline drugs.
ALCOHOLS AND ACETONE BY HEADSPACE GAS CHROMATOGRAPHY Principle of Assay: The alcohols and acetone procedure is used to quantitate methanol, ethanol, isopropanol, and acetone in biological specimens and aqueous solutions using headspace gas chromatography. Substances placed in a closed vial thermostatted at constant temperature will partition into the gaseous phase above the sample based upon the partition coefficient of the substance. Under constant conditions, equilibrium will be reached between the sample phase and gas phase. At this point the concentration of substance in the headspace or gas phase is proportional to the concentration of substance in the sample, and the amount of substance in the sample may be calculated. In this procedure, the sample and an aqueous internal standard solution are combined and thoroughly mixed in a headspace vial. The vial is sealed and placed in the headspace autosampler. The sample is heated for a pre-set time to establish equilibrium of volatile analytes between the liquid and vapor phases. A portion of the headspace is sampled and introduced into the gas chromatograph. Identification of the analyte(s) present in the sample is based on retention time on two different types of columns; quantitation is based on peak areas relative to the internal standard. Equipment: Shimadzu GC-2010 gas chromatographs equipped with flame ionization detectors and two capillary columns such as: RTX BAC-1 and RTX BAC-2 (0.53 mm ID x 30 meters) AOC-5000 Auto Injector headspace autosampler (also allows direct injection and SPME) Computer equipped with the Shimadzu GCSolutions software package Headspace vial, septa, and metal seals (seals must be compatible with magnetic use) Repipet dispenser/diluter Micropipettor - 250ul (SMI) Pipettor, 250 uL
Reagents: n-Propanol, reagent grade Absolute ethanol, 200 proof Methanol, spectrophotometric grade Isopropanol, HPLC grade Acetone, spectrophotometric grade
Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap
Dallas County Institute of Forensic Sciences Toxicology Laboratory 1 Alcohols and Acetone Version 2.2
water or eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Stocks, Standards, Controls, Calibrators: 0.012% n-Propanol (internal standard solution): Transfer 0.72 grams of n-propanol to a 6 liter volumetric flask and dilute to volume with distilled water. 0.015% External Ethanol Control: Pre-made NIST traceable standard. 0.08% External Ethanol Control: Pre-made NIST traceable standard. 0.20% External Ethanol Control: Pre-made NIST traceable standard. 0.05% External Multi-component Alcohol Mix Control: Pre-made NIST traceable standard. This mix contains ethanol, methanol, isopropanol, and acetone. 0.10% External Multi-component Alcohol Mix Control: Pre-made NIST traceable standard. This mix contains ethanol, methanol, isopropanol, and acetone. High Mixed Calibration Standard (0.5% ethanol, 0.5% methanol, 0.125% acetone, 0.125% ispropanol): Transfer 1.25 grams each of acetone and isopropanol and 5.0 grams each of ethanol, and methanol to 1 liter volumetric flask and dilute to volume with deionized water. Alternatively a commercially available NIST traceable standard of this or different concentration may be used. Low Mixed Calibration Standard (0.05% ethanol, 0.05% methanol, 0.0125% acetone, 0.0125% isopropanol): Diltute High Mixed Calibration Standard 1:10. For example, transfer 10 ml High Mixed Calibration Standard to 100ml volumetric flask and dilute to volume with deionized water. High Ethanol Calibration Standard (0.5% ethanol): Transfer 5.0 grams of ethanol to 1 liter volumetric flask and dilute to volume with deionized water. Alternatively a commercially available NIST traceable standard of this or different concentration may be used. Low Ethanol Calibration Standard (0.05% ethanol): Diltute High Ethanol Calibration Standard 1:10. For example, transfer 10 ml High Ethanol Calibration Standard to 100ml volumetric flask and dilute to volume with deionized water. All stock and working standards are kept in the refrigerator. Allow standards to come to room temperature before using. Once a standard solution is made allow it to sit overnight before being put into use. Sample Requirements: The following samples are suitable for analysis: antemortem blood, postmortem blood, serum/plasma, urine, vitreous, bile, gastric, aqueous samples, tissue homogenates, etc.
Dallas County Institute of Forensic Sciences Toxicology Laboratory 2 Alcohols and Acetone Version 2.2
Sample Collection and Preservation: It is recommended that blood samples are preserved with sodium fluoride (gray top tube) and that postmortem samples are collected from femoral vessels. Liquid specimens are usually refrigerated during storage. Solid samples in plastic cups are usually frozen. Glass containers often break if frozen. Analytical Procedure: Calibration: 1. Using the Repipet dispenser, place 4.0 mL of n-propanol internal standard solution in headspace vial. 2. Calibration for ME and H-cases - add appropriate amount of standard using the appropriate Hamilton syringe, micropipetter, or Eppendorf pipetter: a. 0.01% ethanol, 0.01% methanol, 0.0025% acetone, 0.0025% isopropanol i. Add 50 ul of Low Mixed Calibration Standard. b. 0.05% ethanol, 0.05% methanol, 0.0125% acetone, 0.0125% isopropanol i. Add 250 ul of Low Mixed Calibration Standard. c. 0.10% ethanol, 0.10% methanol, 0.025% acetone, 0.025% isopropanol i. Add 50 ul of High Mixed Calibration Standard. d. 0.20% ethanol, 0.20% methanol, 0.05% acetone, 0.05% isopropanol i. Add 100 ul of High Mixed Calibration Standard. e. 0.50% ethanol, 0.50% methanol, 0.125% acetone, 0.125% isopropanol i. Add 250 ul of High Mixed Calibration Standard. 3. Calibration for DWI cases - add appropriate amount of standard using the appropriate Hamilton syringe, micropipetter, or Eppendorf pipetter: a. 0.01% ethanol i. Add 50 ul of Low Ethanol Calibration Standard. b. 0.05% ethanol i. Add 250 ul of Low Ethanol Calibration Standard. c. 0.10% ethanol i. Add 50 ul of High Ethanol Calibration Standard. d. 0.20% ethanol i. Add 100 ul of High Ethanol Calibration Standard. e. 0.50% ethanol i. Add 250 ul of High Ethanol Calibration Standard. 4. Wipe off the tip of the micropipetter (if used) and rinse the plunger and tube in water at least 5 times to clean. 5. Seal the vial with a septum and metal seal ring. 6. Mix the contents with a gentle swirling motion. 7. Analyze by headspace GC. 8. Calibrations are performed by the Shimadzu GCSolutions Workstation; curves are generated using a linear curve fit.
Alcohols and Acetone Version 2.2
Liquid Samples: 1. Using the Repipet dispenser, place 4.0 mL of n-propanol internal standard solution in headspace vial. 2. Add 250 ul of sample (standard, sample, control) using the micropipetter. 3. Wipe off the tip of the micropipetter and rinse the plunger and tube in water at least 5 times to clean. 4. Seal the vial with a septum and metal seal ring. 5. Mix the contents with a gentle swirling motion. 6. Analyze by headspace GC. 7. Calculations are performed by the Shimadzu GCSolutions Workstation; results are reported as percent concentrations (grams/100 milliliters). Tissue Samples: 1. Place 1.0 gram of tissue in the homogenizer with 16 milliliters of internal standard solution and homogenize. 2. Place 4.25 mL of homogenate in a headspace vial using a wide bore serological pipette. 3. Seal the vial with a septum and metal seal ring. 4. Mix the contents with a gentle swirling motion. 5. Analyze by headspace GC. 6. Calculations are performed by the Shimadzu GCSolutions Workstation; results are reported as percent concentrations (grams/100g tissue). Other dilutions must be noted in the case file. Qualitative Identification: The presence of an analyte is determined by matching retention times on two columns of different polarity. Where possible, multiple tubes and/or specimens are analyzed. Isoflurane (an anesthetic gas) has a retention time very similar to isopropanol on both columns and on both instruments. A peak identified as isopropanol is evaluated as follows: 1. When a person consumes isopropanol, both isopropanol and its metabolite, acetone, will typically be present in biological specimens. 2. If isopropanol and acetone are identified on both columns and in both blood and vitreous, isopropanol (and acetone if applicable) will be reported. 3. In the event that isopropanol is identified but the criteria listed above are not met, GC/MS should be used determine whether the peak is isopropanol or isoflurane. 4. Patient history may also be helpful in evaluating the identity of the peak. a. It is common for isopropanol without acetone to be present as a result of transplant services procedures. Isopropanol may be reported without GC/MS in transplant specimens unless otherwise requested or indicated by the Medical Examiner.
Quantitation: The instrument reports alcohols in g/100ml (g% or %). The generated curve uses a linear curve fit. Dilutions: On occasion, samples may require dilution. The result calculated by the instrument represents the concentration of analyte in the diluted sample. To calculate the concentration of analyte in the original sample, multiply by the dilution factor. The dilution process should be documented in the case file. Range of Detection and Reporting Criteria: Limit of Quantitation: The limit of quantitation is defined as the interval from the low calibration standard to the high calibration standard: Compound Linear Range Ethanol 0.01% - 0.50% Methanol 0.01% - 0.50% Isopropanol 0.0025% - 0.125% Acetone 0.0025% - 0.125% Reporting: Where specimen is available, samples are run in duplicate in two independent analytical runs. The lower of the two values is reported and the concentration reported is truncated to two decimal places. Isopropanol and acetone are not reported less than 0.003% due to the significant figures involved in the software reporting parameters. Quality Control: The run is considered acceptable if the standard and control are each within 0.005 g% of the target. o If only one of the standards/controls run falls outside the acceptable range but is within the warning range of 0.005 to 0.01 g% of the target, the assay may be reported and assay performance monitored. o If any two standards or controls fall in this warning range or beyond, the batch should be considered out of control. A supervisor should be notified and action taken to evaluate the assay. Samples should be reprepared and reanalyzed. Each batch of analyses should contain a negative control. Two of the three (0.015%, 0.08%, or 0.20%) external controls and a 0.05%, or 0.10% mixed control should be included in the batch; a 0.20% and two 0.08% external controls are included in DWI batches. Positive samples should be run in duplicate and usually in different batches. The duplicate is usually analyzed on a different specimen tube when available. Positive tissue samples require a second tissue preparation and analysis. Duplicates should agree within 0.01 g% for DWI cases and within 0.02 g% for ME cases. Supervisors must review data exceeding this range prior to reporting. Quality control data is maintained in the Alcohol QC file located in Toxicology.
5 Alcohols and Acetone Version 2.2
In the event that a sample does not inject properly during the initial run, it is acceptable to reinject the affected sample the following day or after the run is complete. A standard or control must be included after the affected sample to confirm the instrument is in proper working order. The correlation coefficient (R2) for all compounds must be 0.995 or greater for the calibration to be successful.
Instrument Operating Procedure: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebook. Instrument Methods: Refer to the Instrument Methods Notebook . References: Genesis Headspace Autosampler Operator's Manual Shimadzu GC Solution Operation Manual CTC Analytics PAL System User Manual Dallas County Institute of Forensic Sciences Alcohols and Acetone Toxicology Laboratory 7 Version 2.1
ALCOHOLS AND ACETONE TRAINING NOTES Currently we are using two columns: BAC-1 and BAC-2. Results are reported from BAC-1 (channel 1). BAC-1 was selected for quantitation because it gives better chromatographic separation of the alcohols. After the sample is added to the aqueous internal standard, mix using a swirling motion. This will improve sample homogeneity and give more consistent results. When adding internal standard solution to the headspace vial with the repipet dispenser, do not allow the initial drop of solution that may be ejected when the plunger is pulled upward to drop on the outside of the vial. It has been observed that wet vials may stick to the autosampler and not drop into the instrument resulting in no injection. In this procedure, units expressed as percent (% or g%) are equivalent to grams ethyl alcohol per 100 mL of sample. For example: a 0.08 % or 0.08 g% standard is 0.08 grams ethyl alcohol per 100 mL of sample. The metal seal must be compatable with the use of a magnet. The AOC-5000 utilizes a magnet to move samples from location to location. The commercially available aluminum rings are not magnetic and will not work with this autosampler. Make sure that the top is crimped correctly. If the metal ring is not properly crimped, it may result in the needle hitting the ring and not properly piercing the septum. Where possible, run two different specimens. Blood and vitreous are the preferred samples for the medical examiner cases. If vitreous is not available, use two separate blood tubes. If only one blood tube is present, run that tube in duplicate. A blank should be run between and after each tissue homogenate and decomposed sample to prevent late eluting compounds from appearing in following samples. If only a small amount of vitreous fluid is available, the chemist should check the case and determine whether electrolytes have been requested. Electrolytes receive priority for analysis unless noted otherwise by the submitter. At equilibrium, vitreous ethanol is expected to exceed that of blood because ethanol distributes primarily into body water and vitreous has a higher water content than blood. At equilibrium, dividing the vitreous ethanol concentration by 1.3 should yield a value similar to the blood ethanol concentration. In general, if the vitreous ethanol concentration deviates from the blood by more than 0.05 g% these results should be reviewed with a supervisor. Also based upon water content of the specimen, the plasma to blood distribution ratio is approximately 1.10 1.35.
Acetone is metabolized to isopropanol and isopropanol is metabolized to acetone. Therefore, it is not unusual to find both of these substances present in the same specimen. It is possible that ethanol and other alcohols may be produced or degraded by bacterial action in postmortem specimens after death and in vitro. This does not occur in properly collected and stored antemortem specimens. The formation of ethanol by microorganisms is inhibited by fluoride; therefore, it is recommended that blood specimens are collected in tubes containing sodium fluoride (gray top). Postmortem formation of ethanol rarely exceeds 0.05 g%. Methanol is often used in embalming fluid and is readily detected in specimens from most embalmed bodies. If the resulting methanol exceeds the upper limit of quantitation you may simply report >0.50% methanol. The result should be confirmed but are not required to agree within +/- 0.02%. Acetone is detected in specimens collected during diabetic or fasting ketoacidosis. If acetone is positive, vitreous electrolytes should also be run even if they were not requested. Verification of Time to Equilibration: The current procedure uses a 15 minute pre-heating period prior to injection on the GC to establish equilibrium of the volatile analytes between the liquid and vapor phase. Testing was performed to document that the 15 minute preheating period used in the new procedure ensures that the volatile substances quantitated in this procedure have reached equilibrium between the headspace and liquid phases prior to injection on GC. In addition, this testing shows that the area ratio for ethanol to n-propanol is essentially the same from 3 minutes forward. If antemortem (hospital) blood is available, an effort should be made to test the earliest suitable specimen. A positive result should be confirmed on the same antemortem tube. Prior to pipetting a blood sample, mix the blood specimen to obtain a homogenous sample. Do not mix vitreous due to particulate materials that may be present.
Broad peaks near ethanol may denote toluene from the previous sample. Run the previous sample using the toluene method and rerun the current affected sample to obtain a chromatogram without the toluene peak. The following table is a list of approximate retention times for toluene and other volatile compounds that may be seen by this method. GC-A Column 1 0.907 Freon 22 in solvent front 1.011 Desflurane 1.013 Diethyl ether 1.239 Methanol 1.458 Hexane(s) Ethanol Hexane(s) Acetone Methylene chloride Isoflurane GC-A Column 2 0.785 Freon 22 GC-B Column 1 0.791 Freon 22 GC-B Column 2 0.832
Freon 22
Methanol Desflurane
0.939 1.039 1.095 1.192 1.396 1.441 1.507 1.561 1.562
Methanol
0.840 0.891
Ethanol Diethyl ether Isopropanol 1.482 Isoflurane Methylene chloride Acetone n-Propanol (IS) Hexane(s) Chloroform Hexane(s) Toluene 1.517 1.644
Desflurane 1.014 Ethanol Isopropanol 1.202 Diethyl ether Isoflurane Methylene chloride Acetone n-Propanol (IS) Hexane(s) Chloroform Hexane(s) Toluene 1.252 1.300 1.402 1.433 1.554 1.843 2.081 2.185 2.351 next sample
Desflurane Diethyl ether Methanol Hexane(s) Ethanol Acetone Hexane(s)
1.016 1.133 1.135 1.309 1.468 1.592 1.603
1.785 1.941 2.184 2.478
Isopropanol 1.704 Isoflurane Methylene chloride n-Propanol (IS) Chloroform Toluene 1.749 1.757 2.657 2.963 2.512 next sample
Isopropanol 1.608 2.457 2.579 1.443 next sample
n-Propanol (IS) 2.604 Chloroform Between Toluene runs
ALKALINE DRUG SCREEN Principle of Assay: The alkaline drug screen is used for the comprehensive screening and quantitation of more than 200 alkaline drugs and metabolites in blood and other biological specimens. Substances detected in this analysis are alkaline (basic) in nature and most contain one or more amine (-NR2) functional groups. The substance to be analyzed is made basic, and the compounds of interest are extracted into n-butyl chloride. The organic phase is transferred to a new tube, made acidic, and the compounds of interest are back extracted into the aqueous phase. After removal of the organic layer, the aqueous phase is made basic and the compounds of interest are extracted into chloroform. This back extraction process selectively removes alkaline substances from the complex biological matrix and concentrates them in an organic phase for analysis; thus decreasing interference from other agents in the matrix and improving assay sensitivity. Quantitation is performed by injecting a portion of the organic phase into a gas chromatograph (GC) fitted with two GC columns of different polarity. Two reference standards are used: alphaprodine is used as the quantitative internal standard and cholestane is used as a secondary external standard to monitor GC performance and to check alphaprodine recovery. Confirmation is performed by injection of the organic phase onto a gas chromatograph/mass spectrometer (GC/MS). Equipment: Hewlett-Packard model 6890 GC, autosampler, and ChemStation: Two capillary columns inserted into same injection port for simultaneous analysis of a sample on two columns such as: DB-1 (cross-linked methyl silicone gum phase) 25m x 0.32mm x 0.52um film thickness DB-5 (cross-linked 5% phenyl silicone gum phase) 25m x 0.32mm x 0.52um film thickness GC/MS Hewlett-Packard model 5973 GC/MS, 7683 series autosampler, and ChemStation with capillary column such as: DB-1, 30m x 0.25mm x 0.25um film thickness Shimadzu model 2010 GC with QP2010 MS with capillary column such as RTX-1MS, 30m x 0.32 mm ID x 0.5um film thickness Screw top culture tubes, 16mm x 125 mm, screw cap with Teflon liner Screw top conical tubes, 5 mL screw cap with Teflon liner Eppendorf pipettes - 50, 150, and 250 volumes SMI micro pipettor - 60 uL, 800 uL Repipet dispenser/diluter - 10.0 mL volume Rotator
Dallas County Institute of Forensic Sciences Toxicology Laboratory Alkaline Drug Screen Version 2.0
Centrifuge Autosampler vials, 32 mm x 11 mm, Teflon lined seals and glass inserts Vortex mixer Vacuum aspirator 5 mL serological pipettes 4 mL volumetric pipettes Reagents: n-Butyl chloride, HPLC grade Hydrochloric acid, concentrated Chloroform, spectrophotometric grade Deionized water Cholestane Alphaprodine Blank blood 1.0 N Hydrochloric acid Add approximately 500 mL deionized water to a 1 liter volumetric flask, carefully add 83 mL of concentrated hydrochloric acid. Mix, allow to cool as necessary, and dilute to volume with deionized water. Ammonium hydroxide, concentrated Ammonia is volatile and evaporation can decrease the concentration of ammonia in the solution. Therefore, refill working bottle with fresh ammonium hydroxide daily to ensure that concentrated ammonium hydroxide is used for proper pH adjustment. Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. n-Butyl chloride is flammable. Use in a vent hood or well-ventilated area. Avoid contact with skin, eyes and mucous membranes. Ammonium hydroxide is a volatile corrosive. Avoid contact with skin, eyes, and mucous membranes; do not inhale vapors. Hydrochloric acid is corrosive. Avoid contact with skin, eyes, and mucous membranes. Chloroform is a chlorinated solvent and is considered a human carcinogen. Use in a vent hood or in a well-ventilated area. Avoid contact with skin, eyes, and mucous membranes.
Alkaline Drug Screen Version 2.0
Stocks, Standards, Controls, Calibrators: 1.0 mg/mL Cholestane Stock Internal Standard: Transfer 50 mg cholestane to a 50 mL volumetric flask and dilute to volume with chloroform. 0.02 mg/mL Cholestane Working Internal Standard: Transfer 2.0 mL cholestane stock internal standard (1.0 mg/mL) to a 100 mL volumetric flask and dilute to volume with chloroform. 1.0 mg/mL Alphaprodine Stock Internal Standard: Add 10 mL deionized water to preweighed alphaprodine free base. 0.1 mg/mL Alphaprodine Working Internal Standard: Transfer 10.0 mL alphaprodine stock standard (1.0 mg/mL) to a 100 mL volumetric flask and dilute to volume with deionized water. 1.0 mg/mL Drug Stock Standard: Transfer 10 mg drug as the base to a 10 mL volumetric flask and dilute to volume with methanol. Some drugs may require the addition of a drop of HCl to get complete dissolution. If the drug is in the form of a salt or contains waters of hydration, the 10 mg must be adjusted accordingly to add 10 mg free base. The calculation to convert the base to the salt is as follows: mg of salt = mg of base x molecular weight of salt / molecular weight of base Note: Occasionally two molecules of drug are included in the salt form. Check the chemical formula prior to making standard. In this case, the molecular weight of the salt should be divided by 2 prior to using the formula above. Note: Make up the cocaine stock using acetonitrile instead of methanol. 6.0 ug/mL Aqueous Dilute Drug Standard: Using reverse pipetting, transfer 150 uL of each drug stock standard (1 mg/L) to a 25 mL volumetric flask partially filled with deionized water. Dilute to volume with deionized water. Make fresh daily; dilute solutions are not stable. Notes: 1. Reverse pipetting is used to improve accuracy when pipetting organic solvents with an Eppendorf pipette. To reverse pipette, push plunger all the way down, fill pipette tip with sample, and dispense by depressing to first stop. 2. The concentration, amount, and volume of the calibrator stock standard and calibration standards may vary proportionally depending on the availability of drug, the expected concentration of the drug, and the needed volume of calibrator stock standard. To make a 1 mg/L drug standard, use 500 uL of dilute drug standard and 1500 uL deionized water in place of the 2 mL of water specified in the alkaline drug procedure. 0.06 mg/mL Alkaline QC Mixture: Transfer 3.0 mL of each of the following 1.0 mg/mL stock standards - methamphetamine, cocaine, lidocaine, citalopram, and trazodone - to a 50 mL volumetric flask and dilute to volume with methanol.
Dallas County Institute of Forensic Sciences Toxicology Laboratory 3 Alkaline Drug Screen Version 2.0
All stock and working standards are kept in the refrigerator. Allow to come to room temperature before using. Sample Requirements: Acceptable specimens: blood, serum/plasma, urine, bile, vitreous, gastric, tissue homogenates, pharmaceutical preparations, etc. Sample Collection and Preservation: It is recommended but not required that blood specimens are preserved with sodium fluoride (gray top) and that postmortem specimens are collected from femoral vessels. Liquids are usually refrigerated during storage. Solid samples in plastic cups are usually frozen for storage. Glass containers often break if frozen. Sample Preparation: Tissues: Homogenize 5 grams tissue in 15.0 mL deionized water. Use 3 mL of homogenate in the analysis. Gastric: Measure the total volume of gastric submitted prior to sampling. If gastric is a homogenous, pipettable liquid, it may be used as is in the analysis. If gastric is non-homogenous and/or thick, homogenize as tissue. For initial analysis, dilute gastric 1 to 100 with water. A significant quantity of drugs will overload the column. If dilution is negative, an undiluted or lesser diluted sample may be run. Other samples: Use 3.0 mL of sample in the analysis. High samples: Gastric, blood, and other specimens may require dilution. In this case, extract less than 3 mL and make up the volume with deionized water Non-standard dilutions and homogenates must be so noted in the case file. Analytical Procedure: 1. 2. Add 2.0 mL deionized water and 50 uL of alphaprodine working internal standard solution (0.10 mg/mL) to a 15 mL screw top culture tube. Vortex briefly. Samples a. Using a wide bore serological pipette, add 3.0 mL of specimen (blood, urine, tissue homogenate, etc.) and vortex the mixture for about 5 seconds. b. For a typical blood "Drug" standard (1.0 mg/L) replace 2mL of the water in step 1 with 500 uL dilute aqueous drug standard (6ug/mL) and 1500 uL of deionized water. Add 3.0 mL blank blood. Vortex for about 5 seconds. c. For the QC sample (1.0 mg/L), add 50 uL of 0.06 mg/mL Alkaline QC Mixture to water and add 3.0 mL blank blood.
3.
4. 5.
6. 7.
8. 9.
10.
Using repipet dispenser, add 10.0 mL n-butyl chloride to each tube and rotate for 5 minutes. Add 250 uL of concentrated ammonium hydroxide and rotate for another 5 minutes. Note: Do not skip rotation of sample at neutral pH. Some drugs such as propoxyphene will extract at neutral pH, but are retained in aqueous phase by protein binding if the sample is alkalinized. Centrifuge. If necessary, attempt to break up any emulsion by using a quick spin of the tube by hand or stirring with a wooden stick; samples should then be re-centrifuged. Using a serological pipette, transfer 9.0 mL of the supernatant (n-butyl chloride layer) into a second screw top culture tube and add 5.0 mL of 1.0 N hydrochloric acid. Rotate for 5 minutes and centrifuge. Discard the top solvent layer using a vacuum aspirator. To a 5 mL centrifuge tube add 60 uL cholestane working internal standard with an SMI pipette, 800 uL concentrated ammonium hydroxide, and 4.0 mL of the hydrochloric acid extract (from step 6) using a volumetric pipette. Cap tube. Vortex for 60 seconds; during this time, invert the tube and shake manually at approximately 15 second intervals. Centrifuge. Using a vacuum aspirator, discard all but about 0.5 mL of aqueous layer. Transfer the chloroform layer to a GC glass insert in an autosample vial for analysis. A small amount of aqueous layer should be left on top of sample to prevent evaporation of the chloroform layer after the vial has been sampled. Inject 2.5 uL into GC. All positive results are confirmed by GC/MS.
Qualitative Identification: The presence of drugs is determined by matching retention times or retention indices of peaks in the sample with retention of known drug standards. Retention time matches on two columns of different polarity provide strong evidence of drug identity; however, confirmation of drug identity is usually made by GC/MS. Quantitation: Typically, drugs are quantitated on the DB-5 column; however, they may be quantitated on the DB-1 column if coelution occurs on the DB-5. Procedures for quantitation follow: 1. Drug standards of known concentration are analyzed, and a drug specific response factor is calculated as follows: Response Factor (RF) = Concentration standard * Area internal standard /Area standard
2.
Concentration of the drug in the sample is calculated using the drug specific RF as follows: Concentration sample = RF * Area sample / Area internal standard
Tissues: The calculated result represents the concentration of drug in the homogenate. To calculate the concentration of drug in the tissue, multiply by the dilution factor. If the standard tissue homogenate is used (1 part tissue + 3 parts water = 4 parts total or 1:4 dilution), multiply the homogenate concentration by 4 to obtain the concentration of drug in the tissue. Results are expressed in mg/Kg. Methods of dilution must be noted in the case file. Gastric: Gastric is reported as mg/total volume of gastric. Other Dilutions: The calculated result represents the concentration of drug in the diluted sample. To calculate the concentration of drug in the original sample, multiply by the dilution factor. For example if two mLs of gastric + 1 mL of water was analyzed instead of three mL of gastric, multiply the calculated concentration by 3/2 to determine the concentration of drug in the undiluted specimen. If the original specimen was a solid and weighed, units are mg/Kg. If the original specimen was a pipettable liquid, units are mg/L. Methods of dilution must be noted in the case file. Quality Control: A quality control sample will be included with each batch of samples for quantitation. Components of the QC sample are selected to be representative of the drug types and retention times analyzed by this method. QC sample results are entered in the Alkaline Screen QC form. Quantitation of QC samples should be 1 mg/L +/- 20% (range 0.80 1.20 mg/L). Cholestane/alphaprodine area ratios are monitored and results entered in the 'Alkaline Screen QC' form. Cholestane/alphaprodine ratio should be between 0.44 and 0.75. QC results outside these ranges must be reviewed by a supervisor who will determine whether results may be reported or repeated. QC results are maintained in the Alkaline Drug Screen file in Toxicology. Instrument Operating Procedure: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebook.
Instrument Methods: Refer to the Instrument Methods Notebook.
References: E. H. Foerster, D. Hatchett, and J.C. Garriott. AA Rapid Comprehensive Screening Procedure for Basic Drugs in Blood or Tissues by Gas Chromatography.@ J. Anal. Toxicol. 2:50-55 (1978).
ALKALINE DRUG SCREEN - TRAINING NOTES Procedural Information: 1. Acceptable ratio of area counts for I.S. 1.1. Cholestane/alphaprodine: 0.44 0.75 1.2. Alphaprodine area: 50-150 2. Acceptable ratios for drugs in the QC range from 0.80 1.20 mg/L. 3. Compounds with more than one GC peak. 3.1. The response factor for each of these compounds is based on addition of all peak areas when they are present (depending upon concentration, all peaks may not be present): 3.1.1. Desipramine (2) 3.1.2. Promethazine (2) 3.1.3. Norpropoxyphene produces 5 peaks; quantitate by adding peaks 2 5 3.1.3.1.Norpropoxyphene produces 5 peaks; the response factor is based on addition of peaks 2-5. Retention times and areas should be cross-checked on both GC columns. 3.1.4. Chlorpromazine (2) 3.1.5. Chlordiazepoxide (3) 3.1.6. Oxycodone (2) 3.1.7. Diltiazem (2) 3.1.8. Thioridazine (2) 3.2. Doxepin produces two peaks by GC, quantitate using the second doxepin peak only. 3.3. Quinidine may produce two peaks on GC/MS; quinidine ethylcarbamate may be formed. 4. Coelution 4.1. Zolpidem and quinidine/quinine coelute with cholestane on DB-5 and should be quantitated on DB-1 when both are present. 4.2. Norcitalopram and norpropoxyphene 4 coelute on DB-5, and norpropoxyphene should be quantitated on DB-1 in the presence of citalopram. 4.3. Atropine and cocaine coelute on both columns. When both are present, cocaine is reported from the cocaine/metabolites GC/MS assay and atropine should be reported as detected and not quantitated. 4.4. Citalopram coelutes with dihydrocoleine and codeine; quantitate on DB-1 when both are present. 4.5. Diphenhydramine coelutes with carisoprodol; quantitate on DB-1 when both are present. 4.6. Metaxalone and methadone coelute; quantitate on DB-1 when both are present. 4.7. Promethazine and bupivacaine coelute; quantitate on DB-1 when both are present. 5. Compounds that are difficult to identify or distinguish by GC/MS: 5.1. Quinidine/Quinine--detected only in high concentrations and cannot be distinguished, report quinidine/quinine 5.2. Hydroxychloroquine 5.3. Mesoridazine--some of this compound converted into thioridazine 5.4. Fluphenazine
5.5. Perphenazine 5.6. Hydroxyzine--detected only in high concentrations; may be reported if GC peaks match on two columns and hydroxyzine related is present by GC/MS 5.7. Alprazolam--detected only in high concentrations 5.8. Haloperidol--detected only in high concentrations 5.9. Ephedrine/pseudoephedrine not detected at low concentration 5.9.1.Ephedrine and pseudoephedrine cannot be distinguished by GC without derivatization; report ephedrine/pseudoephedrine detected and send to referral lab for quantitation if needed. 5.10. Atropine detection may be difficult at low concentration 5.11. Erythro-dihydrobupropion and threo-dihydrobupropion are isomers which separate by GC; however, the GC/MS library spectra may identify only dihydrobupropion. 6. If postmortem and antemortem blood are submitted, a drug screen is usually performed on postmortem blood. If requested, the drug screen may also be performed on antemortem blood. 7. If a complete GC/MS is obtained for phencyclidine but the concentration is less than 0.02 mg/L, results are reported as <0.02 mg/L phencyclidine. 8. Once the analytical process is begun, it should be followed to conclusion in a timely manner. The extraction process should not be left mid-stream for extended periods of time because sample degradation may occur with some chemicals at specific stages of the extraction process. 9. The lower limit of quantitation for this assay is 0.02 mg/L for most substances. Substances are not usually reported below this concentration unless a corresponding parent drug or metabolite is present at 0.02 mg/L or higher. 10. Substances present in excess of 5 mg/L are usually diluted and reanalyzed. 10.1. Compounds with high RF such as carbamazepine may be reported at higher concentration (10 mg/L). 10.2. Lidocaine is usually quantitated to 10 mg/L or reported as >10 mg/L without dilution. 10.3. Propoxyphene exceeding 1 mg/L is diluted and rerun. 11. Decomposed samples contain many amines. A special search should be made for methamphetamine and amphetamine by GC and GC/MS. 12. All peaks should be evaluated by comparison to the alkaline standards retention time table. The automated retention time computer matching does not replace this process. 13. Blood collected in a gray top tube containing sodium flouride as preservative is preferable for analysis. Sodium fluoride inhibits (but does not eliminate) action of plasma esterases in vitro. Plasma esterases decrease concentration of some drugs such as cocaine during storage. 14. Femoral blood is recommended for analysis where it is available. Some alkaline drugs, such as tricyclic antidepressants, undergo postmortem redistribution after death. Femoral blood is less susceptible to this change than heart or subclavian blood which is in contact with internal body organs. 15. Due to poor chromatography, chloroquine and hydroxybupropion are not quantitated but reported as detected when present. 16. Due to low therapeutic concentration, fentanyl is typically sent to a reference laboratory for quantitation and reporting. 17. When a substance is identified by GC/MS but a standard is not available for quantitation, its presence is noted as detected.
18. Reporting of opiates: 18.1. Codeine, hydrocodone, and hydromorphone are typically reported from the opiate drug screen not the alkaline drug screen. 18.1.1. Results from both assays should be compared; some variation may occur between specimen tubes. 18.2. Other opiates such as oxycodone and dihydrocodeine are reported from the alkaline drug screen. 18.3. Hydrocodone may be reported from the alkaline screen if specimen decomposition makes it unsuitable for analysis using the opiate GC/MS assay. 19. The following benzodiazepines are typically identified by the alkaline screen but quantitated using the Benzodiazepine GC/MS method: lorazepam, temazepam, clonazepam, alprazolam, and triazolam. 20. Cocaine is identified using the alkaline screen; it is typically quantitated using the Cocaine and Metabolites GC/MS method. 21. When homogenates or dilutions are used, a summary of the dilution process should be included on the chromatogram for example, 1 ml blood + 2 ml water or 5 g tissue + 15 ml DI water => homogenize => 3 ml to assay. The calculated result must be multiplied by the dilution factor for example x3 in the first example above and x4 in the second example. 22. Screw caps may sometimes be an external source of hydrocarbons. Hydrocarbons can be removed by blowing out or washing cap tops in the appropriate solvent (n-butyl chloride for large cap or chloroform for small caps). 23. Caffeine is detected on both the alkaline and acid/neutral drug screens. Since it is routinely present in biological specimens, it is not quantitated or reported unless its concentration exceeds 10 mg/L. Caffeine is quantitated using the alkaline not acid/neutral drug screen. Matrix Effects: Matrix effects: Spiked samples of blood, various autopsy bloods (e.g., decomposed, thin, thick), tissue homogenates, urine, bile, and vitreous have been analyzed. The response factors obtained for blood can be used with all these specimens. Use of Two Columns Fitted into One Injector: In this procedure, samples are analyzed on two different columns simultaneously to increase accuracy of analyte identification. Both columns are placed into the same injector. Therefore, the GC inlet split ratio should be set to double the desired split ratio (i.e. set to 1:15 instead of 1:30); actual sample split is half the split ratio entered into the GC. Response Factors: The alkaline procedure uses historical response factors for quantitation. These historical response factors have been shown to remain constant among instruments, among similar columns from different manufacturers, between chemists, and over time. The response factors are verified for each GC batch by running a multi-component quality control sample containing drugs of several
representative chemical types. Response factors are routinely calculated for new drugs and checked for existing drugs. When running response factors, there are several things to consider. You will be correlating new data with old data, data between two instruments, and data from two columns. The group that you will be working on is designed so that there should be no coelution of analytes. Each group should have three independent extractions, and each extraction should be injected on both instruments. You will need to make up fresh stock standards for each drug that is available. If the drug is not available in the safe use the stock standard that is already made and order new drug. Below is a guideline for making up stock standards: 1.0 mg/mL drug stock standard: Transfer 10 mg of the drug as the base to a 10 mL volumetric flask and dilute to volume with methanol (some drugs may require the addition of a drop of HCl to get complete dissolution). If the drug is in the form of a salt or contains waters of hydration, the 10 mg must be adjusted accordingly. The calculation to convert the base to the salt is: mg of salt = weight of base x formula weight of salt / mol. wt. of base Since weighing 10.0 mg in a volumetric may be very difficult, weigh out an amount that is very close to 10.0 mg. Remember that 10.0 mg is the target and 11.0 mg or 9.0 mg would not be considered close. Once you have a weight, use that weight to calculate the actual concentration of the drug. amount weighed mg / 10.0 mL = standard concentration The 1.0 mg/mL concentration is the target value for most drugs but may vary for some. You will need to record this information on the drug standard worksheet and put it in the proper location in the logbook. Guidelines for data evaluation: The C/A ratio must be between 0.44 and 0.75 The new response factors should be within +/- 10% of the old response factor; if not, advise a supervisor.
The response factors should be within +/- 10% between instruments and columns; if not, advise a supervisor. Changes in Area Counts with Column Change Calculation of Linear Velocity:
11
When a new column is installed a change in area counts may be observed; this is to be expected. Because there are variations in the inner diameter of the columns, flows through these columns may be slightly different even though the dimensions are said to be the same. A good way to check this is to check linear velocity *. Columns of the same internal diameter should have the same linear velocity. If there is a difference in the internal diameter, the larger column will have more flow; and therefore, more sample will go onto the column. The area counts on the column with larger internal diameter (the one with the highest linear velocity) should be greater than the one with the smaller internal diameter. *Linear velocity = column length (cm) / retention time of non-retained peak (sec) Updating Retention Indices on the Gas Chromatograph: See Documentation for Retention Index Calculations in this Manual to learn more about the use of retention indices. 1. 2. In a 15 mL extraction tube, place 4 mL 1N HCl. Add 100 ul of each of the following drug stock standards (1mg/mL) in methanol: amphetamine phendimetrazine chlorpheniramine desipramine demethyldiazepam zolpidem alprazolam verapamil mesoridazine Add 1.5 mL alphaprodine working internal standard solution in water (0.2 mg/L) Add 2 mL chloroform. Add 800uL ammonium hydroxide and cap the tube. Vortex for 60 seconds; invert and shake manually at 15 second intervals during the vortexing period. Centrifuge. Transfer chloroform and some aqueous phase to a 5 mL conical test tube. Transfer a small amount (about 50 uL) of the organic layer to an autosampler vial and inject on both instruments. Remaining solution may be stored for future checks. Print the chromatograms and store for future reference. Go to TOX on the main menu and select update retention times. Select the instrument for which you have retention time data and type in the retention times for each compound. You must enter retention times and NOT retention indices. Save your results by clicking the save button and exit.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
12
Documentation for Retention Index Calculations Overview: Retention Index is a measure of retention of a compound on a chromatography column based upon the relative elution of that compound to a set of reference compounds. Using retention index instead of retention time to identify components in a chromatogram alleviates the problem of updating tables of retention times whenever column changes occur either from deterioration of the column or from physical changes such as replacement or shortening of column. The calculation of retention index is incorporated as part of the ChemStation report generation and is described in this document. Note: When Retention Index was initially enabled, the gas chromatograph was configured with a FID and a NPD detector. This configuration has since been changed to two FIDs. References to NPD in this document and in the macros controlling the retention index calculations were not changed and now refer to and control the second FID. The ChemStation software makes available so-called hooks at certain places during the normal operation of the standard software. These hooks allow the user to make modification to the operation of the software without altering the software. The PostQuant hook is used in the alkaline drug screen procedure to add retention index to the report. According to the ChemStation documentation: The PostQuant hook is called during Run Method after standard Data Analysis does the quantification and during the interactive Print Report from the menu in the Data Analysis view. The quantification results in ChromRes register are fully available at that time and any additional result calculation should be done here and added to ChromRes register because this is the latest point before report formatting starts. Retention Index is calculated via the PostQuant Hook using the Peak table in the ChromRes register. This table has headings for measured retention time, expected retention time, area, height, width, symmetry, etc. The macro enabled for the PostQuant Hook calculates the RI and places the result in the expected retention time column ExpRetTime. The Expected retention time is added to the report layout and its header is changed from Exp.RT to RI. The PostQuant Hook also changes the annotation on the chromatogram peaks from retention time to retention index. A temporary table is created for each signal during the calculation which is later used to create a preliminary report identifying and quantitating the compounds found. (See separate documentation for preliminary report generation). Retention Index is calculated based on relative retention to normal hydrocarbons where the index is 100 times the number of carbons in the reference compound. For example, a compound eluting at the same time as octane has a retention index of 800. Since hydrocarbons are not detected on nitrogen phosphorous detector, a set of ten alternate compounds were selected as reference compounds. These were assigned Retention Index values from 1 to 10 (to avoid confusion with standard Rentention Index values) in the following sequence: amphetamine, phendimetrazine,
Dallas County Institute of Forensic Sciences Toxicology Laboratory 1 Documentation for Retention Index Calculation Version 2.0
alphaprodine, chlorpheniramine, desipramine, demethyldiazepam, zolpidem, alprazolam, verapamil, and mesoridazine. During the linear temperature program (up to zolpidem; RI 7), RI is calculated by linear interpolation of the retention time between two adjacent reference compounds: RI = RefLow + (RT - RefLowRT) / (RefHighRT-RefLowRT) where: RI = Retention Index of component RT = Retention time of component RefLow = Retention Index of reference compound with retention time <= that of RT RefLowRT = Retention time of RefLow compound RefHighRT = Retention time of RefHigh compound Past the linear temperature program, starting with zolpidem, logarithmic interpolation is used between adjacent reference compounds. This is accomplished by converting all retention times in the formula above to RT = log(RT - RT6) where:RT is retention time of component or reference compound RT6 is retention time of demethyldiazepam (RI=6) The retention times for the reference compounds are stored in four separate tables which are updated using the custom menu item Update Reference RT=s under the main menu Tox. The retention times for the reference compounds are stored in a user generated register RefTime and saved in the file d:\hpchem\core\reftimes.reg. There is a table for each column (signal) and for each instrument. These tables are named FID1, NPD1, FID2, and NPD2. The column headings are Date, and R0 through R11. Each row in the table contains the date that the reference mixture was run in YYYYMMDD format. Each R column contains the retention time for the corresponding reference compound. R0 is equal to 0 and R11 = 33 representing the length of the chromatographic run. The PostQuant macro locates the last row with a date equal to or less than the run date of the chromatogram being integrated and uses the retention time in that row for RI calculations. The macros in the file d:\hpchem\core\reftimes.mac and d:\hpchem\core\swifs.mac are automatically loaded during HPChemStation initialization by the macro user.mac. The PostQuant Hook macro usrPostQuantHook is enabled with the macro command: SetHook PostQuant. usrPostQuantHook and the custom menu is enabled with the command: SetHook PreViewMenu, usrSwifsMenu These commands are also found in the user.mac. To disable RI calculation and RI annotation of chromatograms, the PostQuant SetHook statement must be removed from the user.mac file and the ChemStation must be restarted. To disable RI annotation of chromatograms, place a ! before the line:
Dallas County Institute of Forensic Sciences Toxicology Laboratory 2 Documentation for Retention Index Calculation Version 2.0
usrChangeAnnotation in the usrPostQuantHook macro. To remove RI column from the report, delete ExpRT from the detail section of the report layout. Macros: The file d:\hpchem\core\reftimes.mac contains the following macros: usrPostQuantHook usrChangeAnnotation usrGetDate$ usrCalcRI usrSaveRefTimes usrEditRefTimes usrCreateRefTable The first four macros are used as part of the RI calculation. The next two may be used to view, update or edit the retention times of the reference compounds in the reference tables. The last was used to create the tables in the RefTimes.reg. This macro should not be run unless the RefTimes register gets accidentally deleted. The file d:\hpchem\cor\dialog.mac contains the following macros: usrSelectInstr usrEnterRefTimes These macros are used to update reference compound retention times via the custom menu. General Comments: All macro names start with usr. This prevents accidentally replacing a build in command or macro. All variables are declared as local to the macro. This assures that global variables which may be used in other macros are not changed and guarantees that they are removed from active work space when the macro is finished executing. If a variable needs to be global, it is prefixed with usr. See the attached listings of the below described macros. usrPostQuantHook ! Rows is the number of peaks identified in the current chromatogram and is obtained from the Peak table of the ChromRes[1] register. ! Date$ is the injection date of the current chromatogram in YYYYMMDD format. See usrGetDate function macro for details. ! If _MethFile$....EndIF statements " Set the RefTab$ variable to establish which of the four reference tables will be used in the RI calculation. " Create a blank AlkData register from AlkData.reg and set ResultTab$ variable storing results for Preliminary Report.
Dallas County Institute of Forensic Sciences Toxicology Laboratory Documentation for Retention Index Calculation Version 2.0
! ! ! !
DelReg makes sure that the next command does not append to an existing register. LoadObj loads the four reference tables into the RefTimes register. RowIndex is the row in the reference table with a date equal to or less than the analysis date of the current chromatogram when searched from the last entry to the first. For Row ... Next Row loop cycles through each entry in the Peak table of the ChromRes[1] register and " Obtains the measured retention time . " Obtains the RI using the function macro usrCalcRI. " Replaces the ExpRetTime value with the RI. " Gets amount (Area Ratio) for each component " if Amount <> 0: an integrated peak not rejected by integration parameters Inserts a row in results table and enters RI and Amount in table DelReg removes RefTimes register.
usrGetDate$: This function macro is called by usrPostQuantHook macro. It obtains the date from the currently loaded chromatogram and formats in YYYYMMDD format. ! DelReg makes sure that the LoadSignal statement does not append to an existing register. ! File statement specifies the name of the raw data file to be loaded in the next statement. ! Load Signal statement loads the signals from the raw data file to the SignalReg. ! DateTime$ obtains the injection date from the signal register. This is in the format of MM/DD/YY HH:MM AM/PM format ! Date$ holds the date portion extracted from the DateTime$. It is the first substring delimited by . ! Yr$ holds the year portion of Date$. It is the 3rd substring delimited by / in Date$. ! If ... EndIf statement convert Yr$ from 2-digit year to 4-digit year for Y2K compliance. ! Mo$ holds the month portion of Date$. It is the 2nd substring delimited by /in Date$ ! If ... EndIf statements make sure that Mo$ is in MM format. ! Day$: similar to Mo$ ! Date$ recombine date in YYYYMMDD format. ! Delete SignalReg ! Return Date$ to calling macro. usrCalcRI: This function macro calculates the RI given the parameters retention time (RT), reference table (RefTab$), and the RowIndex into the RefTab$. ! MaxCols is the number of columns in the RefTab$ to be checked. (The NPD tables have one less entry than the FID tables). ! RefLow is the index number of compound less than or equal to that of RT ! RefLowRT if retention time of RefLow ! For Col ... Next Col loop cycles through the columns until the RT is bracketed between two reference compounds.
Dallas County Institute of Forensic Sciences Toxicology Laboratory Documentation for Retention Index Calculation Version 2.0
! ! ! !
ColName$ returns the column name of the current column being tested (R0 to R11. Column names had to start with a character. Numbers (0) were not allowed. " RefRT of current column. " If RefRT <= RT ...Else....EndIf statements set the Reference number and the retention times of those reference numbers below and above the current RT. Once the upper limit is set, Col is set to MaxCol so that For loop is exited. " RefLow and RefHigh are indexes extracted from the integer portion of ColName$. RT6 is retention time of reference compound 6 (demethyldiazepam) If RefLow > RT6 ... EndIf statements convert all retention times to logarithms of the retention time minus that of RT6. RI calculates the retention index as a fractional interpolation between two reference compounds. Return RI: returns the calculated RI to the calling program. "
Documentation for Retention Index Calculation Version 2.0
ARSENIC BY VGA FLAME ATOMIC ABSORPTION Purpose: Arsenic (As) is the twentieth most abundant element in the earths crust. It is a trace element and is routinely present in all human tissues. Arsenic readily binds to sulfhydryl groups; and therefore, is bound to proteins in vivo and concentrates in hair and nails. Samples are acid digested to break down organic constituents. Arsenic is converted to the trivalent state by the addition of potassium iodide (KI). Arsenic is then reduced to arsine (AsH3) gas and transported to the heated quartz absorption tube in the atomic absorption spectrometer using a vapor generation accessory (VGA). Atomic absorption is an analytical process in which an element is volatilized in a sample chamber. An element specific lamp emits a constant intensity of light of known wavelength through the sample chamber. Arsenic atoms absorb light at 193.7 nm, and the amount of light absorbed is proportional to the concentration of arsenic in the sample. Equipment: Varian SpectrAA-220FS Sample Preparation System SPS5 Test tubes, 25 mL for autosampler Centrifuge tubes for standards rack Micropipettors ranging from 50L to 1000L Volumetric flask Digestion tubes Pipets Urine Metals Control, such as BioRad Level 1 Reagents: Nitric Acid (HNO3,), concentrated trace metal grade Sulfuric Acid (HSO4), concentrated - trace metal grade Hydrochloric Acid (HCl), concentrated trace metal grade Arsenic Stock Standard 1000g/mL (As) suitable grade for Atomic Absorption use for trace metals, purchased. Metals Control, such as BioRad Level 1 or 2 follow instructions shipped with the reference material for reconstitution and storage. Potassium Iodide (KI) trace metal grade or equivalent Urea (NH2CONH2) Ultrapure or Trace metal grade
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Sodium Borohydride (NaBH4) 98% assay minimum Sodium Hydroxide (NaOH) Antifoam Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eyewash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Sulfuric, nitric, and hydrochloric acids are corrosive. Avoid contact with skin, eyes, and mucous membranes. Avoid inhaling vapors. Sodium hydroxide is a corrosive base. Avoid contact with skin, eyes, and mucous membranes. Arsenic is a known human carcinogen and can cause multiple organ system toxicity. It is corrosive; avoid contact with skin, eyes, and mucous membranes. Avoid inhaling vapors. Potassium iodide is an irritant. Avoid contact with eyes, skin, or mucous membranes. Urea is harmful if swallowed or inhaled and causes irritation to skin, eyes and respiratory tract upon contact. Use with adequate ventilation. Sodium borohydride is reactive with water or acid liberating flammable hydrogen gas. It is hydroscopic and may causes burns. Use extreme caution when storing or handling. Store in dessicator. Preparation of Reagents, Calibrators, Controls, and Standards: 2500 g/L Intermediate Arsenic Standard A 6N HCl (make 2 times) Into a 50 mL volumetric, pipet 125L Stock Arsenic Standard Dilute to volume with deionized H20. Expires 1 week from prep date. Fill a 200 ml volumetric flask with about 75 ml deionized water. Add 100mL of concentrated HCl and swirl. Allow to cool and bring to volume with deionized water. In a hood using eye protection, place 50mL of concentrated nitric acid into a glass stoppered container. Add 50mL of concentrated sulfuric acid. Carefully swirl for good mixing of the two acids. Allow to cool before use.
Nitric : Sulfuric Acid (50:50) (digestion acid)
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20% Urea (w/v)
Place 20g of urea into a 100mL volumetric flask. Bring to volume with deionized water. Endothermic Reaction Make fresh for each set.
20% Potassium Iodide (w/v)
Place 10g of KI into a 50mL volumetric flask. volume with deionized water. Endothermic Reaction Make fresh for each set.
Bring to
0.5 % NaOH / 0.6 % NaBH4 Reductant
Place 0.5g NaOH and 0.6g NaBH4 in a 100 mL volumetric flask and bring to volume with deionized water. Make fresh for each set.
Negative Control
Add 2.5mL of blank blood to a 50mL digestion tube
Positive Control
Add 2.5mL of blank blood to a 50mL digestion tube, spike with 50L of Arsenic Standard A swirl to mix.
Positive Control Urine Metal Control Level 1
Add 2.5mL of Urine Metal Control Level 1 or 2 to a 50mL digestion tube
Sample Requirements: Acceptable specimens: blood, serum/plasma, urine, bile, vitreous, gastric, tissues, food, hair, nails, etc. Sample Collection and Preservation: It is recommended but not required that blood specimens are preserved with sodium fluoride (gray top) and that postmortem specimens are collected from femoral vessels. Liquids are usually refrigerated during storage. Solid samples in plastic cups are usually frozen for storage. Glass containers often break if frozen. To collect hair, select a pencil size bundle of hair on the head. Tie at intervals down the hair bundle. Pull from the scalp (postmortem) or cut close to the scalp. Mark the head end of the bundle. Measure the length of the bundle. The hair bundle is cut into sections at known
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distances from the head. Sections may be analyzed separately to assist in determining time of exposure. Nails may be sectioned and analyzed for the same reason. Sample Preparation: Tissues: Weigh a known amount of tissue. Mince and place into digestion tube. Other samples: Place a known volume or weight of other samples into the digestion tube. Mince if appropriate to aid acid digestion. Dilutions and homogenates must be so noted in the case file. Analytical Procedure: Use acid washed glassware. Clean glassware with 30% HNO3. 1. Preparation of Calibrators: Place the following into a 50mL digestion tube: Negative control - 2.5mL of blank blood 20 g/L Arsenic Standard-20L of Arsenic Standard A (2500g/L)+2.5mL blank blood 50 g/L Arsenic Standard-50L of Arsenic Standard A + 2.5mL of blank blood 75 g/L Arsenic Standard-75L of Arsenic Standard A + 2.5mL of blank blood 100 g/L Arsenic Standard - 100L of Arsenic Standard A + 2.5mL of blank blood Swirl each tube. 2. Preparation of Samples: Place 2.5 mL of each sample into an individual 50 mL digestion tube. Preparation of Controls: Place 2.5 mL of each control into an individual 50 mL digestion tube. A typical batch will use one or two positive and negative controls and one external (BioRad) QC. Add 5mL of the digestion acid (50:50 - Nitric:Sulfuric) to each digestion tube. Allow to stand 4-6 hours or overnight under the hood. (This usually prevents excess foaming.) In a hood, heat samples at about 90 deg C in water bath until straw colored. Add 5mL of deionized water and heat for additional 30 minutes. Remove from heat and cool. Add 5mL of 20% urea to each tube, swirl gently, and wait 1 minute. Add 2.5mL of 20% KI. Swirl gently. Dilute to volume with 6N HCl.
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3.
4. 5.
6. 7. 8. 9. 10. 11.
12.
If forming is present in the reaction chamber, add 1 drop of antifoam emulsion to samples and gently invert to mix. The autosampler unit may be used and loaded as follows, or manual introduction of sample may be used. SPS-5 Rack Type A (Standards Rack 1-12) Slot# 1 2 3 3 4 Solution 0 g/L 20 g/L 50 g/L 75 g/L 100 g/L
13.
SPS-5 Rack Type 25 (Sample Rack 1-60) 1 2 3 4 5 Negative Control Positive Control BioRad QC Sample Sample etc.
Quality Control: A negative control, a positive control, and an external control such as the BioRad must be run at the beginning and end of every set. In general, a negative control and positive control should be run every 5-6 samples. Control values should be +/- 15% of the target for the positive control, and the negative control should be representative of the calibration zero. The external control should match manufacturers range or be within +/- 15% of target. Instrument Operating Procedures: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebook. Instrument Parameters: Program name Instrument Mode Calibration Mode Lamp Current (mA)
Arsenic in Blood Template Absorbance Concentration 10.0

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Slit Width (nm) Slit Height Wavelength (nm) Flame Delay Time Constant (sec) Replicates Background Correction Air Flow Acetylene Qualitative Identification:
0.5 Normal 193.7 Air/ Acetylene 45 sec 60 sec 3 BC On 13.5 2.00
Elemental arsenic is known to absorb light at 193.7 nm. Calculations: Data is truncated to two decimal places. Results are reported from the instrument in ug/L. Liquids are usually reported as ug/L. To calculate the concentration of a solid sample, multiply ug/1000ml (ug/L) reported by the instrument by 2.5 ml to obtain ug. Divide by the weight of the sample in Kg to obtain ug/Kg. If other dilutions were made, reported result represents the concentration of arsenic in the diluted sample. To calculate the concentration in the original sample, multiply by the dilution factor. References: Varian Applications Note: Arsenic Determination by Hydride Generation. Interpretation of Results: The following is provided as a general guide for interpretation; interpretation of individual case data requires case specific evaluation. Normal concentrations: Hair 0 -1.92 mg/kg Blood 0 0.06 mg/L Nails 0 1.70 mg/kg Liver 0 0.09 mg/kg Toxic concentrations: Blood 0 9.3 mg/L Liver 2.0 120 mg/kg
Arsenic Version 2.0
BENZODIAZEPINE DERIVATIVES BY GC/MS Principle of Assay: Benzodiazepines are a class of structurally and pharmacologically similar compounds widely used as antidepressants, anticonvulsants, anxiolytics and hypnotics. Because many benzodiazepines are used in low dosages, drug metabolites are analyzed in addition to parent drug. In this assay, a liquid-liquid extraction using n-butylchloride is performed to separate benzodiazepines, related metabolites, and deuterated internal standards from the sample matrix. The residue remaining after drying the n-butylchloride extract is derivatized using MTBSTFA1%TBDMCS. After the tert-butyldimethylsilyl derivatives are formed, a portion of the extract is analyzed by GS/MS with selected ion monitoring (SIM). Equipment: HP model 6890 series II gas chromatograph with HP autosampler Hewlett-Packard model 5973 mass selective detector Screw top culture tubes, 15 mL, screw cap with Teflon liner Screw top conical tubes, 5 mL, screw cap with Teflon liner Eppendorf pipets - 50 uL and 20-200 uL adjustable volumes Hamilton Gas-Tight syringe - 25 uL, 50 uL Rotator Centrifuge Heating Block, 900C Vortex mixer Autosampler vials, 32 x 11 mm, Teflon lined seals and glass inserts Water bath \ Evaporator 2 mL Graduate pipett Disposable Pastuer pipett Reagents: N-Methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide + 1% tert-butyldimethyl-chlorosilane (MTBSTFA + 1% TBDMCS) n-Butyl Chloride Ethylacetate 40% Sodium Hydroxide Dissolve 40g NaOH in approximately 80 mL of deionized water. Allow to cool. Bring to 100 mL total volume with deionized water. pH 11, 0.5M Phospate Buffer Dissolve 8.7 g K2HPO4 (potassium phosphate dibasic) in approximately 80 mL deionized water. Adjust pH to 11 with 40% NaOH. Bring volume to 100 mL with deionized water.
Dallas County Institute of Forensic Sciences Toxicology Laboratory 1 Benzodiazepines Version 2.0
Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or at an eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident Immediately to a supervisor. Seek medical attention as necessary. n-Butylchloride is a volatile solvent. Avoid contact with skin; do not breathe vapors. MTBSTFA with 1% TBDMCS is corrosive. Avoid contact with skin, eyes, and mucous membranes. Sodium hydroxide is a corrosive base. Avoid contact with skin, eyes, and mucous membranes. Stocks, Standards, Controls, Calibrators: Internal Standard Solution: Oxazepam-d5 Stock Internal Standard (100 ug/mL): Prepared by manufacturer. Store in freezer. Alprazolam- d5 Stock Internal Standard (100 ug/mL): Prepared by manufacturer. Store in freezer. Working d5 Internal Standard (10 ug/mL): Using an appropriate pipet or syringe, transfer 1.0 mL of each of the above listed d5 benzodiazepines to a 10.0 ml volumetric flask and dilute to volume with methanol. Store in refrigerator. Drug Standard Solutions: Drug Stock Standards: Prepared by manufacturer. Store in refrigerator: Mix 1: Oxazepam 1.0mg/mL Temazepam 1.0mg/mL Lorazepam 1.0mg/mL Alprazolam 1.0mg/mL -Hydroxyalprazolam 0.1mg/mL Mixed Drug Standards (10 ug/mL): Two mixed drug standard solutions are made: Drug Mix 1 and Drug Mix 2 containing drugs as noted above. Into a 5.0 mL volumetric flask labeled Mix 1 or Mix 2 as appropriate, place 50 uL of each appropriate 1.0 mg/mL stock drug standard using a 50 uL Eppendorf pipette
Mix 2: Flunitrazepam 1.0mg/mL 7-Aminoflunitrazepam 0.1mg/mL Clonazepam 1.0mg/mL 7-Aminoclonazepam 0.1mg/mL Triazolam 1.0mg/mL -Hydroxytriazolam 0.1mg/mL
500 uL of each appropriate 0.1 mg/mL drug standard Dilute to volume with deionized water. Store in the refreigerator Sample Requirements: Acceptable specimens: blood, serum/plasma, urine, bile, vitreous, gastric, tissue homogenates, pharmaceutical preparations, etc. Sample Collection and Preservation: It is recommended but not required that blood specimens are preserved with sodium fluoride (gray top) and that postmortem specimens are collected from femoral vessels. Liquids are usually refrigerated during storage. Solid samples in plastic cups are usually frozen for storage. Glass containers often break if frozen. Sample Preparation: Tissues: Homogenize 5 grams tissue in 15.0 mL deionized water. Use 2 mL of homogenate in the analysis. Gastric: If gastric is a homogenous, pipettable liquid, it may be used as is in the analysis. If gastric is non-homogenous and/or thick, homogenize as tissue. For initial analysis, dilute gastric 1 to 100 with water. A significant quantity of most drugs will still overload the column. If dilution is negative, an undiluted or lesser diluted sample may be run. Other samples: Use 2.0 mL of sample in the analysis. High samples: Gastric, blood, and other specimens may require dilution. In this case, extract less than 2 mL and make up the volume with deionized water. Non-standard dilutions and homogenates must be so noted in the case file. Analytical Procedure: 1. Preparation of Calibration Standards: Calibrator (20 ng/mL): Transfer 2.0 mL blank blood to a 15 mL screw top culture tube. Add 4.0 uL of the appropriate Drug Mix Standard (10 ug/mL)and vortex. Calibrator (50 ng/mL): Transfer 2.0 mL blank blood to a 15 mL screw top culture. Add 10 uL Drug Mix Standard (10 ug/ml) and vortex. Calibrator (200 ng/mL): Transfer 2.0 mL blank blood to a 15 mL screw top culture tube. Add 40 uL Drug Mix Standard (10 ug/ml) and vortex.
Calibrator (1000 ng/mL): Transfer 2.0 mL blank blood to a 15 mL screw top culture tube. Add 200 uL Drug Mix Standard (10 ug/ml) and vortex. Negative Control: Transfer 2.0 mL blank blood to a 15 mL screw top culture tube and vortex. 2. Preparation of Controls: BioRad Control (2TCA): This external control is used for the Mix 1 group only; it contains alprazolam. Reconstitute the lyophilized serum per manufacturers direction. Place 2.0 mL of the reconstituted control in a 15 mL screw top culture tube and vortex. The target concentration for the control sample is noted by the manufacturer. Alltech Control: The appropriate controls are used for a Mix 1 assay or Mix 2 assay. Stock drug solutions are used as received from the manufacture. from Mix 1 Working Solution: volume to add Oxazepam 1.0 mg/mL 20 uL Temazepam 1.0 mg/mL 20 uL Lorazepam 1.0 mg/mL 20 uL Alprazolam 0.25 mg/mL 80 uL Alpha-hydroxyalprazolam 200 uL 0.1 mg/mL Mix 1 Stock Manufacturer Mix 2 Stock from Manufacturer Mix 2 Working Solution: volume to add 20 uL 20 uL 20 uL 20 uL 200 uL
Flunitrazepam 1.0 mg/mL 7-Aminoflunigrazepam 1.0 mg/mL Clonazepam 1.0 mg/mL 7-Aminoclonazepam 1.0 mg/mL Triazolam 0.1 mg/mL
Alpha-hydroxytriazolam 0.1 mg/mL 200 uL Alltech Working Solution: o To a 5.0 mL volumetric flask transfer the volume of stock noted in Column 2 or 4 above. o Dilute to volume with deionized water. Preparation of Control: o Place 2.0 mL of blank blood into a 15 mL screw top culture tube. o Add 50 uL of the Alltech working solution and vortex. o The target concentration for the Alltech control sample is 100 ng/mL or 0.100 mg/L.
3. Preparation of Samples: Place 2 mL of each sample into a 15 mL screw top culture tube.
4. To each tube (calibrator, control, and sample), add 20 uL deuterated internal standard and vortex. 5. Add 5 mL n-butyl chloride to each tube. 6. Rotate at a medium setting for five minutes. 7. Remove from rotator and add 1 mL pH 11, 0.5M phosphate buffer to each tube. 8. Rotate at a medium setting for five minutes. 9. Centrifuge on high for five minutes. 10. Transfer organic layer to a 5 mL conical tubes and evaporate to dryness at ~40C. 11. Add 50 uL MTBSTFA-1%TBDMCS and 50 uL ethylacetate to each conical tube. Cap and vortex for 30 seconds and heat at 90C for 30 minutes. 12. Transfer to labeled autosampler vials with inserts, cap, and inject on GC/MS using the appropiate SIM method. Calculations: The GC/MS results are reported in ug/L. Convert to mg/L by dividing ug/L by 1000 ug/mg. For example, 10 ug/L equals 0.010 mg/L Other Dilutions: The calculated result represents the concentration of drug in the diluted sample. To calculate the concentration of drug in the original sample, multiply by the dilution factor (total volume extracted in mL /mL original sample specimen added). For example if 1 mL of urine + 1 mL of water was analyzed instead of 2.0 mL of sample, multiply the calculated concentration by 2 (2mL total volume/1 mL urine) to determine the concentration of drug in the undiluted specimen. If the original specimen was a solid and weighed, units are mg/Kg. If the original specimen was a pipettable liquid, units are mg/L. Methods of dilution must be noted in the case file. Quality Control: QC results must meet manufacturers specifications or be within +/- 20% of target. QC data is maintained in the Toxicology Laboratory. The sample results for alprazolam should also be compared with the results obtained from the alkaline screen and should be similar. The lower limit of quantitation for the assay is 0.02 mg/L for each analyte. Linearity of the assay has been verified from 0.020 1.000 mg/L for all analytes. Ion qualifiers must match within +/- 20%; otherwise, the sample must be reviewed with a supervisor or repeated. Instrument Operating Procedure: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebook. Instrument Methods:
Refer to the Instrument Methods Notebook in the Toxicology Laboratory. Reporting Guidelines: Limits of quantitation: 0.02 - 1.00 mg/L Alprazolam should have full scan spectra from alkaline screen. If result is >1.00 mg/L, report as such or dilute. See supervisor for review.
Benzodiazepines Version 2.0
References: Urine Benzodiazepine Confirmation by Gas Chromatography/Mass Spectrometry. Sedgwick County Regional Forensic Science Center, Wichita, Kansas. Analysis of Benzodiazepines in Blood. Texas Department of Public Safety Crime Laboratory, Austin, Texas.
BENZODIAZEPINE TRAINING NOTES 1. A setting of 6 on the old evaporator gives a temperature of ~40C. 2. Upon drying down, if a sample yields a highly oily/fatty residue DO NOT continue sample analysis; the sample is unsuitable for analysis. Injecting fats into the instrument will greatly deteriorate peak shape. 3. If the instrument has not been used recently, it is recommended to ramp the GC oven temperature to 300 degrees and hold for at least ten minutes prior to the first injection to bake out the system. 4. The SIM ions for select benzodiazepines, metabolites, and internal standards are listed below: SIM GC/MS Methods: Mix 1 Method: BZDSIM.M Oxazepam-d5: 462, 464, 519 Oxazepam: 457, 459, 514 Temazepam: 357, 283, 255 Lorazepam: 491, 493, 513 Alprazolam-d5: 284, 209, 313 Alprazolam: 279, 204, 308 -Hydroxyalprazolam: 381, 382, 383 Mix 2 Method: BZDSIM2.M Oxazepam-d5: 462, 464, 519 Flunitrazepam: 312, 286, 266 7-Aminoflunitrazepam: 283, 255, 254 Clonazepam: 372, 374, 326 7-Aminoclonazepam: 342, 344, 399 Alprazolam-d5: 284, 209, 313 Triazolam: 313, 238, 342 -Hydroxytriazolam: 415, 417, 380
5. Cholesterol in blood coelutes with triazolam and may preclude analysis of triazolam in blood by this method. However, urine does not typically contain cholesterol and may be a suitable specimen for analysis in this situation. 6. Benzodiazepine metabolism varies with the subclass of drug and may occur by glucuronide conjugation, nitrogen reduction, or hydroxylation. 7. Derivatization with MTBSTFA-1%TBDMCS forms tert-butyldimethylsilyl derivatives at the active hydrogen in hydroxyl groups, carboxylic acids, and primary and secondary amines. Compounds without active hydrogens, such as alprazolam, do not form derivatives; see below.
Functional Group Derivitzation Sites: Mix 1: Oxazepam amide and hydroxyl Temazepam hydroxyl Lorazepam amide and hydroxyl Alprazolam none -Hydroxyalprazolam - hydroxyl Mix 2: Flunitrazepam none 7-Aminoflunitrazepam none Clonazepam amide 7-aminoclonazepam amide Triazolam none -Hydroxytriazolam - hydroxyl
Below is a drawing of oxazepam showing the amide and hydroxyl groups at which derivatization occurs. Primary amines are active sites for derivatization by silination, however, for 7aminoflunitrazepam and 7-aminoclonazepam, derivatization does not occur at these sites. The decreased reactivity may be resulting from the amine being directly attached to an aromatic ring, see below.
Oxazepam
7-Aminoflunitrazepam
CANNABINOIDS BY GC/MS Principle of Assay: See Cannabinoids in Urine by GC/MS for analysis of urine Delta-9-tetrahydrocannabinol (THC) is the primary active component of marihuana. It is very lipid (fat) soluble and is rapidly distributed into body fat during the smoking of marihuana. The most commonly analyzed metabolite of THC is 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (COOHTHC) which is water soluble. The extraction and analysis of substances of differing solubility from an aqueous medium such as blood or urine presents an analytical challenge. In this assay, solid phase extraction columns are used to trap THC and COOH-THC and their deuterated internal standards from the sample. The column is then washed with a sequence of solvents designed to sequentially remove matrix components, THC, and COOH-THC. The residue from the dried column eluate is derivatized with BSTFA-1%TMCS and analyzed using GC/MS with selected ion monitoring (SIM). Equipment: Extraction columns, such as Clean Screen by Worldwide Monitoring #CSTHC206 Vacuum manifold such as Varian VacElut SPS24 Hewlett-Packard model 6890 gas chromatograph Hewlett-Packard model 7673, 6890, or 7683 autosampler Hewlett-Packard model 5973 gas chromatograph/mass spectrometer (GC/MS) Capillary column: such as DB-1 or DB-5 (cross-linked methyl silicone gum phase) 12m to 25m Conical tubes, 5 ml, screw cap with Teflon liner Culture tubes, 15 ml, 16mm x 125 mm, screw cap with Teflon liner Test tubes, 13 x 100 mm Syringes to measure standards 10 ul and 50 ul volumes Centrifuge Autosampler vials, 32 mm x 11 mm, Teflon lined seals and glass inserts Vortex mixer Eppendorf pipets 50 ul and 100 ul volumes Rotator Heating block for derivatizing Water bath/evaporator Reagents: Sodium acetate Hydrochloric acid, concentrated Methanol Hexane Ethyl acetate
Dallas County Institute of Forensic Sciences Toxicology Laboratory THC and COOH-THC by GC/MS Version 2.0
Acetic acid, glacial Acetonitrile BSTFA with 1%TMCS 5% Methanol in Buffer: Place 13.6g sodium acetate in a 1000 ml volumetric flask. Add water (less than to volume), pH to 6.0 with 1N HCl, and bring to volume. Mix. Remove 50 ml of buffer from the flask and discard. Add 50 ml of methanol to the flask. Mix.
Eluants: 1. THC Eluant - Hexane:ethyl acetate: Mix 95 ml hexane with 5 ml ethyl acetate. 2. COOH-THC Eluant - Hexane:ethyl acetate:acetic acid: Mix 75 ml hexane, 24 ml ethyl acetate, and 1 ml glacial acetic acid. 3. Methanol:water (50:50): Mix 100 ml water and 100 ml methanol. Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Hydrochloric and acetic acids are corrosive. membranes. Avoid contact with skin, eyes, and mucous
Methanol, hexane, ethyl acetate, and acetonitrile are volatile solvents. Avoid contact with skin; do not breathe vapors. BSTFA with 1% TMCS is corrosive. Avoid contact with skin, eyes, and mucous membranes. Stocks, Standards, Controls, Calibrators: THC Stock Standard (1 mg/ml): Prepared by manufacturer. COOH-THC Stock Standard (100 ug/ml): Prepared by manufacturer. Working Mix Standard: (1000 ng/ml): Prepare two working mix standards - one for the calibrators and one for the quality control. Transfer 10 ul THC Stock Standard and 100 ul COOH-THC Stock Standard to a 10 ml volumetric flask and dilute to volume with methanol. THC-D3 Stock Internal Standard (100 ug/ml): Prepared by manufacturer. COOH-THC-D3 Stock Internal Standard (100 ug/ml): Prepared by manufacturer.
Working Internal Standard (0.5 ug/ml): Add 50 ul of the THC-D3 Stock Internal Standard and 50 ul of the COOH-THC-D3 Stock Internal Standard to a 10 ml volumetric flask and dilute to volume with methanol. Concentration is 0.5 ug/ml for each deuterated analyte. Store in the freezer; light sensitive. External QC Sample: Prepared by manufacturer such as Biorad. Sample Requirements: Acceptable specimens: blood, serum, plasma, vitreous, gastric, etc. Refer to the Cannabinoids in Urine by GC/MS procedure for analysis of urine. Note: Tissue homogenates are not suitable for this procedure since they do not flow well through the extraction columns. Sample Collection and Preservation: It is recommended but not required that blood specimens are preserved with sodium fluoride (gray top) and that postmortem specimens are collected from femoral vessels. Liquids are usually refrigerated during storage. Solid samples in plastic cups are usually frozen for storage. Glass containers often break if frozen. Sample Preparation: Sample dilutions must be noted in the case file. Analytical Procedure: Preparation of Calibrators: To 15 ml screw cap vials add the following: 2 ng/ml calibrator: Add 2 ul working mix standard to 1 ml blank blood. 25 ng/ml calibrator: Add 25 ul working mix standard to 1 ml blank blood. 50 ng/ml calibrator: Add 50 ul working mix standard to 1 ml blank blood. 100 ng/ml calibrator: Add 100 ul working mix standard to 1 ml blank blood. Preparation of Controls: To 15 ml screw cap vials add the following: 20 ng/ml QC control: Add 20 ul of the QC working mix standard to 1ml blank blood. Negative control: Extract 1 ml blank blood without standard added. Preparation of Samples: To a 15 ml screw cap vial, add 1 ml of sample blood. 1. Add 50 ul working internal standard (blood) to each tube. 2. Add 2 ml acetonitrile to each tube.
Cap and vortex. Rotate for 10 minutes. Centrifuge for ~5 minutes at medium speed. Decant supernatant into a 15 ml culture tube and add 5 ml buffer. Vortex. Set up one extraction column for each specimen in the vacuum manifold. Place one 13 x 100 test tube in the collection position for each column. Secure the top on the vacuum manifold and ensure that it is in the Waste position. Condition columns with a) 2 ml methanol (allow to flow by gravity) b) 2 ml buffer (allow to flow by gravity) 7. Add the buffer/sample mixture to the columns and allow drain by gravity. 8. Rinse columns with 1 ml buffer and allow to drain by gravity. 9. Dry under full vacuum for 10 minutes. Wipe out any moisture in columns with a cotton swab; use a separate swab for each tube. Wipe bottom tips of column to remove excess water. 10. Collect the THC fraction: Shift vacuum manifold lid to the Collect position. Elute each column with 2.5 ml hexane:ethyl acetate (95:5). 11. Transfer the THC fraction eluate from the collection test tube to a 5 ml conical tube and process beginning with step 16. Return collection tube to vacuum manifold. 12. Position the manifold lid in the Waste position. Rinse columns with 5 ml methanol: water (50:50). 13. Dry under full vacuum for 10 minutes. Remove excess water as in step 9. 14. Position the vacuum manifold lid in the Collect position. Elute with carboxy-THC eluate with 2.5 ml hexane:ethyl acetate:acetic acid (75:24:1). 15. Transfer the carboxy-THC eluate fraction to a fresh 5 ml conical tube. 16. Evaporate all conical tubes in a water bath and remove immediately when dry. 17. Add 50 ul BSTFA with 1% TMCS. Vortex and place in heating block at 70 oC for 30 minutes. 18. Transfer to autosampler vials and analyze by GC/MS using method THC.M for THC and COOH-THC.M for carboxy-THC. Calculations: The GC/MS reports the results in ug/L which must then be converted to mg/L by the chemist performing the assay. To convert ug/L to mg/L, divide by 1000. Other Dilutions: The calculated result represents the concentration of drug in the diluted sample. To calculate the concentration of drug in the original sample, multiply by the dilution factor. For example if 0.5 mLs of gastric + 0.5 mL of water was analyzed instead of 1.0 mL of gastric, multiply the calculated concentration by 1/0.5 or 2 to determine the concentration of drug in the undiluted specimen. If the original specimen was a solid and weighed, units are mg/Kg. If the original specimen was a pipettable liquid, units are mg/L. Methods of dilution must be noted in the case file. Quality Control: A 20 ng/ml (0.02 mg/L) quality control sample and external control (Biorad) is included in each run. Results should be within range noted by manufacturer or within +/- 20% of target. QC sample results
3. 4. 5. 6.
are maintained in the Toxicology Laboratory. The lower limit of quantitation of the assay is 0.002 mg/L, and the assay is linear up to 0.100 mg/L for both analytes. Qualifier ions should be within +/- 20% of target. Instrument Operating Procedure: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebook. Instrument Methods: Refer to the Instrument Methods Notebook. References: Varian Applications Note: Extraction of Drugs of Abuse using Bond Elut Certify THC and Carboxy-THC in Serum, Plasma, or Whole Blood. United Chemical Technologies Application Note: THC and Carboxy THC in Whole Blood, Serum or Plasma for GC/MS Using: 200 mg Clean Screen Extraction Column
THC and COOH-THC by GC/MS Version 2.0
TETRAHYDROCANNABINOL AND METABOLITE IN BLOOD BY GC/MS TRAINING NOTES 1. Silyl derivatives are used to improve chromatographic properties of acids, alcohols, thiols, amines, and other functional groups containing reactive hydrogens. BSTFA forms silyl derivatives of both THC and COOH-THC; see attached schematic. TMCS is present to enhance reactivity. BSTFA is unstable in the presence of moisture. It is important that sources of moisture are removed prior to derivatization examples include, drying tips of the extraction column before final elutions, drying column eluates before adding derivatizing, etc. It is also important to protect the bulk derivatization reagent from moisture during storage; for this reason, BSTFA is usually purchased and used in single use containers which are not reused. It is important to notice the position of the vacuum manifold lid. The Waste position sends column effluent into the bottom of the manifold. The Collect position sends column effluent into the collection tube. Tissue homogenates are not suitable for column extraction because they do not flow well through the column. Prior to this assay, specimens are usually screened by another assay such as ELISA.
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3.
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THC and COOH-THC by GC/MS Version 2.0
CARBOXY-TETRAHYDROCANNABINOL IN URINE BY GC/MS Principle of Assay: 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (COOH-THC or carboxy-THC) is the primary indicator of marijuana use in the urine. COOH-THC is present in urine in the conjugated form; therefore, alkaline hydrolysis of the urine is performed first. In this assay, COOH-THC is extracted from urine using liquid-liquid extraction, derivatized with BSTFA-1%TMCS and analyzed using GC/MS with selected ion monitoring (SIM). Equipment: Hewlett-Packard model 6890 series II gas chromatograph Hewlett-Packard model 5973 mass selective detector Hewlett-Packard model 7673, 6890, or 7683 autosampler Capillary column: such as DB-1 or DB-5 (cross-linked methyl silicone gum phase) 12 m to 25 m. Culture tubes, 13 x 15 mm, screw cap with Teflon liner Conical tubes, 5 ml, screw cap with Teflon liner Eppendorf pipettes - 50, 100, 200, and 400 ul volumes Hamilton syringe - 50 ul volume Rotator Centrifuge Water bath/evaporator, approximately 600C Heating block, 700C Vortex mixer Autosampler vials, 32 x 11 mm, Teflon lined seals and glass inserts pH paper Wooden applicators Reagents: Hexane Ethyl acetate Potassium hydroxide - (KOH) BSTFA with 1% TMCS (N,O-bis-(trimethylsily)trifluoroacetamide\Trimethylchlorosilane) Hydrochloric acid, concentrated 10N Potassium Hydroxide: Transfer 56 grams potassium hydroxide (KOH) to a 100 ml volumetric flask and add sufficient water to dissolve; mix. Cool and dilute to volume with deionized water. Hexane/Ethyl Acetate (7:1): Mix 140 ml of hexane and 20 ml ethyl acetate. Mix and store at room temperature. Make fresh each assay.
COOH-THC in Urine by GC/MS Version 2.0
Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Hydrochloric acid is corrosive. Avoid contact with skin, eyes, and mucous membranes. Hexane and ethyl acetate are volatile solvents. Avoid contact with skin; do not breathe vapors. Potassium hydroxide is a corrosive base. Avoid contact with skin, eyes, and mucous membranes. BSTFA with 1% TMCS is corrosive. Avoid contact with skin, eyes, and mucous membranes.
Stocks, Standards, Controls, Calibrators: COOH-THC Stock Standard (100 ug/ml): Prepared by manufacturer. THC Stock Standard (100 ug/ml): Prepared by manufacturer. Working Mix Standard: (1000 ng/ml): Prepare two working mix standards - one for the calibrators and one for the quality control. Transfer 10 ul THC Stock Standard and 100 ul COOH-THC Stock Standard to a 10 ml volumetric flask and dilute to volume with methanol. COOH-THC-D3 Stock Internal Standard (100 ug/ml): Prepared by manufacturer. THC-D3 Stock Internal Standard (100 ug/ml): Prepared by manufacturer. Working Internal Standard (0.5 ug/ml): Add 50 ul of the THC-D3 Stock Internal Standard and 50 ul COOH-THC-D3 Stock Internal Standard to a 10 ml volumetric flask and dilute to volume with methanol. Store in the freezer; light sensitive. External QC Sample: Prepared by manufacturer such as Biorad. Sample Requirements: Acceptable specimens: urine Sample Collection and Preservation: Liquids are usually refrigerated during storage.
Sample Preparation: Sample dilutions must be noted in the case file.
Analytical Procedure: Preparation of Calibrators: To a 15 ml screw cap tube add the following: Calibrator (10 ng/ml): Add 20 ul working mix standard (1 ug/ml) to 2.0 ml drug free urine. Vortex. Calibrator (25 ng/ml): Add 50 ul working mix standard (1 ug/ml) to 2.0 ml drug free urine. Vortex. Calibrator (50 ng/ml): Add 100 ul working mix standard (1 ug/ml) to 2.0 ml drug free urine. Vortex. Calibrator (100 ng/ml): Add 200 ul working mix standard (1 ug/ml) to 2.0 ml drug free urine. Vortex. Calibrator (200 ng/ml): Add 400 ul working mix standard (1 ug/ml) to 2.0 ml drug free urine. Vortex. Preparation of controls: To a 15 ml screw cap tube add the following: 25 ng/ml QC Control: Add 50 ul QC working mix standard (1 ug/ml) to 2.0 ml drug free urine. Vortex. Negative Control: Extract 2.0 ml drug free urine without standard added. Preparation of Samples: To a 15 ml screw cap tube, add 2 ml of sample urine. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Add 100 ul Working Internal Standard (1.0 ug/ml) to each tube with a 100 ul Eppendorf pipet. Vortex. Add 200 ul of 10N KOH to each tube using an Eppendorf pipet. Cap tubes, vortex for a few seconds, and place capped vials in a water bath (50-600C) for 15 minutes. Allow to cool before proceeding. In the hood, add 7 drops concentrated hydrochloric acid with a disposable Pasteur pipet and vortex to mix. Check the pH of each tube to ensure it is acidic. If not, add more concentrated hydrochloric acid, vortex, and check pH again. Add 8 ml hexane/ethyl acetate solvent. Cap tube and rotate for about 15 minutes. Centrifuge for about 5 minutes. Transfer the organic layer (top) to a 5.0 ml screw cap conical tube and evaporate to dryness using a water bath (50-600C). Heat uncapped in oven at 75 deg C for about 5 minutes; allow to cool. Using 50 ul Eppendorf pipet, add 50 ul BSTFA-1%TMCS to each tube. Cap and vortex for about one minute.
3 COOH-THC in Urine by GC/MS Version 2.0
11. 12.
Derivatize in heating block at 700C for 30 minutes. Allow capped vials to cool and transfer to an autosampler vial with insert, cap and run by GC/MS using method file THCURINE.M.
Calculations: The GC/MS reports the results in ug/L which must then be converted to mg/L by the chemist performing the assay. To convert ug/L to mg/L, divide by 1000. Other Dilutions: The calculated result represents the concentration of drug in the diluted sample. To calculate the concentration of drug in the original sample, multiply by the dilution factor. For example if 1.0 mL of urine + 1.0 mL of water was analyzed instead of 2.0 mL of urine, multiply the calculated concentration by 2/1 or 2 to determine the concentration of drug in the undiluted specimen. If the original specimen was a pipettable liquid, units are mg/L. Methods of dilution must be noted in the case file. Quality Control: A 25 ng/ml (0.025 mg/L) quality control sample and external control (Biorad) is included in each run. The results are monitored and filed for future reference. Results should be within range noted by manufacturer or within +/- 20% of target. QC sample results are maintained in the Toxicology Laboratory. The lower limit of quantitation of the assay is 0.01 mg/L, and the assay is linear up to 0.200 mg/L for COOH-THC. Qualifier ions should be within +/- 20% of target. Instrument Operating Procedure: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebook. Instrument Methods: Refer to the Instrument Methods Notebook.
CARBOXY-TETRAHYDROCANNABINOL IN URINE BY GC/MS TRAINING NOTES 1. 2. 3. 4. 5. 6. 7. Conjugated COOH-THC is hydrolyzed by heating the urine in 10N KOH. Refer to the THC blood procedure for additional training information. Use reverse pipetting technique for all controls, calibrators, and standards. Rinse Eppendorf pipet dispenser with deionized water after use of KOH. Add HCl in hood to avoid vapors. It is very important to check acidity in Step 5. Ethyl acetate/hexane solvent should be made fresh each use.
CARBOXYHEMOGLOBIN Principle of Assay: Whole blood is treated with sodium dithionite which reduces methemoglobin to reduced hemoglobin; methemoglobin is present in significant quantities in postmortem blood and, at high concentration, interferes with analysis of carboxyhemoglobin (CO). This process also reduces oxyhemoglobin to reduced hemoglobin. The sample is centrifuged and aspirated into the NOVA CCX. The specimen is automatically mixed with lysing agent (postmortem blood is already hemolyzed), and drawn into an optical cuvette. Light from a thallium/neon hollow cathode lamp passes through the cuvette. Absorbance is measured at seven specific emission lines. Based on absorbances at four specific wavelengths, the amount of reduced (deoxy) hemoglobin, oxyhemoglobin, carboxyhemoglobin, and methemoglobin are calculated. Turbidity and sulfhemoglobin are also detected; warnings are printed when thresholds of turbidity and sulfhemoglobin are exceeded in unsuitable samples. Equipment: NOVA Critical Care Xpress (NOVA CCX) Centrifuge Pasteur Pipets Microcentrifuge cups Spatula Cotton Swabs Reagents: Sodium dithionite (Na2S2O4)-working bottle contents should be replaced every 6 months. Triton X-100 (p-tert-octylphenoxypolyethoxyethanol) NOVA CO-Oximeter Calibrator Cartridge NOVA CO-Oximeter Deproteinizing Solution NOVA Controls 7, 8 & 9 NOVA Total Hemoglobin (tHb) Calibrator Preparation of Reagents: 10% Triton X-100: Measure 10 mL Triton X-100 into a 100 mL volumetric flask; Triton X-100 is viscous, allow the pipette to drain. Bring to volume with deionized water. Add a stir bar and mix for approximately 10 minutes. Allow to stand overnight before using.
Carboxyhemoglobin Version 2.0
Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Controls, Calibrators: NOVA Controls 7, 8, 9 Sample Requirements: Types of samples for analysis include whole blood and blood-containing fluid from tissues such as spleen. Instrument Preparation: 1. Inspect sample inlet port and clean if necessary using a clean swab moistened with deionized water. Refer to page 3-2 in instrument manual for instructions. 2. Press SYSTEM MENU, MAINTENANCE, FLOWPATH MAINTENANCE, DEPROTEINIZE COOX FLOWPATH buttons; follow instructions which appear on the screen. Calibration Procedure: 1. Verify adequate volume in the cartridges by touching the reagent graph on the top right corner of the screen. Replace any cartridges if 10% or less of the cartridge is left. To replace a cartridge pack, press SYSTEM MENU, MAINTENANCE, REPLACE/INSTALL CARTRIDGE and select pack to be replaced. Follow the instructions on the screen. 2. To calibrate the instrument press SYSTEM MENU, CALIBRATE, INTERNAL COOX CALIBRATION. 3. External COOX Calibration (tHb Calibrator) should be performed once per month, as needed, or if the controls are repeatedly out of range using an ampoule of the Total Hemoglobin Calibrator (tHb Calibrator). To perform COOX External Calibration: press SYSTEM MENU, CALIBRATE, EXTERNAL COOX CALIBRATION, present tHb calibrator ampoule to probe. Perform calibration procedure 3 times. Instrument will prompt you to enter the lot number. Note: The complete lot number is not on the ampoule; it may be found on the tHb Calibrator box. 4. Print out the days calibration: SYSTEM MENU, CALIBRATE, DATA. Then perform one of the following:
Dallas County Institute of Forensic Sciences Toxicology Laboratory Carboxyhemoglobin Version 2.0
1) 2)
Select PRINT DIAGNOSTIC REPORT, or Select COOX CALIBRATION DATA; select MENU, PRINT
Quality Control Procedures: 1. Controls are run each day the instrument is used; all three controls are analyzed. Controls are kept at room temperature. 2. Press QC, Select Level: 7, 8 or 9 with proper lot number (NOT internal). 3. Press ANALYZE. Present control to probe and press ANALYZE again. 4. Compare results to the approved range on your print out. If a control is out of range, repeat analysis. If a control continues to be out of range, follow troubleshooting methods in the instrument manual. 5. If there are no problems, and the controls are within range, then the analyst may begin to run samples. 6. Enter the QC results into the instrument maintenance log. Analytical Procedure: 1. 2. 3. 4. Allow specimens to come to room temperature. Add approximately 300 L of 10% Triton X-100 to a centrifuge cup. Add approximately 50 mg of sodium dithionite using a spatula. Add aproximately 300L of specimen to make an approximate 1:1 dilution with the 10% Triton X-100. 5. Gently mix the zeroing solution, sodium dithionate, and specimen. Cap the centrifuge cup and centrifuge for approximately 2 minutes. 6. Remove the centrifuge cups from the centrifuge. 7. Select Test Panel: COOX 8. Container should be defaulted to OTHERS. 9. Enter sample information: A. Sample Type: Postmortem Blood. B. Patient ID: Type sample name. (Ex. 05M2198 or 05H0192) 10. Press ANALYZE, present sample to probe and press ANALYZE again. 11. To repeat same sample for duplicate analysis, press ANALYZE during the results screen and press REPEAT. 12. If moving on to a new sample, press NEW. 13. After the last sample as been analyzed, deprotenize as described under the Instrument Preparation Section, Step 2. Calculations and Reporting Criteria: Manufacturers Linear Range: Total Hb: 5-30 g/dL Carboxyhemoglobin: 0-100% saturation
Report NOVA CCX results for % carboxyhemoglobin. Truncate to the nearest whole number. Report all concentrations below 1% as <1%. For example: a carboxyhemoglobin of 2.1% and 2.9% would both be reported as 2% carboxyhemoglobin. Report the lower value of the duplicate analysis. Quality Control: All specimens should be run in duplicate. Controls must meet manufacturers specifications for the analytes of interest. If analytes are out of range, repeat the control. If analytes are still out of range, perform instrument calibration. QC results are maintained in the Toxicology Laboratory. Instrument Operating Procedure: The instrument operating procedures may be found near the instrument; refer to instrument manuals and/or instrument procedure notebook. Reporting Criteria: 1. Values for carboxyhemoglobin for duplicate analyses should agree within +/- 3 units. 2. Methemoglobin should be less than 20% (g/dL). If this is not the case, the specimen should be reanalyzed using more sodium dithionite. If methemoglobin remains high the specimen may be decomposed and unsuitable for analysis. Note: Instrument does not flag. 3. High methemoglobin may also be caused by the presence of sulfhemoglobin in decomposed specimen. In this case the specimen is unsuitable for analysis. Note: Instrument will flag. 4. Total hemoglobin should be greater than 5% (g/dL); otherwise, there is not enough hemoglobin for accurate analysis. This will be indicated on the screen by the message sample not detected. The analyst should evaluate whether another specimen tube is more acceptable for analysis. It is acceptable to use red blood cells from the bottom of a tube to obtain a higher total hemoglobin concentration. 5. Oxyhemoglobin should be near zero. If this is not the case, the specimen should be reanalyzed using more sodium dithionite. The specimen may also be decomposed and unsuitable for analysis. 6. Sulfhemoglobin should be below 1.5% or the status code SulfHgb High will appear. Results with this status code must be reviewed by a supervisor prior to reporting. Interpretation: Carboxyhemoglobin saturation in normal, non-smokers is usually less than 3%; in normal, smokers it may rise to 10%. Carboxyhemoglobin saturation in excess of 50% is generally considered life threatening. CPR will reduce carboxyhemoglobin concentrations as will moving a living person into fresh air.
There are several mechanisms of death in a fire situation including exposure to elevated concentrations of carbon monoxide and/or cyanide and exposure to elevated temperature and fire. Methylene chloride is metabolized, in part, to carbon monoxide. Individuals exposed to methylene chloride may have carboxyhemoglobin concentrations in the range of 10 - 15%. References: Kunsman GW, Presses CL, and Rodriguez P. Carbon Monoxide Stability in Stored Postmortem Blood Samples. J Anal Tox 24:572-578, 2000. NOVA CCX Instrument Manuals
CARBOXYHEMOGLOBIN TRAINING NOTES 1. Sodium dithionite may occasionally oxidize when exposed to air for a long period of time. When this occurs, the sodium dithionite will not reduce the methemoglobin and will cause a high methemoglobin. Replace working sodium dithionite if this is suspected. 2. The specimen for analysis must be clear of debris after centrifugation. The probe should be placed in the center of the liquid for aspiration to prevent aspirating debris on the surface and on the bottom of the tube. A cotton swab may be used to remove fat or other debris floating at the surface. 3. Specimen, sodium dithionite, and diluent should be mixed gently to avoid producing oxyhemoglobin. 4. Status codes and error messages should be corrected by following troubleshooting procedures found in the instrument manual. 5. To enter a new QC lot number on the CO-Oximeter, select SYSTEM MENU, SETUP, QC. Under the lot information tab, select ADD. Enter: lot number, level, expiration date. Select DONE. Enter the lower and upper limit for each test. Recheck entered values. Select SAVE. 6. To change the cartridge on the CO-Oximeter, select SYSTEM MENU, MAINTENANCE, REPLACE/INSTALL CARTRIDGE, CARTRIDGE #4-COOX. Follow the on-screen directions. 7. To flush the lines on the CO-Oximeter, select SYSTEM MENU, MAINTENANCE, FLOWPATH MAINTENANCE, FLUSH COOX FLOWPATH. Follow the on-screen directions.
COCAINE AND METABOLITES by GC/MS Principle of Assay: Cocaine and cocaethylene are detected and may be quantitated using the Alkaline Drug Screen. Cocaethylene is formed as a metabolite of cocaine when ethanol and cocaine are present in the body at the same time. However, two major metabolites of cocaine ecgonine methyl ester and benzoylecgonine are too water soluble to be extracted using the Alkaline Drug Screen. The determination of these metabolites is important due to the short half life of cocaine and cocaethylene and the continued degradation of cocaine in a body after death and in vitro. Cocaine (COC) and its metabolites cocaethylene (CE), ecgonine methyl ester (EME), and benzoylecgonine (BE) and the corresponding deuterated compounds are extracted from biological fluids using solid phase extraction. Sample pretreatment consists of dilution with water and centrifugation to remove solid particulates. The supernatant is treated with phosphate buffer and extracted using a solid phase extraction column. BE and EME present in the residue from the dried column eluate are derivatized with pentafluoropropionic acid anhydride and pentafluoropropanol. The compounds are analyzed by gas chromatography/mass spectrometry (GC/MS) using selective ion monitoring (SIM). Equipment: Vacuum manifold, such as Varian VacElut SPS24 Extraction columns such as Clean Screen extraction columns, Worldwide Monitoring, Cat. No. CSDAU206 Hewlett-Packard model 6890 N gas chromatograph Hewlett-Packard model 7683 autosampler Hewlett-Packard model 5973 mass selective detector Capillary column such as: HP-1 MS (cross-linked methyl silicone gum phase) 30m x 0.25mm x 0.40um film thickness Conical tubes, 5 ml with Teflon lined screw cap Culture tubes, 16mm x 125 mm with Teflon lined screw cap Test tubes, 13x100 mm Syringes and pipettes to measure standards Centrifuge Autosampler vials, 32 mm x 11 mm with Teflon lined seals and glass inserts Vortex mixer 1 ml serological pipettes Oven or heating block for derivatization Nitrogen, compressed gas cylinder Disposable pipettes Water bath/evaporator Reagents: Potassium Phosphate, monobasic (KH2PO4): F.W. 136.09
Dallas County Institute of Forensic Sciences Toxicology Laboratory 1 Cocaine and Metabolites Version 2.1
Sodium Hydroxide, NaOH: F.W. 40 Ammonium Hydroxide (NH4OH): concentrated (14.8 M) Hydrochloric Acid (HCl): concentrated (12.1M) Pentafluoropropionic Acid Anhydride (PFAA or PFPA) Pentafluoropropanol (PFPOH) Methanol Methylene Chloride Isopropanol Acetonitrile Ethyl Acetate Phosphate Buffer, 0.1M, pH 6.0: Dissolve 13.6 g KH2PO4 in 800 ml deionized H20. Adjust to pH 6.0 by addition of 40% NaOH. Dilute to 1000 ml using deionized H20. Mix. Sodium Hydroxide, 40%: Dissolve 40g NaOH in deionized H20. Cool. Dilute to 100 ml with deionized H20. Methylene Chloride/Isopropanol/Ammonium Hydroxide (78/20/2): To prepare 200 mL, combine 4 mL ammonium hydroxide, 156 mL methylene chloride, and 40 mL isopropanol. Prepare fresh daily. Mix reagents in the order listed. Hydrochloric Acid, 0.1M: To 400 ml deionized H20 add 4.2 ml concentrated HCl. Dilute to 500 ml with deionized H20. Mix. Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Sodium hydroxide is a corrosive base. Avoid contact with skin, eyes, and mucous membranes. Ammonium hydroxide is a volatile, corrosive base. Avoid contact with skin, eyes, and mucous membranes; do not inhale vapors. Hydrochloric and phosphoric acids are corrosive. Avoid contact with skin, eyes, and mucous membranes. Methanol, methylene chloride, isopropanol, acetonitrile, ethyl acetate, and pentafluoropropanol are volatile solvents. Avoid contact with skin; do not breathe vapors. Pentafluoropropionic acid anhydride is a strong corrosive acid. Avoid contact with skin, eyes, and mucous membranes.
Cocaine and Metabolites Version 2.1
Stocks, Standards, Controls, and Calibrators: COC, CE, EME, and BE standards (1.0 mg/ml): purchase from vendor such as Cerilliant. Corresponding deuterated standards (100 ug/ml): purchase from vendor such as Cerilliant. Working Internal Standard Mixture (10 ug/ml): Transfer 1.00 ml of each deuterated standard to a 10 ml volumetric flask. Dilute to volume with acetonitrile. Working Standard Mixture (10 ug/ml; 50 ug/ml for BE): Transfer 100 uL of COC, CE, EME, and 500 uL of BE to a 10 ml volumetric flask. Dilute to volume with acetonitrile. Working QC Mixture: Made the same as Working Standard Mixture but prepared independently. External QC: purchase from vendor such as BioRad; reconstitute as noted by manufacturer; check package insert for target concentration. Sample Requirements: Acceptable specimens: blood, serum/plasma, urine, bile, vitreous, gastric, etc. Sample Collection and Preservation: It is recommended but not required that blood specimens are preserved with sodium fluoride (gray top) and that postmortem specimens are collected from femoral vessels. Liquids are usually refrigerated during storage. Solid samples in plastic cups are usually frozen for storage. Glass containers often break if frozen. Sample Preparation: Gastric: If gastric is a homogenous, pipettable liquid, it may be used as is in the analysis. If gastric is non-homogenous and/or thick, homogenize as tissue. For initial analysis, dilute gastric 1 to 100 with water. A significant quantity of most drugs will still overload the column. If dilution is negative, an undiluted or lesser diluted sample may be run. Other samples use 1.0 mL of sample in the analysis. High samples: Gastric, blood, and other specimens may require dilution. In this case, extract less than 1 mL and make up the volume with deionized water. Analytical Procedure: 1. Add 4 ml water to each 15 ml culture tube in the batch. 2. Standards: Prepare 20, 100, 500, 1000, and 2000 ug/L standards of COC, CE, and EME and 100, 500, 2500, 5000, and 10,000 ug/L standard of BE by adding 2, 10, 50, 100, and
200 uL of Working Standard Mixture to each standard tube using a syringe or Eppendorf pipette. 3. QC: Prepare 250 ug/L COC, CE, and EME control and 1250ug/L BE control by adding 25 uL of Working QC Mixture to the QC tube using a syringe. 4. Add 50 uL Internal Standard to each tube using Eppendorf pipette. 5. Add 1 ml blank blood to the blank, each standard, and QC sample tube; add 1 ml of sample to each sample tube; add one ml of external control to tube. Vortex and let stand 5 minutes. 6. Centrifuge for about 10 minutes at approximately 2000 rpm, decant supernatant into a 15 mL culture tube, and add 2 mL buffer. Vortex. 7. Set up one extraction column for each specimen in the vacuum manifold. Place one 13x100 mm test tube in the collection position for each column. Secure the top on the vacuum manifold and ensue that it is in the Waste position. Condition columns with: a. 3 mL methanol (allow to flow by gravity) b. 3 mL water (allow to flow by gravity) c. 1 mL phosphate buffer (allow to flow by gravity) 8. Add the buffer/sample mixture to the columns and allow to flow by gravity. 9. Wash columns with: a. 2 mL water (allow to flow by gravity) b. 2 mL 0.1 M HCl (allow to flow by gravity) c. 2 mL methanol (allow to flow by gravity) 10. Dry under full vacuum for 10 minutes. Wipe out any moisture in the columns with a clean, cotton swab; use a separate swab for each tube. Wipe bottom tips of column to remove excess water. 11. Shift vacuum manifold lid to the Collect position. Elute each column with 3 mL methylene chloride/isopropanol/ammonium hydroxide (78/20/2). 12. Transfer eluted samples to conical tubes and dry to residue in a water bath at about 50-55 degrees C while blowing with air. 13. Heat in an oven at approximately 100 oC for about five minutes to remove any moisture from the tubes. 14. Let cool to room temperature then derivatize by adding 50uL pentafluoropropionic acid anhydride (PFAA or PFPA) and 50 ul pentafluoropropanol (PFPOH) to each tube; cap tubes; heat at approximately 70 degrees C for about 15 minutes. 15. Dry to residue in the water bath under air then reconstitute with 100 uL ethyl acetate. 16. Transfer to autosampler vials and analyze by GC/MS SIM method for cocaine analysis. Calculations: The GC/MS calculates the results based on a 250 ug/L internal standard and reports the results in ug/L which must then be converted to mg/L by the chemist performing the assay. To convert ug/L to mg/L, divide by 1000. Other Dilutions: The calculated result represents the concentration of drug in the diluted sample. To calculate the concentration of drug in the original sample, multiply by the dilution factor. For example if 0.5 mL of gastric + 0.5 mL of water was analyzed instead of 1.0 mL of gastric, multiply the calculated concentration by 1/0.5 or 2 to determine the concentration of drug in the
Dallas County Institute of Forensic Sciences Toxicology Laboratory 4 Cocaine and Metabolites Version 2.1
undiluted specimen. If the original specimen was a solid and weighed, units are mg/Kg. If the original specimen was a pipettable liquid, units are mg/L. Methods of dilution must be noted in the case file. Quality Control: A 250 ug/L control and external control (Biorad) are included in each run; results should be within range noted by manufacturer or within +/- 20% of target. The sample results for cocaine and cocaethylene are also compared with the results from the alkaline screen if performed and should be very similar. Internal 250 ug/L QC sample should fall within +/- 20% of target. QC sample results are maintained in the Toxicology Laboratory. Linearity of the assay has been verified from 20 ug/L to 2000 ug/L for cocaine, ecgonine methyl ester, and cocaethylene and from 100 ug/L to 10,000 ug/L for benzoylecgonine. Qualifier ions should be within +/- 20% of target. Reporting Guidelines: EME, COC, and CE: Reporting Range 0.02 - 2.00 mg/L If result is >0.01 mg/L, but less than 0.02 mg/L and qualifiers match, report as <0.02 mg/L. If result is <0.01 mg/L, report as negative. If result is >2.00 mg/L, report as such or dilute. See supervisor for review. BE: Reporting Range 0.10 - 10.00 mg/L If result is >0.05 mg/L, but less than 0.10 mg/L and qualifiers match, report as <0.10 mg/L. If result is <0.05 mg/L, report as negative. If result is >10.00 mg/L, report as such or dilute. See supervisor for review. Note: Quantitative results are valid for split EME peaks and can be reported if the peaks are approximately equal and the qualifiers match. Split peaks may be manually integrated as one peak for both D3 internal standard and EME if necessary. Instrument Methods: Refer to the Instrument Methods Notebook. Instrument Operating Procedure: Refer to the Instrument Operating Procedure located near the GC/MS and/or applicable instrument manuals.
References: Varian Application Note: Extraction of Drugs of Abuse Using Bond Elut Certify United Chemical Technologies Application Note: Cocaine and Benzoylecgonine in Serum, Plasma, or Whole Blood for HPLC 200 mg Clean Screen Extraction Column.
COCAINE METABOLITES by GC/MS TRAINING NOTES 1. Acetylated derivatives are produced to improve the chromatographic properties of alcohols, phenols, amines, and carboxylic acids. In this assay, benzoylecgonine and ecgonine methyl ester are derivatized and the products of the acetylation derivatization process are described in the Toxicology Training Manual. 2. Anhydrides such as PFAA are moisture sensitive. Therefore, moisture must be dried from the sample prior to derivatizing. 3. In aqueous solution, cocaine hydrolyzes to ecgonine methyl ester. This degradation occurs in the body after death and in vitro. Collection of specimens in gray top tubes containing sodium fluoride retards but does not eliminate the degradation process. Cocaine analyses should be performed on blood collected in gray top tubes if available. 4. During life, cocaine is metabolized to ecgonine methyl ester primarily by action of plasma cholinesterases and benzoylecgonine primarily by non-enzymatic hydrolysis. Ecgonine methyl ester is rapidly metabolized to ecgonine, and concentrations of ecgonine methyl ester are usually low during life. After death, cocaine continues to break down into ecgonine methyl ester and less to benzoylecgonine, and metabolism of ecgonine methyl ester to ecgonine is slowed. Therefore ecgonine methyl ester builds up after death. A perimortem concentration of cocaine can be estimated by converting ecgonine methyl ester present at autopsy to a molar equivalent of cocaine and adding in the cocaine concentration. Further information regarding this process is attached. 5. After the column has been cut or changed, the 500 ug/L calibration should be run as a sample in the cocscan method to determine correct windows for the EME, BE, COC, and CE.
CYANIDE - ION SPECIFIC ELECTRODE Principle of Assay: The sample is mixed with acid in a Conway microdiffusion dish. Cyanide is released from the specimen matrix, diffuses via the headspace in the Conway dish, and is trapped in a 0.1N NaOH solution. Cyanide is quantitated in this basic solution using an ion specific electrode. Both normal and toxic concentrations can be measured by this method. Equipment: Conway microdiffusion dishes, with lids Volumetric flask, 100 mL Eppendorf micropipettor, 1.0 mL Hamilton syringe, 50 ul Mixer such as IKA-VIBRAX-VXR Rotating Platform Cyanide ion specific electrode Reference electrode pH/Ion specific electrode meter Beakers for electrode solutions Reagents: Potassium cyanide Sodium hydroxide Sulfuric acid, concentrated Lead acetate Reference electrode filling solution 0.10 N Sodium Hydroxide: Transfer 4.0 grams sodium hydroxide to a one liter volumetric flask and dilute to volume with deionized water. Store at room temperature. 6.0 N Sulfuric Acid: Add approximately 500 mL deionized water to a one liter volumetric flask and then add 167 mL concentrated sulfuric acid. Mix and allow to cool. Dilute to volume with deionized water. Store at room temperature. Saturated Lead Acetate: Transfer 1.0 g lead acetate to a 10 mL volumetric flask and dilute to volume with 1.0N sodium hydroxide. Store at room temperature.
Cyanide Version 2.0
Safety Precautions: Specific safety issue: Caution should be taken NOT to mix cyanide salts with an acid. Cyanide gas is given off with this reaction and the results may be fatal! The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes, which have been splashed with commonly, used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eyewash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Sulfuric acid is corrosive. Avoid contact with skin, eyes, and mucous membranes. Sodium hydroxide is a corrosive base. Avoid contact with skin, eyes, and mucous membranes. Biological exposure to lead may cause various health effects. Avoid accidental ingestion or exposure; wear gloves. Potassium cyanide can be fatal and causes multiple organ toxicity. Cyanide prevents cells from using oxygen. Avoid contact with eyes, skin, and mucous membranes. NOTE: Mixing potassium cyanide and acid will release toxic cyanide gas. Stocks, Standards, Controls, Calibrators: Potassium Cyanide Stock Standard (1000 ug/mL): Transfer 250 mg of potassium cyanide to a 100 mL volumetric flask and dilute to volume with 0.1N sodium hydroxide. Stable for 6 months at room temperature. Do not leave uncapped for a prolonged period of time. Potassium Cyanide Working Standard (100 ug/mL): Prepare two working mix standards one for the calibrators and one for the quality control. Using an Eppendorf pipet, transfer 1.0 mL cyanide stock standard (1000 ug/mL) to a 10 mL volumetric flask and dilute to volume with 0.1N sodium hydroxide. Stable for one day only. Sample Requirements: Acceptable specimens: blood, serum/plasma, urine, bile, gastric, vitreous, tissue homogenates, food, etc. Sample Collection and Preservation: It is recommended but not required that blood specimens are preserved with sodium fluoride (gray top. For postmortem samples, femoral blood is preferable. Liquid specimens (blood, urine, etc.)
Cyanide Version 2.0
should be refrigerated during storage; solid specimens (gastric, tissues, etc.) in plastic cups should be frozen during storage; food is usually stored refrigerated. Cyanide concentrations are reported to vary greatly upon storage; samples should be analyzed as soon as possible. Sample Preparation: Tissues: Gastric: Homogenize 5 grams tissue in 15.0 mL deionized water. For initial analysis dilute 1 part gastric with 99 parts water (1 to 100 dilution). Any significant quantity of a drug will be detected. If diluted sample is negative, an undiluted or less diluted sample may be run. Non-homogenous gastric should be homogenized prior to use. Other samples: Dilute and homogenize as necessary. Consult a supervisor for assistance. Dilutions must be noted in the case file. Analytical Procedure: See attached diagram of the Conway diffusion dish. 1. 2. 3. 4. 5. Prepare the 100 ug/mL potassium cyanide working standard. Label Conway dishes for samples,controls, and four standards (0.3, 0.5, 1.0, 2.0 mg/L). Fill the outside chamber of each Conway dish with approximately 1 mL of 6N sulfuric acid. Using a 1.0 mL Eppendorf, pipette 2.0 mL of 0.1N sodium hydroxide into the center well of each Conway dish. For each standard, use a 1.0 mL Eppendorf to pipette 2.0 mL of 0.1N sodium hydroxide into the sample chamber of the standard Conway dishes. Using a Hamilton syringe, add the following amounts of potassium cyanide working standard (100 ug/ML) to the sodium hydroxide solution in the sample chamber: 0.3 mg/L 6.0 ul 0.5 mg/L 10.0 ul 1.0 mg/L 20.0 ul 2.0 mg/L 40.0 ul Preparation of Controls: use a 1.0 mL Eppendorf to pipette 2.0 mL of 0.1N sodium hydroxide into the sample chamber of the control Conway dishes. 1.0 mg/L QC control: Using a Hamilton syringe, add 20 uL of potassium cyanide working standard (100 ug/mL) to the sodium hydroxide solution in the sample chamber. Negative control: Do not add standard to the sodium hydroxide solution in the sample chamber. 7. For each sample, pipette 2.0 mL of blood into one half of the sample chamber of the Conway dish.
6.
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8. 9. 10. 11.
12.
13. 14.
15.
Pipette 2.0 mL of 6.0N sulfuric acid into the sample chamber away from the sample or standard. Take care not to mix the two liquids in the sample chamber. Place lid on Conway dishes and rotate lid to distribute acid around the outer well to seal the dish. Carefully rotate the entire Conway dish to mix the acid with the sample or standard. Place all Conway dishes on rotator platform mixer for 1 hour. Approximately 1 hour before the Conway dish mixing is complete, fill the reference electrode with Reference Electrode Filling Solution and place the electrode in a beaker containing 50 mL 0.10N sodium hydroxide. Add two drops of the potassium cyanide working standard (100 ug/mL). Switch the meter to the mV setting. After one hour of mixing, analyze the center well of each standard dish in sequence. Leaving the Conway dish on the mixer, remove the lid from a Conway dish and place the electrode in the center well. (NOTE: The electrode should not touch the bottom of the dish but should be completely immersed in the 0.1 N NaOH. Readings should be taken while samples are being rotated on the mixer.) After three minutes record the mV reading. Repeat this step with each cyanide standard. Place the electrode in standby, rinse it with deionized water and blot the tip with a kimwipe before taking the next sample reading. Repeat Step 11 and 12 above for each sample with one additional step: after taking the first 3 minute mV reading, add one drop of lead acetate solution to the center well and take a second mV reading after 3 minutes. When all analyses are complete empty reference electrode, rinse at least two times with deionized water and blot the tip of electrode with a Kimwipe. Enter the mV readings observed for the cyanide standards and the samples using the Cyanide Calculation procedure found in the TOX file on the QC computer.
16.
Calculations: The concentration of cyanide is calculated using the Excel spreadsheet ACyanide Calculation@ procedure found in the TOX file on the QC computer. Input the mV readings obtained for the four standards. Input the names and mV readings obtained for each lead acetate treated sample analyzed. The concentrations for the standards and samples will automatically be calculated and can be recorded off the screen. To calculate the concentration of cyanide manually, use semi-log graph paper to plot mV of standards on log scale (y) versus concentration on regular scale (x). Concentration of samples is read directly from the graph using mV readings of the lead acetate treated sample. NOTE: The results for the lead acetate treated and non-lead acetate treated sample should be compared. If results are substantially lower for the lead acetate treated sample, interference from hydrogen sulfide should be considered. Consult a supervisor if this situation occurs.
Cyanide Version 2.0
Dilutions: The calculated result represents the concentration of drug in the diluted sample. To calculate the concentration of drug in the original sample, multiply by the dilution factor. Quality Control: The lower limit of quantitation is 0.3 mg/L and the assay is linear up to 2 mg/L. Concentrations outside this range require dilution. Concentrations below 0.3 mg/L are reported as negative. Instrument Operation: Refer to the GHB training notes for instructions regarding operation of the pH/ion specific electrode meter. Interpretation: Cyanide is found in low levels in normal, healthy individuals related to metabolism, diet, and/or smoking. The normal value for nonsmokers is 0.01 mg/L while smokers may reach 0.05 mg/L. The lethal concentration for cyanide is about 2.0 mg/L. A study involving 34 fatal cases showed an average cyanide concentration of 12.4 mg/L with the range of 1.1 - 53 mg/L. Cyanide is generated in patients on nitroprusside therapy. Early signs of cyanide toxicity were noticed in patients receiving nitroprusside; blood concentrations of cyanide in these individuals were 0.50 - 0.65 mg/L. Cyanide is produced during the burning of plastics and is also found in the blood of many fire victims. Cyanide concentrations in postmortem samples have been reported to increase and decrease after death due to a variety of mechanisms including bacterial action (increase and decrease) and evaporation of cyanide (decrease). Interference: Hydrogen sulfide, which is produced by protein decomposition, is also detected by the cyanide ion specific electrode and may falsely elevate cyanide results in postmortem samples. Lead acetate is added to bind the sulfide ion and reduce any sulfide interference. The difference between the sample readings before and after addition of lead acetate provides and indication whether sulfide interference should be considered in interpretation of results.
Cyanide Version 2.0
References: McAnally B, Lowry W, Oliver R, and Garriott J. Determination on Inorganic Sulfide and Cyanide in Blood Using Specific Ion Electrodes: Application to the Investigation of Hydrogen Sulfide and Cyanide Poisoning. J. Analytical Toxicology: 3 (May/June): 111-114, 1979. Notes: If the cyanide electrode continuously drifts and a constant mV reading cannot be obtained then the electrode needs to be polished. The polishing procedure and materials can be found with the instructions on how to operate the electrode.
Cyanide Version 2.0
Conway Diffusion Dish
Cyanide Version 2.0
CYANIDE TRAINING NOTES Prior to analysis, rinse Conway dishes with 6 N sulfuric acid followed by deionized water. In the event blood is not available and liver and skeletal muscle are the only tissues available, liver should be used.
Cyanide Version 2.0
ELECTROLYTES, UREA NITROGEN, AND GLUCOSE Principle of Assay: Electrolytes (Na+, K+, Cl-), urea nitrogen, and glucose are determined by using the NOVA Critical Care Express analyzer. Sodium (Na+), potassium (K+), and chloride (Cl-) are measured directly by ion selective electrodes. Urea nitrogen (VUN or BUN) is converted to ammonia by urease bound in the BUN membrane. The ammonia is detected by an ammonia selective electrode; the concentration of ammonia is proportional to the concentration of urea in the sample. Glucose is converted to hydrogen peroxide and gluconic acid by glucose oxidase bound in the glucose membrane. Hydrogen peroxide is detected by an amperometric electrode; the concentration of hydrogen peroxide is proportional to the concentration of glucose in the sample. Vitreous humor, serum, or plasma can be analyzed for electrolytes, urea nitrogen, and/or glucose. Equipment: NOVA Critical Care Express (Nova CCX) analyzer Sample cups Eppendorf 200 uL pipetter and disposable pipette tips Pipettor, 100 uL Micro-centrifuge and micro-centrifuge cups Cotton swabs Reagents: NOVA Calibrator Cartridge NOVA Chemistry Controls 4 & 5 NOVA Performance Check Solution (glucose and BUN over-range check solution) NOVA Glucose and BUN Membranes NOVA Deproteinizing Solution/ABG (Preheater Deproteinizing Solution), #12704 Deionized water Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Sample Requirements: The following specimen may be analyzed by this method: vitreous humor, serum, or plasma.
Dallas County Institute of Forensic Sciences Toxicology Laboratory Electrolytes Version 2.0
Instrument Preparation: Inspect sample inlet port for cleanliness. If necessary, clean sample inlet port using a clean swab moistened with deionized water. Refer to page 2-12 of the Nova CCX instrument manual for addional information. Calibration Procedure: 1. Verify adequate volume in the cartridges by touching the reagent graph on the top right corner of the screen. Replace any cartridges if 10% or less of the cartridge is left. To replace a cartridge pack, press SYSTEM MENU, MAINTENANCE, REPLACE/INSTALL CARTRIDGE and select pack to be replaced. Follow the instructions on the screen. 2. To calibrate select SYSTEM MENU, CALIBRATE, ABG/CHEM CALIBRATION. Uncalibrated analytes will be crossed out at the top of the screen. If a sensor fails to calibrate, an appropriate error code is generated. If needed, refer to the troubleshooting section of the instrument manual. 3. To print the calibration report, select SYSTEM MENU, CALIBRATE, DATA, PRINT DIAGNOSTIC REPORT. This will release the calibration report to print. Quality Control Procedures: 1. Allow the Chemistry Perf Check Solution to thaw to room temperature (kept in freezer). 2. Before opening, shake ampule for about 10 seconds. 3. Select QC, Select Level: Perf Check (NOT internal). Be sure to check that lot numbers match. Press ANALYZE. Present control to probe and press ANALYZE again to initiate aspiration. Compare results to the approved range on your print out. 4. Locate Controls 4 and 5. Thaw ampules to room temperature. Shake ampule for 10 seconds. 5. Press QC, Select Level: Level 4 (NOT internal), press ANALYZE to start the analysis process. Present Control 4 to probe and press ANALYZE again. 6. Press QC, Select Level: Level 5 (NOT internal), press ANALYZE to start the analysis process. Present Control 5 to probe and press ANALYZE again. If a control is out of range, arrows will be next to the data indicating a high or low result. If QC is out of range, repeat analysis. If QC continues to be out of range, follow troubleshooting methods in the instrument manual. 7. If there are no problems, and the controls are within range, then the analyst can begin to run the samples. 8. Enter the QC results into the instrument maintenance log. 9. Check results by reading directly from the printout. If the QC samples are out of range, then the instrument will automatically flag the result by placing a < next to a test that was below the acceptable range and > next to the test that was above the acceptable range. This may indicate a problem with the instrument; follow troubleshooting methods in the instrument manual if an error code or status code is present.
Electrolytes Version 2.0
10. If there are no problems, and the controls are within range, then the analyst can begin to run the samples. 11. Enter the QC results into the instrument maintenance log. 12. Enter the Lot # and expiration date of all the QC samples used in the Reagent Log Book. Analytical Procedure: 1. 2. 3. 4. Transfer at least 500L of the sample into a sample cup or microcentrifuge cup. Test panel is already defaulted to: ELECTROLYTES. Select container should be defaulted to OTHERS. Enter sample information: A. Sample Type should be defaulted to: Vitreous. B. Type sample name. (Ex. 05M2198 or 05H0192) Press ANALYZE. Present sample to probe and press ANALYZE again. To repeat same sample for duplicate analysis, press ANALYZE duing the results screen and press REPEAT. If moving on to a new sample press NEW. At the conclusion of testing, pour non-diluted vitreous samples back into the appropriate morgue tube to conserve specimen.
5. 6. 7. 8.
Calculations and Reporting Criteria: Manufacturers Linear Range: Analytes Sodium Potassium Chloride Glucose BUN Serum (& Vitreous) 80-220 mmol/L 1.0-20.0 mmol/L 50-200 mmol/L 15-500 mg/dL 3.0-100.0 mg/dL
Results are reported as indicated by the instrument when results fall within the manufacturers linear range. Instrument results are truncated and reported as follows: Na, Cl, glucose, and BUN truncated to a whole number (for example, 120 mmol/L) K truncated to one decimal place (for example, 3.5 mmol/L) Results outside the manufacturers linear range are reported as greater than or less than the applicable endpoint of the linear range.
Results reporting should also take into consideration the following: 1. Low glucose - It is very common that postmortem vitreous humor contains less than 15 mg/dL of glucose. In this situation, the NOVA will print an error message glucose low range error. Glucose results should be reported as <15 mg/dL. 2. High glucose - If glucose exceeds 500 mg/dL the NOVA will print glucose high range error. The specimen should be diluted with deionized water and reanalyzed. The glucose result must be multiplied by the dilution factor for reporting. (For example when 0.5 mL vitreous mixed with 1.0 mL deionized water is analyzed, the NOVA result should be multiplied by 3 and reported.) Other analytes must be reported from the nondiluted specimen run. 3. High BUN If BUN exceeds 100 mg/dL, the NOVA will print BUN high range error. BUN may be reported as >100 mg/dL or a dilution with deionized water may be performed; results must be multiplied by the dilution factor for reporting. Other analytes must be reported from the non-diluted specimen run. 4. Low BUN If the NOVA reports BUN low range error then the sample should be reported as unsuitable for analysis. 5. Na+ duplicates should vary no more than 5 mmol/L or a third sample should be analyzed. 6. Decomposed specimens may be unsuitable for analysis; if this is the case, they should be reported as such. 7. Minimum sample volume is approximately 150 ul for a single panel and 300 ul for duplicate analysis. When sufficient specimen is not available for analysis, this should be noted on the report. 8. If a sample dilution is made, multiply the instrument output by the dilution factor and note on the instrument output. 9. Quality control samples must meet guidelines set by manufacturer. QC results are maintained in the Toxicology Laboratory.
Instrument Operating, Maintenance, and Troubleshooting Procedure: 1. The instrument operating procedures may be found near the instrument; refer to instrument manual and/or instrument procedure notebook. 2. Instrument prompts to perform maintenance and warnings regarding errors in operation will appear on the Nova CRT screen. Refer to the instrument operation manual for response.
Dallas County Institute of Forensic Sciences Toxicology Laboratory Electrolytes Version 2.0
3. The user will be prompted to change the BUN or GLU membranes approximately every 2 weeks. 4. Deproteinization of the ABG (arterial blood gas) flowpath is done on an as needed basis using the NOVA Deproteinizing Solution/ABG (also called Preheater Deproteinizing Solution). Deproteinization ruins the BUN membrane and should be performed just before changing the BUN membrane. (Note: A more stringent cleaning of the flowpath is described on page 4-17 of the NOVA instrument manual. The BUN and glucose membranes must be replaced after this process.) 5. Instrument maintenance and repair must be documented in the NOVA Instrument Maintenance Log. Interpretation: Electrolyte analysis may be used to assess hydration/dehydration status, control of diabetes, postmortem interval, etc. The following compares 2700 vitreous electrolytes performed at IFS with normal vitreous electrolytes reported by Coe: IFS Results of 2700 vitreous analyses performed in 1996-1997 (includes all cases, not just normals) Sodium Potassium Chloride Glucose VUN Average 140 9.9 123 38 21 Median 142 8.6 124 0 12 Std. Dev. 9.3 3.8 8.9 97.1 29.7 Coe* Normal values 10.5 29 hours postmortem Sodium Potassium Chloride Average 141 8.7 118 Std. Dev. 4 1.1 7
Glucose 51 28
VUN 18 8
*Reference: John I. Coe. Postmortem Chemistry, ASCP National Meeting, 1984 References: NOVA CCX Instrument Manuals Coe JI. Postmortem Chemistry Update. Am J Forensic Med Pathol. 14(2):91-117, 1993.
ELECTROLYTES, UREA NITROGEN, GLUCOSE TRAINING NOTES 1. The NOVA CCX has two flow paths: the AGB/CHEM (arterial blood gas/chemistry) flow path and the CO-Oximeter flow path. This procedure uses the ABG/CHEM flow path. The act of removing the glucose and/or BUN sensors ruins the membranes and requires that the membranes be replaced. If the sample contains particulate material, it should be centrifuged prior to analysis. Error messages should be evaluated, and manufacturers maintenance and troubleshooting recommendations should be followed as applicable. See the instrument maintenance log for required routine maintenance. Approximately three calibrators must be run before the NOVA meets calibration specifications. Results are routinely reported as described for the following error codes: Glucose Error 258 Error 95 Glucose Low Range Error report <15 mg/dl Glucose High Range Error glucose exceeds 500 mg/dl; dilute with deionized water and rerun BUN Low Range Error report as sample unsuitable BUN High Range Error BUN exceeds 100 mg/dl; dilute with deionized water and multiply result by dilution factor
2.
3. 4.
5. 6.
7.
BUN
Error 261 Error 123
UNIVERSAL ELISA METHOD Principle of Assay: The acronym ELISA stands for Enzyme Linked Immuno-Sorbent Assay. This technique is used as a screening procedure for opiates, carboxy-tetrahydrocannabinol, and other drugs. Positive results from ELISA assays are confirmed using other techniques such as gas chromatography/mass spectrometry (GC/MS). The procedure is based upon the competitive binding between antigen (drug) and an antibody to the antigen. The high affinity, purified polyclonal antibody is fixed to the walls of micro-plate wells. Both sample containing drug (antigen) and reagent containing enzyme-labeled drug (drug attached to Horseradish peroxidase) are added to a microplate well. The labeled and unlabeled drug (antigen) compete for binding to the antibody. Binding of each antigen type (labeled or unlabeled) is proportional to the concentration of the antigen type in the reaction mixture. The wells are washed, and a chromogenic substrate (3, 3, 5, 5 tetramethylbenzidine and urea peroxidase, TMB) is added. This forms a blue color reaction. This reaction is stopped by the addition of 1 N hydrochloric acid. The intensity of the color developed is inversely proportional to the concentration of the drug in the sample. Equipment: EVO Tecan Miniprep Workstation Tecan Spectra Reader Columbus Washer Appropriate computer with Tecan and Magellan Software Transfer pipets capable of approximately 500 uL volume 12 x 75; 16x100 ; 16x125 glass culture tubes Various pipettes and/or dispensers capable of 20uL-1000mL Screw top test tubes Centrifuge Various size volumetrics Syringes capable of delivering 2.5 to 50 ul Vortex Reagents: Distilled water Methanol Specific ELISA kits containing the following: - Microplates - Conjugate - TMB Chromogenic Substrate - Stop Reagent (1 N hydrochloric acid)
ELISA Version 2.1
Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eye wash station. Refer to appropriate MSDSs and the kit inserts for additional information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Stocks, Standards, Controls, Calibrators: Preparation of stocks, standards, and controls is described in the Analyte and Method Specific Parameters section of this method. Each instrument method will contain the following in the first three positions: Negative Control (NC): Use approximately 500 uL to 1mL of blank urine or blank blood. Low Control (LC): Use approximately 1 mL total volume Positive Control (PC): Use approximately 1 mL total volume Sample Requirements: Types of samples for analysis: blood, urine, bile, vitreous, gastric, tissue homogenates, other liquid specimens, etc. Sample Collection and Preservation: It is recommended but not required that blood specimens are preserved with sodium fluoride (gray top) and that postmortem specimens are collected from femoral vessels. Liquids are usually refrigerated during storage. Solid samples in plastic cups are usually frozen for storage. Glass containers often break if frozen. Sample Preparation: 1. Dilute each sample and control. a. Mix 100 uL of sample or control and 900 uL of deionized water in a 13 x 100 glass culture tube. 2. Tissues and other solid or semi-solid samples a. Homogenize 5 grams tissue in 15.0 mL deionized water. b. Mix 100 uL of sample and 900 uL of deionized water in a 13 x 100 mL glass culture tube. i. For homogenates that will not pipet, centrifuge a portion (1-2mL) in a test tube. Mix 100uL supernatant and 900 uL deionized water in a 13 x 100 mL glass culture tube. Instrument Setup:
ELISA Version 2.1
1) Fill deionized water container and ensure that the end of the tubing is at the bottom of the container. Tubing has a tendency to float in the container. Be sure to secure the tubing so that it is submerged. a) If bubbles are present in the tubing line, see Section Flushing for instructions on Flushing the lines. 2) Check that all fittings on instrument tubing are finger tight. 3) Check that the Acid Stop level is sufficient for decontamination and as a stop solution. 4) Click on the Shortcut to EVO75 Shell (also called NaviTrak) icon from the desktop. Wait for NaviTrak to load. 5) Click Define Your Panel of Test. The last panel of tests that was run will load automatically. a) click Recall Panel 01 for the general screen (OPI, THC, COC) b) click Recall Panel 02 for the SA (sexual assault) screen (BARB, FLUZ) c) click Recall Panel 03 for the methamphetamine screen (METH) 6) A pop-up menu will confirm the recall of the test, click Yes or Abort. 7) Click Pipet this Configuration. 8) Enter Sample IDs. a) Use the barcode scanner to scan control labels (side of instrument) and the sample IDs from the sample tubes. b) As an alternative you may enter the ID manually and then press TAB. c) When all the labels are entered, click the Store list 01. d) NOTE: Samples 1, 2, and 3 will be NC, LC, and PC respectively 9) A pop-up menu will confirm, click Yes or Abort. a) List names are formatted with the date and a letter identifier. Ex. 0127A (MMDDA). Click OK. 10) Click Run this list. 11) Click Print Sample List. 12) Confirm the sample list has printed, and there are no errors. 13) Check entire liquid path from system liquid container to end of probe tip to ensure that there are no air bubbles in line and syringe. Flush the lines if any air bubbles are present; see section on Flushing.. 14) Place the samples, conjugates, and microplates into position. Check that all racks are pushed all the way back and on the positioning pins; the shield must be in the down position. 15) Place the conjugate (approx. 6 mL of conjugate ~ 40 tests) in test tube rack in the following order: a) (1 OPI, 2 THC, 3 COC ) b) (1 BARB, 2 FLUN) c) (1 METH) 16) Click Start Evoware, Pipet this configuration, ## samples, Samples run in singlicate. 17) Navitrak will now close. 18) A notepad page will pop up with some general instructions from the manufacturer. These can be ignored. EVOware Operation: 1. Click the green triangle icon. Wait for EVOware to load.
Dallas County Institute of Forensic Sciences Toxicology Laboratory 3 ELISA Version 2.1
Menu screen will open: choose Edit an Existing Script. A new window will open. Click on Favorites. From the list box choose 1tip_96 Click green arrow New Screen will open ignore. Click on green arrow in tool bar. Screen will reduce. 6. Click green arrow in the new window. 7. The instrument will now begin to pipet the diluted samples to each microplate well. 8. The instrument will then add 100 uL of the appropriate conjugate to the appropriate plate. 9. When the conjugates have been added to all wells, remove the plates and place them in the drawer to incubate for one hour. The EVOware onscreen timer will start automatically after conjugate is added. The timer countdown is one hour. 10. At the end of incubation, the Tecan will begin a decontamination step. The shield will be locked until a pop-up screen appears. 11. Retrieve the microplates for washing. Wash all plates. Washing the Microplate: 1. When the one hour incubation is nearly complete, prime the microplate washer. a. Run 1: ELISA will be on the screen of the washer, choose Other. b. Prime: Ch 1 appears. c. Choose Yes. d. Choose Yes to the next two prompts, and the instrument will prime itself. 2. Lift the manifold and check that all nozzles have good water flow. Ensure that the inlet tubing is not crimped and that the waste tubing has an unrestricted path to the sink. 3. Place the microplate (notch toward the top right) on the washer. a. Choose Yes 4. Run1: YYYYYYYYYYYY is on the display a. Use the arrow (< or >) key to space over. Use the square or red key to change the number of strips to wash from Y(yes) to n (no). i. Ex. 5 strips are to be washed. YYYYYnnnnnnn should be on the display. ii. Ex. if every other strip is to be washed YnYnYnYnYnYn should be the display. 5. Answer Yes at the prompts. 6. Repeat for any other microplates. TMB ADDITION: 1. Place the washed microplate on the deck and check the amount of TMB solution is sufficient in the side rack. Close the shield, and click OK. TMB will be added to all of the plates. 2. Place the TMB in proper position of the side rack (see notes for analyte conjugate position). 3. Timer 2 will begin counting down after TMB is added.
2. 3. 4. 5.
4. 5. 6. 7.
DO NOT REMOVE MICROPLATES FROM DECK AFTER TMB IS ADDED. The Tecan will automatically add acid stop to the plates once Timer 2 runs down. Window will read Remove all Microplates and Read. Select OK to continue. Select OK. Tecan will decontaminate and the shield will unlock.
Reading the Microplates: 1. Click on the Magellan6 M icon on desktop. 2. The tray will open on the spectrophotometer. 3. A pop-up will come up asking What do you want to do? a. Select Start Measurement and click green arrow. 4. Select Use Predefined Method and choose the proper method a. (Ex. _BARB, _FLUN, _COC, etc.) 5. Click green arrow. 6. Under Workspace type in MMDDATHC (month, date, letter, analyte). 7. Then click green arrow. a. Tray will close for measuring. Print is automatically generated. 8. Close plate reading window. 9. Repeat procedure for other plates starting from step 3. 10. When assay is complete click Exit Magellan. Completing Assay: 1. To exit EVOware, click File then exit, then unload drivers. 2. Empty the acid stop and TMB from the troughs. 3. Place any unused conjugate back into their proper kits. Flushing Lines: 1. 2. 3. 4. 5. Click Evoware Standard (green arrow). Click Run an Existing Script then Start (green arrow). Click Favorites: pick Flush_1Tip, Start (green arrow). New window will open; click Run (green arrow). Pop-up will ask to flush again, enter 1 for yes or 0 for no.
Analyte and Method Specific Parameters: I. Stocks, Standards, and Controls A. Barbiturate (BARB) Controls: 1. Stock - 1 mg/mL secobarbital stock solution in MeOH. a) NOTE: This standard is also used in the Acid Neutral Screen; refer to that procedure for preparation instructions. 2. Working Standard, 10 ug/mL a) Add 100 uL of 1 mg/mL secobarbital stock to a 10 mL volumetric and bring to volume with methanol (MeOH). 3. Barbiturate LC or Cutoff, 300 ng/mL
B.
C. D.
E.
F.
a) In a test tube, mix 970 uL of blank urine and 30 uL of the working standard. 4. Barbiturate PC, 1500 ng/mL a) In a test tube, mix 850 uL of blank urine and 150 uL of the working standard. Flunitrazepam Metabolite (FLUZ) Controls: 1. Stock - 1 mL ampule of 100 ug/mL 7-amino flunitrazepam 2. Working Standard, 1 ug/mL a) Add 100 uL of 100 ug/mL 7-amino flunitrazepam stock to a 10 mL volumetric flask and bring to volume with methanol. 3. FLUZ LC or Cutoff, 5 ng/mL a) In a test tube, mix 995 uL of blank urine and 5 uL of the working standard. 4. FLUZ PC, 25 ng/mL a) In a test tube, mix 975 uL of blank urine and 25 uL of the working standard. BARB and FLUZ Negative Control (NC): 1. Use approximately 500 uL to 1mL of blank urine. BARB and FLUZ Notes 1. The BARB and FLUZ cutoff (LC) can be prepared in the same test tube by mixing 965 uL of blank urine, 30 uL BARB working standard and 5 uL FLUZ working standard. 2. The BARB and FLUZ PC can be prepared in the same test tube by mixing 825 uL of blank urine, 150 uL BARB working standard and 25 uL of the FLUZ working standard. 3. Expiration of the LCs and PCs is 2 weeks from preparation date. Opiate Controls (OPI): 1. Stock - 1mL ampule of 1 mg/mL morphine 2. Working Standard, 1 ug/mL a) Add 10 uL of 1 mg/mL morphine stock to a 10 mL volumetric flask and bring to volume with methanol. 3. OPI LC, 25 ng/mL a) In a test tube, mix 975 uL of blank blood and 25 uL of the working standard. 4. OPI PC, 100 ng/mL a) In a test tube, mix 900 uL of blank blood and 100 uL of the working standard. THC Metabolite (THC) Controls 1. Stock - 1mL ampule of 100 ug/mL N-nor-9-carboxy-delta-9-THC 2. Working Standard, 1 ug/mL a) Add 100 uL of 100ug/mL carboxy-THC to a 10 mL volumetric flask and bring to volume with methanol. 3. THC LC, 20 ng/mL a) In a test tube, mix 980 uL of blank blood and 20 uL of the working standard. 4. THC PC, 50 ng/mL
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a) In a test tube, mix 950 uL of blank blood and 50 uL of the working standard. G. Cocaine Metabolite (COC) Controls: 1. Stock -1 mL ampule of 1 mg/mL benzoylecgonine 2. Working Standard, 10 ug/mL a) Add 100 uL of 1 mg/mL benzoylecgonine stock to a 10 mL volumetric flask and bring to volume with methanol. 3. COC LC, 100 ng/mL a) In at test tube, mix 990 uL of blank blood and 10 uL of the working standard. 4. COC PC, 500 ng/mL a) In a test tube, mix 950 uL of blank blood and 50 uL of the working standard. H. OPI, THC, and COC Negative Control (NC): 1. Use approximately 500 ul to 1mL of blank blood. I. OPI, THC, and COC Notes 1. The OPI, COC, and THC LC can be prepared in the same test tube by mixing 945 uL of blank blood, 25 uL OPI working standard, 20 uL THC working standard, and 10 uL of COC working standard. 2. The OPI, COC, and THC PC can be prepared in the same test tube by mixing 800 uL of blank blood, 100 uL OPI working solution, 50 uL THC working solution, and 50 uL COC working solution. 3. Expiration of OPI, THC, and COC LCs and PCs is 2 weeks from preparation date. J. Methamphetamine (Meth) Controls: 1. METH Stock a) Option 1 Use the 2 mL bottle of 50 ng/mL d-methamphetamine sent with ELISA kit b) Option 2 (1) Prepare a 1mg/mL d-methamphetamine in methanol solution. (a) NOTE: This standard is also used as an ALK response factor; refer to that procedure for preparation instructions. (2) Stock (50ng/mL solution) - In a 100 mL volumetric use 5 uL of the 1mg/mL d-methamphetamine in methanol solution and dilute to volume with blank urine or deionized water 2. METH LC, 20 ng/mL a) Mix 600ul blank urine or deionized water with 400 uL of 50 ng/mL dmeth stock with for the d-meth low control (LC). 3. METH PC, 50 ng/mL a) Use 0.5mL of the ELISA METH stock. II. Tecan Methods General Screen SA Panel METHAMPHETAMINE for methamphetamine/MDMA analysis III. Conjugate Microplate Positions
Position 1 OPI BARB METH
Position 2 THC FLUZ
Position 3 COC
IV. Conjugate Positions A. Barbiturate - 1 B. Opiate 1 C. Cocaine 2 D. THC 3 E. Flunitrazepam 2 F. Methamphetamine 1 V. Cut Off (LC) Limits A. Barbiturates - 300 ng/mL secobarbital B. Cocaine -100 ng/mL benzoyleconine C. Flunitrazepam - 5 ng/mL 7-amino flunitrazepam D. Opiates - 25ng/mL morphine E. THC - 20 ng/mL carboxytetrahydrocannabionol F. Methamphetamine 20 ng/mL d-methamphetamine VI. Magellan Methods A. Methods are listed in a generic format: _METHOD B. The following prefixes are used: BARB barbiturates COC cocaine metabolite FLUN flunitrazepam metabolite METH methamphetamine OPI opiate THC tetrahydrocannabinol metabolite Quality Control: 1. The low control concentration sets the cutoff limits for each assay. 2. The positive control must be more positive than the low control and should calculate as a positive. 3. The negative control should be negative. 4. Results of the negative, low, and high controls are included in the case file. Instrument Operating Procedure: The instrument operating procedure may be found near the instrument; refer to instrument manual. References: Flunitrazepam Direct ELISA kit insert from Immunalysis Barbiturate Direct ELISA kit insert from Immunalysis
Cocaine Metabolite (BE) Direct ELISA kit insert from Immunalysis Opiate Direct ELISA kit insert from Immunalysis THC Direct ELISA kit insert from Immunalysis Methamphetamine Direct ELISA kit insert from Immunalysis.
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UNIVERSAL ELISA TRAINING NOTES 1. 2. 3. 4. 5. 6. 7. Discard TMB chromogenic substrate reagent if it turns blue. Do not freeze reagents. Do not mix reagents from different lot numbers. Keep reagents out of direct sun light and excessive heat. Bring all reagents to room temperature before use. Check the expiration date of the kit before use; do not use kits after the expiration date. A drop of greater than 50% in the A0 (zero-standard absorbance reading) for a constant incubation time indicates deterioration of the antibody plate, enzyme conjugate, or chromogenic substrate. 8. Always remember to remove the microplate from the spectrophotometer after the microplate is read; the TMB solution will cause corrosion to the internal parts of the spectrophotometer. 9. If the Tecan stops during the pipetting process, it may be because there is not enough sample in the tube. Click on Go to Z Max on the window that pops up. 10. In the Sample and Conjugate Additions section: If the one hour incubation time is exceeded, the TMB incubation time should be shortened. For example, if the first incubation time is 2 hours, the TMB color development time should be shortened from 25 minutes to 15 minutes; otherwise, color development will be too strong. 11. Sampled homogenates may be centrifuged to allow for easier sampling. 12. Positives in the opiate, THC, cocaine, flunitrazepam methods should be placed on assay lists for confirmation by GC/MS methods. 13. Methamphetamine or MDMA only positives from the alkaline screen should be confirmed by a second extraction of another specimen or by ELISA method if another specimen is unavailable. 14. Cross-reactivity in the Flunitrazepam Direct ELISA Kit has been noted to produce false positives in samples that contain diazepam and/or 7-amino clonazepam and/or high concentrations of numerous benzodiazepines. 15. Strong opiate positives with a negative GC/MS should be evaluated to determine if they should be sent to a referral lab for buprenorphine testing.
10
ELISA Version 2.1
ETHCHLORVYNOL QUANTITATION Principle of Assay: Ethchlorvynol is a sedative hypnotic that is detected and identified in the acid/neutral screen. Because ethchlorvynol is volatile, some of the drug may be lost during the evaporation step in the acid/neutral procedure. Therefore when ethchlorvynol is detected in the acid/neural screen, it is quantitated using this method. In this analysis, the specimen is made acidic and ethchlorvynol is extracted into chloroform. Quantitation is performed using gas chromatography. Identification of ethchlorvynol by GC/MS is performed as a part of the standard acid/neutral procedure. Equipment: Hewlett-Packard model GCD GC/MS Hewlett-Packard model 7673 autosampler Capillary columns : such as DB-5 (cross-linked methyl silicone gum phase) 12 m x 0.20 mm x 0.33 um film thicknness Culture tubes, 15 ml, screw cap with Teflon liner Conical tubes, 5 ml, screw cap with Teflon liner Eppendorf pipet - 100ul 5 ml serological pipets 1 ml serological pipets Vortex mixer Centrifuge Autosampler vials, 32mm x 11mm, Teflon lined seals and glass inserts Reagents: Hydrochloric acid, concentrated Chloroform Ethchlorvynol standard is light sensitive; standard and stock should be stored, light protected, in the refrigerator. 3N Hydrochloric Acid: Fill a 500 ml volumetric flask approximately half full with deionized water. Add 125 ml concentrated hydrochloric acid. Mix carefully and allow to cool if necessary. Fill to the mark with deionized water. Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used
Dallas County Institute of Forensic Sciences Toxicology Laboratory Ethchlorvynol Version 2.0
chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eyewash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Hydrochloric acid is corrosive. Avoid contact with skin, eyes, and mucous membranes. Chloroform is a chlorinated solvent and is considered a human carcinogen. Use in a vent hood or in a well-ventilated area. Avoid contact with skin, eyes, and mucous membranes. Standards: 1.0 mg/ml Thymol Internal Standard: Weigh 10 mg thymol and transfer to a 10 ml volumetric flask. Dilute to volume with methanol. 10 mg/ml Ethchlorvynol Stock Standard (such as Alltech cat. 01733, 100 uL vials): Weigh 100 mg ethchlorvynol (will take two vials) and transfer to a 10 ml volumetric flask. Dilute to volume with methanol. Store in light protected tube and below room temperature. 10 mg/L Ethchlorvynol Working Standard: To a 15 ml screw cap tube, add 5ul ethchlorvynol stock standard (10 mg/ml) to 1 mL deionized water. Add 4 ml blank blood and rotate for 5 minutes. 50 mg/L Ethchlorvynol Working Standard: To a 15 ml screw cap tube, add 25ul ethchlorvynol stock standard (10 mg/ml) to 1 mL deionized water. Add 4 ml blank blood and rotate for 5 minutes. 100 mg/L Ethchlorvynol Working Standard: To a 15 ml screw cap tube, add 50ul ethchlorvynol stock standard (10 mg/ml) to 1 mL deionized water. Add 4 ml blank blood and rotate for 5 minutes. Ethchlorvynol Stock QC __mg/mL: Weigh contents of one vial of Ethchlorvynol standard (approx. 60-70 mg) in 10 mL volumetric flask. Dilute to volume with methanol. Store in light protected tube in refrigerator. Concentration in mg/mL will be the mg of ethchlorvynol added divided by 10; for example if 60 mg is used in the stock, the stock concentration will be 6 mg/ml. Working QC __mg/L: Add 50 uL Ethchlorvynol Stock QC (approx. 60-70 mg/mL) to 1 mL deionized water. Add 4 mLs blank blood and rotate for 5 minutes. Concentration of working QC in mg/L will be 10 times the stock concentration; for example if a 6 (mg/ml) stock is used, the working QC will be 60 mg/L. Negative Control: Use 500 uL of blank blood.
Ethchlorvynol Version 2.0
Sample Requirements: Acceptable specimens: blood, serum/plasma, urine, bile, gastric, vitreous, tissue homogenates, pharmaceutical preparations Sample Collection and Preservation: It is recommended but not required that blood specimens are preserved with sodium fluoride (gray top); however, most acid and neutral drugs are not as susceptible to in vitro enzymatic degradation as alkaline drugs. Femoral blood is preferable; however, acid and neutral drugs are typically less susceptible to postmortem redistribution than alkaline drugs. Liquid specimens (blood, urine, etc.) should be refrigerated during storage; solid specimens (gastric, tissues, etc.) in plastic cups should be frozen during storage. Ethchlorvynol degrades over time in biological specimens. Specimens should be refrigerated upon receipt and analyzed as soon as possible. Sample Preparation: Tissues: Homogenize 5 grams tissue in 15.0 mL deionized water. Use 500 uL of the homogenate for analysis Gastric: For initial analysis dilute 1 part gastric with 99 parts water (1 to 100 dilution). Any significant quantity of a drug will be detected. If diluted sample is negative, an undiluted or less diluted sample may be run. Non-homogenous gastric should be homogenized prior to use. Other samples: Use 500 uL in the analysis. Non-standard dilutions must be noted in the case file. Analytical Procedure: 1. 2. 3. 4. 5. 6. Place 500 uL of each sample and standard in a 5 mL conical tube. Add 100 uL of thymol internal standard. Vortex. While vortexing the conical tube, add 100 uL 3N HCl. Note: HCl must be added while vortexing or the blood in the tube will clot and not mix well. Add 1 mL chloroform. Vortex. Centrifuge on high for about 5 minutes. Transfer chloroform layer (bottom) to an autosampler vial for GC analysis.
Ethcholrvynol Version 2.0
Quality Control: Controls should match manufacturers range or fall within +/- 20% of target concentration. If not, consult a supervisor or repeat the assay or sample. QC results are maintained in the Toxicology Laboratory. Qualifier ions should be within +/- 20% of target. Instrument Operating Procedure: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebook. Training Notes: Refer to Valproic Acid Quantitation
Ethcholrvynol Version 2.0
ETHOSUXIMIDE QUANTITATION Principle of Assay: Ethosuximide is an anticonvulsant drug that is detected in the acid/neutral screen. Because ethosuximide is volatile, some of the drug may be lost during the evaporation step in the acid/neutral procedure. Therefore when ethosuximide is detected in the acid/neural screen, it is quantitated using this method. In this analysis, the specimen is made acidic and ethosuximide is extracted into chloroform. Quantitation is performed using GC/MS. Initial dentification of ethosuximide is performed as a part of the standard acid/neutral procedure. Equipment: Hewlett-Packard model GCD GC/MS Hewlett-Packard model 7673 autosampler Capillary columns : such as DB-5 (cross-linked methyl silicone gum phase) 12 m x 0.20 mm x 0.33 um film thicknness Conical tubes, 5 ml, screw cap with Teflon liner Culture tubes, 15 ml, screw cap with Teflon liner Eppendorf pipet - 100ul 5 ml serological pipets 1 ml serological pipets Vortex mixer Centrifuge Autosampler vials, 32mm x 11mm, Teflon lined seals and glass inserts Reagents: Hydrochloric acid, concentrated Chloroform 3N Hydrochloric Acid: Fill a 500 ml volumetric flask approximately half full with deionized water. Add 125 ml concentrated hydrochloric acid. Mix carefully and allow to cool if necessary. Fill to the mark with deionized water. Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or
Ethosuximide Version 2.0
eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Hydrochloric acid is corrosive. Avoid contact with skin, eyes, and mucous membranes. Chloroform is a chlorinated solvent and is considered a human carcinogen. Use in a vent hood or in a well-ventilated area. Avoid contact with skin, eyes, and mucous membranes. Standards: 1.0 mg/ml Thymol Internal Standard: Weigh 10 mg thymol and transfer to a 10 ml volumetric flask. Dilute to volume with methanol. 10 mg/ml Ethosuximide Stock Standard: Weigh 100 mg ethosuximide and transfer to a 10 ml volumetric flask. Dilute to volume with methanol. 10 mg/L Ethosuximide Working Standard: To a 15 ml screw cap tube, add 5ul ethosuximide stock standard (10 mg/ml) to 1 mL deionized water. Add 4 ml blank blood and rotate for 5 minutes. 50 mg/L Ethosuximide Working Standard: To a 15 ml screw cap tube, add 25ul ethosuximide stock standard (10 mg/ml) to 1 mL deionized water. Add 4 ml blank blood and rotate for 5 minutes. 100 mg/L Ethosuximide Working Standard: To a 15 ml screw cap tube, add 50ul ethosuximide stock standard (10 mg/ml) to 1 ml deionized water. Add 4 ml blank blood and rotate for 5 minutes. Quality Control 25 mg/L ethosuximide: To a 15 ml screw cap tube, add 12.5ul ethosuximide QC stock standard (10 mg/ml) to 1 ml deionized water. Add 4 ml blank blood and rotate for 5 minutes. Negative Control Use 500 uL of blank blood. Sample Requirements: Acceptable specimens: blood, serum/plasma, urine, bile, gastric, vitreous, tissue homogenates, pharmaceutical preparations Sample Collection and Preservation: It is recommended but not required that blood specimens are preserved with sodium fluoride (gray top); however, most acid and neutral drugs are not as susceptible to in vitro enzymatic degradation as alkaline drugs. Femoral blood is preferable; however, acid and neutral drugs are typically less susceptible to postmortem redistribution than alkaline drugs. Liquid specimens (blood, urine, etc.) should be refrigerated during storage; solid specimens (gastric, tissues, etc.) in plastic cups should be frozen during storage.
Dallas County Institute of Forensic Sciences Toxicology Laboratory Ethosuximide Version 2.0
Sample Preparation: Tissues: Homogenize 5 grams tissue in 15.0 mL deionized water. Use 500 uL of the homogenate for analysis Gastric: For initial analysis dilute 1 part gastric with 99 parts water (1 to 100 dilution). Any significant quantity of a drug will be detected. If diluted sample is negative, an undiluted or less diluted sample may be run. Non-homogenous gastric should be homogenized prior to use. Other samples: Use 500 uL in the analysis. Non-standard dilutions must be noted in the case file. Analytical Procedure: 1. 2. 3. 4. 5. 6. Place 500 uL of each sample and standard in a 5 mL conical tube. Add 100 uL of thymol internal standard. Vortex. While vortexing the conical tube, add 100 uL 3N HCl. Note: HCl must be added while vortexing or the blood in the tube will clot and not mix well. Add 1 mL chloroform. Vortex. Centrifuge on high for about 5 minutes. Transfer chloroform layer (bottom) to an autosampler vial for GC/MS analysis. Samples are run in full scan mode with quantitation by SIM.
Quality Control: Controls should match manufacturers range or fall within +/- 20% of target concentration. If not, consult a supervisor or repeat the assay or sample. QC results are maintained in the Toxicology Laboratory. Qualifier ions should be within +/- 20% of target. Instrument Operating Procedure: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebook. Training Notes: Refer to Valproic Acid Quantitation
Ethosuximide Version 2.0

ETHYLENE GLYCOL ANALYSIS Principle of Assay: Ethylene glycol is the principal constituent of many automotive antifreeze preparations. In this procedure ethylene glycol (EG) and the internal standard, 1,2-butanediol (BD), are derivatized during extraction with n-butylboronic acid (BBA). BBA combines with difunctional hydroxyl containing compounds to form five- and six- member rings. The reactions are rapid, occur at room temperature in a variety of solvents, and are essentially complete in a very short period of time. The derivatives are analyzed using GC/MS. Equipment: Hewlett-Packard GCD gas chromatograph/ mass spectrometer Hewlett-Packard model 7673 autosampler Capillary column : such as DB-1 (cross-linked methyl silicone gum phase) 25m x 0.32mm x 0.52um film thickness. Adjustable pipette -20 to 200uL Rotator Centrifuge Autosampler vials, 32 mm x 11 mm, Teflon lined seals and glass inserts Vortex mixer Disposable glass Pasteur pipets Volumetric flasks - 5mL and 10mL Microcentrifuge tube 1.5mL Culture tubes, 15 ml, screw top with Teflon liner Reagents: Acetone, analytical grade Ethyl acetate, analytical grade 1-Butaneboronic acid, 99+% 1% n-Butylboronic Acid (derivatizing agent): Place 50 mg of n-butylboronic acid into a 5 mL volumetric flask. Add 10-15 drops of acetone and mix. Gradually bring to volume with ethyl acetate. 1,2-Butanediol, 98% Ethylene glycol Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or
Ethylene Glycol Version 2.0

eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Solvents used in this procedure are volatile. Avoid exposure to skin, eyes, and mucous membranes. Stocks, Standards, Calibrators and Controls: Ethylene Glycol Working Standard (22.26 mg/mL): Place 200 uL of ethylene glycol into a 10 mL volumetric flask and bring to volume with deionized water. (Density of ethylene glycol at 20 deg C = 1.1135 g/mL.) 1,2-Butanediol Working Internal Standard (20.12mg/mL): Place 20 uL of 1,2-butanediol into a 10 mL volumetric flask and bring to volume with deionized water. (Density at 20 deg C = 1.0060 g/mL.) Ethylene Glycol Calibrators and Controls: In a 15 ml conical tube, place the following and rotate for about 10 minutes to mix: Calibrator 1 (56 mg/L): 10uL ethylene glycol working standard and 4mL of blank blood Calibrator 2 (278 mg/L): 50uL ethylene glycol working standard and 4mL of blank blood Calibrator 3 (557 mg/L): 100uL ethylene glycol working standard and 4mL of blank blood Control (167 mg/L): 30uL ethylene glycol working standard and 4mL of blank blood Control (Negative): 4 mL blank blood Sample Requirements: Acceptable specimens: blood, serum/plasma, urine, gastric and, consumer products Sample Collection and Preservation: Liquid specimens (blood, urine, etc.) should be refrigerated during storage; solid specimens (gastric, tissues, etc.) in plastic cups should be frozen during storage. Food and product items are usually stored refrigerated.
Sample Preparation: Gastric: For initial analysis dilute 1 part gastric with 99 parts water (1 to 100 dilution). Any significant quantity of a drug will be detected. If diluted sample is negative, an undiluted or less diluted sample may be run. Non-homogenous gastric should be homogenized prior to use. Other samples: Dilute and homogenize as needed. Analytical Procedure: 1. 2. 3. 4. 5. In a microcentrifuge tube, place 250 ul of sample, calibrator, or control and 20 uL of working internal standard and vortex. Add 250uL of 1% n-butylboronic acid to each tube. Vortex for about 30 seconds. Centrifuge in the microcentrifuge for 3 min. Note: If phase separation does not occur, place tubes in the freezer for about 10 minutes then re-centrifuge. Transfer organic supernatant (ethyl acetate top layer) to autosampler vial and inject on GC/MS.
Calculations: Results are reported in mg/L. Other Dilutions: The calculated result represents the concentration of drug in the diluted sample. To calculate the concentration of drug in the original sample, multiply by the dilution factor. For example if 0.5 mLs of gastric + 0.5 mL of water was analyzed instead of 1.0 mL of gastric, multiply the calculated concentration by 1/0.5 or 2 to determine the concentration of drug in the undiluted specimen. If the original specimen was a solid and weighed, units are mg/Kg. If the original specimen was a pipettable liquid, units are mg/L. Methods of dilution must be noted in the case file. Quality Control: The lower limit of quantitation for the assay is 56 mg/L ethylene glycol. Linearity of the assay has been verified from 56-557 mg/L. Ion qualifiers must match within +/- 20%; otherwise, the sample must be reviewed with a supervisor or repeated. One control sample is run with each batch and the results should be within +/- 20% of target. QC results are maintained in the Laboratory. Instrument Methods:

Refer to the Instrument Methods Notebook. Instrument Operating Procedure: Refer to the Instrument Operating Procedure located near the instrument. References: McCurdy HH and Solomons ET. An Improved Procedure for the Determination of Ethylene Glycol in Blood. J Anal Tox 6: 253-254, 1982. Ethylene Glycol Analysis, Bexar County Forensic Science Center, San Antonio, Texas.
ETHYLENE GLYCOL TRAINING NOTES Use ETHGLY3 method on GC/MS. When preparing derivatizing agent (1% n-butylboronic acid) dissolve in 10 15 drops of acetone and gradually bring to volume with ethyl acetate.
FENTANYL by GC/MS Principle of Assay: Fentanyl is a short acting, synthetic narcotic analgesic of high potency. The half life of the drug reportedly ranges from 3-12 h. The drug is available as an injectable solution or as a transdermal patch. Fentanyl is generally used as an adjunct to surgical anesthesia and for pain management in prehospital transport. Therapeutic concentrations are relatively low, necessitating the 2.5 ng/mL (0.0025 mg/L) limit of quantitation of this method. Fentanyl also can be abused, with toxicity resulting in respiratory depression, seizures, coma or death. Fentanyl and the deuterated analog, fentanyl-d5 (internal standard), are extracted from biological fluids using solid phase extraction. pH is adjusted using acetate buffer and centrifugation of samples removes solid particulates. The supernatant is introduced into the conditioned solid phase extraction (SPE) columns. The column eluate is evaporated to dryness and reconstituted with chloroform. The compounds are analyzed by gas chromatography/mass spectrometry (GC/MS) using selective ion monitoring (SIM). Equipment: Hewlett-Packard model 6890 series II gas chromatograph Hewlett-Packard model 6890 autosampler Hewlett-Packard model 5973 mass selective detector Capillary column: DB-5MS, DB-1MS, 15m x 0.25m x 0.25 mm, or equivalent Screw top conical tubes, 15 mL, screw cap with Teflon liner 25 uL gas tight syringe 50 uL pipettor 100 uL pipettor 5 mL serological pipets Repipet dispenser Centrifuge Vortex mixer United Chemical Technologies Clean Screen solid phase extraction columns (CSDAU206) Vacuum manifold, such as Varian VacElut SPS24 Test tubes, 13x100 mm 5 mL conical test tubes Water bath/evaporator Disposable pipettes Autosampler vials, 32 mm x 11 mm, Teflon lined seals and glass inserts Reagents: 0.1 M Acetate buffer, pH 4.5 Deionized water Methanol 0.1 M Acetic acid
Dallas County Institute of Forensic Sciences Toxicology Laboratory 1 Fentanyl by GC/MS Version 2.0
Hexanes Ammonium hydroxide Isopropyl alcohol Dichloromethane Chloroform Blank blood 0.1M Acetic acid: To a 1000 mL volumetric flask, add 5.7 mL of glacial acetic acid and bring to volume with deionized water; mix well. 0.1M Sodium acetate buffer, pH 4.5: To a 1000 mL volumetric flask containing approximately 500 mL deionized water, add 8.2 grams of sodium acetate (anhydrous), OR 13.6 grams of sodium acetate trihydrate. Swirl until dissolved. Add 8.0 mL of glacial acetic acid and bring to volume with deionized water. Check the pH. If necessary, adjust with NaOH or acetic acid to a final pH of 4.4 - 4.6. Column elution solution (make fresh daily): To a 100 mL graduated cylinder add 18 mL of isopropyl alcohol and 2 mL of concentrated ammonium hydroxide; bring to volume with dichloromethane. Note: n-propanol may be substituted for isopropyl alcohol. Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Acetic acid is corrosive. Avoid contact with skin, eyes, and mucous membranes. Ammonium hydroxide is a volatile, corrosive base. Avoid contact with skin, eyes, and mucous membranes; do not inhale vapors. Methanol, hexanes, isopropyl alcohol, and dichloromethane are volatile solvents. Avoid contact with skin; do not breathe vapors. Chloroform is a chlorinated solvent and is considered a human carcinogen. Use in a vent hood or a well-ventilated area. Avoid contact with skin, eyes, and mucous membranes. Stocks, Standards, Controls, Calibrators: Stock fentanyl standard (1mg/mL): This standard is typically obtained commercially but may also be prepared in-house. Fentanyl working standard (1000 ng/mL): Transfer 10 L of fentanyl stock (1mg/mL) to a 10 mL volumetric flask. Dilute to volume with methanol.
Fentanyl QC working solution (100 ng/mL): A 100 g/mL stock solution is obtained commercially. Transfer 10 L of fentanyl QC stock to a 10 mL volumetric flask. Dilute to 10 mL with methanol for a 100 ng/mL working QC solution. OR Prepare a 1 mg/mL stock solution by adding 15.7 mg fentanyl citrate to a 10 mL volumetric flask. Transfer 10 L stock solution to a 10 mL volumetric flask. Dilute to 10 mL with methanol for a 100 ng/mL working QC solution. Fentanyl-d5 internal standard working solution (1000 ng/mL): A 100 g/mL fentanyl-d5 stock solution is obtained commercially. Transfer 100 l of stock solution to a 10 mL volumetric flask to prepare a 1000 ng/mL working internal standard solution. Sample Requirements: Acceptable specimens: blood and urine. Sample Collection and Preservation: It is recommended but not required that blood specimens are preserved with sodium fluoride (gray top), and that postmortem specimens are collected from femoral vessels. Liquids are usually refrigerated during storage. Sample Preparation: Samples: Use 1 mL of sample in the analysis High concentration samples may require dilution. In this case, use less than 1 mL of sample and add deionized water to make a total volume of 1.0 mL. Sample dilutions must be noted in the case file. Analytical Procedure: 1. Label disposable 15 mL screw top test tubes for each standard, control and specimen. 2. Add 4 mL deionized water to each tube. 3. Preparation of calibrators and controls: Calibrators: 0.0025 mg/L (2.5 ng/mL) calibrator: Add 2.5 uL fentanyl working solution. 0.005 mg/L (5 ng/mL) calibrator: Add 5 uL fentanyl working solution. 0.010 mg/L (10 ng/mL) calibrator: Add 10 uL fentanyl working solution. 0.050 mg/L (50 ng/mL) calibrator: Add 50 uL fentanyl working solution. 0.100 mg/L (100 ng/mL) calibrator: Add 100 uL fentanyl working solution. Controls: 0.010 mg/L (10 ng/mL) QC control: Add 100 uL of the QC working solution. Negative control: No addition of standard. 4. Add 50 uL of fentanyl-d5 internal standard working solution to all tubes. 5. Add 1 mL blank blood to all calibrator and control tubes.
Add 1 mL of sample to each corresponding sample tube. Add 2.0 mL 0.1M acetate buffer, pH 4.5 to all tubes. Vortex mix. Centrifuge for 10-12 minutes on high. Load the appropriate number of labeled Clean Screen extraction columns (CSDAU206) for each sample onto the vacuum manifold. Secure the top on the vacuum manifold and ensure that it is in the Waste position. 10. Condition the columns by adding: a. 2 mL methanol; aspirate completely. b. 2 mL deionized water; aspirate. c. 2 mL of 0.1M acetic acid; aspirate. 11. Introduce samples into columns and aspirate slowly. 12. Wash the columns by adding: a. 2 mL 0.1 M acetic acid into columns; aspirate. b. 2 mL deionized water into columns; aspirate. c. 2 mL hexanes; aspirate and dry for approximately 8 minutes under full vacuum. d. 2 mL methanol; aspirate and dry for approximately 8 minutes under full vacuum. 13. Prepare extraction manifold for specimen collection. Place appropriately labeled 13x100 mm test tubes into collection position. Wipe tips clean and replace lid; verify the SPE tips are securely placed inside collection tubes. Secure the top on the vacuum manifold and change it to the Collect position. 14. Add 2 mL column elution solution to columns; allow to elute by gravity. 15. Transfer the eluant to a 5 mL conical tube. 16. Evaporate each specimen to dryness in a water bath with air at 40C. 17. Reconstitute each specimen with 50 uL of chloroform. 18. Transfer to labeled autosampler vials and analyze by GC/MS SIM method for fentanyl. 19. Dispose of SPE waste in the appropriate hazardous waste containers. Quality Control: One positive control and one negative control are run in each batch. The positive control should be within +/- 20% of target, and qualifier ions must be within +/- 20% of target. The negative control should be negative. If not, consult a supervisor or repeat the assay or sample. QC results are maintained in the Toxicology Laboratory. Instrument Operating Procedure: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebook. Instrument Methods: Refer to the Instrument Methods Notebook. Reference: United Chemical Technologies Application Note: Fentanyl in Whole Blood for GC/MS Using: 200 mg Clean Screen Extraction Column.
6. 7. 8. 9.
FENTANYL TRAINING NOTES 1. Extracted specimens will evaporate after 36-48 hours, so specimens should be capped tightly and injected in a timely manner. 2. Extracted specimens that have dried up may be reconstituted with 50 L chloroform. 3. Make sure the temperature of the water bath is 60C. 4. For cleaning purposes, make sure that the VacElute is turned on to a hiss even when pulling through by gravity.
Fentanyl by GC/MS Version 2.0
GAMMA-HYDROXYBUTYRATE by GC/MS Principle of Assay: Gamma hydroxybutyrate (GHB) is an endogenous constituent of the brain and may function as a neurotransmitter. GHB is a metabolite of GABA, the primary inhibitory neurotransmitter within the CNS. GHB is a drug of abuse and has been implicated as a knock-out drug to facilitate sexual assault. It is common that normal postmortem blood samples contain GHB; therefore, the lower limit of reporting for postmortem samples is 25 mg/L. The lower limit of reporting for other specimens is 10 mg/L. GHB and the deuterated compound (GHBD6) are extracted from biological fluids using solid phase extraction. Sample pretreatment consists of addition of acetone and centrifugation of samples to remove solid particulates. The supernatant is evaporated to dryness and reconstituted with phosphate buffer. Samples are introduced into the solid phase extraction columns. The column eluate is evaporated to dryness and derivatized with BSTFA with 1% TCMS and ethyl acetate. The compounds are analyzed by gas chromatography/mass spectrometry (GC/MS) using selective ion monitoring (SIM). Equipment: Hewlett-Packard model 6890 series II gas chromatograph Hewlett-Packard model 6890 autosampler Hewlett-Packard model 5973 mass selective detector Capillary column: such as DB-5 (cross-linked methyl silicone gum phase) 12 m x 0.20 mm x 0.33 um film thickness United Chemical Technologies Clean Screen GHB extraction columns (CSGHB206) Screw top conical tubes, 5 ml, screw cap with Teflon liner 50 uL syringe 50 uL pipettor 100 uL pipettor 200 uL pipettor 1000 uL pipettor Centrifuge Autosampler vials, 32 mm x 11 mm, Teflon lined seals and glass inserts Vortex mixer Evaporator Test tubes, 13x100 mm Disposable pipettes Nitrogen, compressed gas cylinder Vacuum manifold, such as Varian VacElut SPS24 Reagents: Potassium phosphate buffer, 0.1 M, pH 6.0 Sodium hydroxide, 40%
Dallas County Institute of Forensic Sciences Toxicology Laboratory 1 GHB by GC/MS Version 2.1
Acetone Methanol Ethyl acetate BSTFA with 1% TMCS Deionized water Ammonium hydroxide Potassium phosphate buffer, 0.1 M, pH 6.0: Dissolve 13.6 g KH2PO4 in 800 ml deionized water. Adjust to pH 6.0 by addition of 40% NaOH. Dilute to 1000 ml using deionized water. Mix. Sodium hydroxide, 40%: Dissolve 40g NaOH in deionized water. Cool. Dilute to 100 ml with deionized water. Methanol/Ammonium hydroxide solution (99/1): To a 100 ml graduated cylinder add 1 ml of concentrated ammonium hydroxide, and fill to 100 ml with methanol. Prepare fresh daily. Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Ammonium hydroxide is a volatile, corrosive base. Avoid contact with skin, eyes, and mucous membranes; do not inhale vapors. Sodium hydroxide is a corrosive base. Avoid contact with skin, eyes, and mucous membranes. Methanol and ethyl acetate are volatile solvents. Avoid contact with skin; do not breathe vapors. BSTFA with 1% TCMS is corrosive. Avoid contact with skin, eyes, and mucous membranes. Stocks, Standards, Controls, Calibrators: Stock GHB standard (1mg/mL): Obtained commercially. GHB working standard (200 mg/L): Transfer 2.0 ml of GHB stock (1mg/mL) to a 10 ml volumetric flask. Dilute to volume with methanol. GHB quality control (200 mg/L): Transfer 2.0 ml of GHB stock (1mg/mL) to a 10 ml volumetric flask. Dilute to volume with methanol. This is the same concentration as the working standard but prepared independently. GHB deuterated internal standard (100 ug/ml): Obtained commercially.
GHB by GC/MS Version 2.1
Sample Requirements: Acceptable specimens: blood, serum/plasma, and urine. Note: Blood and serum/plasma are analyzed using a curve and QC samples made in blank blood. Urine samples are analyzed using a curve and QC samples made in deionized water. Response is dependent upon the matrix. Sample Collection and Preservation: It is recommended but not required that blood specimens are preserved with sodium fluoride (gray top), and that postmortem specimens are collected from femoral vessels. Collection of postmortem blood in gray top tubes combined with refrigeration retards but does not eliminate postmortem formation of GHB. Liquids are usually refrigerated during storage. Sample Preparation: Sample dilutions must be noted in the case file. Analytical Procedure: Preparation of Calibrators: Note Calibrators and controls are made in deionized water when analyzing urine samples. Calibrators and controls are made in blank blood when analyzing blood samples. See training note #4. To a 5 ml conical tube add the following: 10 mg/L calibrator - Add 10 uL GHB working standard to 200 uL deionized water or blood. 25 mg/L calibrator - Add 25 uL GHB working standard to 200 uL deionized water or blood. 50 mg/L calibrator - Add 50 uL GHB working standard to 200 uL deionized water or blood. 100 mg/L calibrator - Add 100 uL GHB working standard to 200 uL deionized water or blood. 200 mg/L calibrator - Add 200 uL GHB working standard to 200 uL deionized water or blood. Preparation of Quality Control Samples: To a 5 mL conical tube add the following: 50 mg/L control - Add 50 uL GHB working control to 200 uL deionized water or blank blood. Negative control - Add 200 uL deionized water or blank blood. Preparation of Samples: blank blank blank blank blank
1. 2. 3. 4.
Add 200 uL of blood or urine to a 5 ml conical tube. Add 50 uL of deuterated GHB internal standard to all tubes. Vortex. Add 1 mL of acetone to each tube, cap and vortex, let sit for 5 minutes. Centrifuge for 10 minutes at approximately 2000 rpm and transfer liquid into clean labeled 5 ml conical tubes. 5. Evaporate to dryness in water bath at about 50-55 C while blowing with air (about 15 to 20 minutes). 6. Reconstitute dried extracts with 400 uL of potassium phosphate buffer, 0.1 M, pH 6.0, vortex, and transfer samples into labeled sample tubes. 7. Using a cotton tip applicator, tap down the packing in the extraction columns. 8. Set up one extraction column for each specimen in the vacuum manifold. Place one 13x100 mm test tube in the collection position for each column. Secure the top on the vacuum manifold and ensure that it is in the Waste position. Condition columns with: a. 3 mL methanol (allow to flow by gravity) b. 3 mL water (allow to flow by gravity) c. 1 mL phosphate buffer (allow to flow by gravity) 9. Add the buffer/sample mixture to the columns and allow to drain by gravity. 10. Dry under full vacuum for 10 minutes. Wipe out any moisture in each column with a clean cotton swab; use a clean swab for each tube. Wipe bottom tips of column to remove excess water. 11. Shift vacuum manifold lid into the Collect position. Elute each column with 2.0 mL methanol/ammonium hydroxide solution (99:1). 12. Transfer eluted samples to conical tubes and dry to residue in the water bath at about 50-55 degrees C while blowing with air. 13. Reconstitute each sample with 75 uL of ethyl acetate and 75 uL of derivatizing reagent (BSTFA with 1% TMCS). 14. Vortex, cap tubes, and centrifuge. 15. Transfer extract to labeled autosampler vials. Note: leave precipitate and a little of the sample in the bottom of the conical vial. 16. Analyze by GC/MS SIM method GHBD6 for urine samples and GHBD6B for blood samples. Reporting Criteria: Although GHB is an endogenous substance, it is usually not detectable in normal blood or urine of living persons. Sexual assault samples: GHB has been implicated as a means of incapacitating a sexual assault victim. In these cases, urine is screened first if available. GHB is reported as detected in urine if the concentration is greater than 10 mg/L. If the urine is positive, then blood is tested. Report GHB if the blood concentration is greater than 10 mg/L. GHB has a short half life; therefore, GHB in blood is expected to remain detectable for only a few hours after use, and GHB in urine may remain detectable for 12 hours or so. Medical Examiner samples: GHB is routinely formed after death in postmortem blood samples. Therefore in postmortem cases, GHB is reported only if the concentration in blood is greater than
Dallas County Institute of Forensic Sciences Toxicology Laboratory 4 GHB by GC/MS Version 2.1
25 mg/L. Postmortem formation of GHB in autopsy blood has been reported in excess of 150 mg/L even in a refrigerated, sodium fluoride preserved specimen. Urine is not as subject to postmortem formation of GHB and may be a useful specimen for autopsy toxicology. Quality Control: One control sample and a negative control are run in each batch. The control should match target within +/- 20%, and qualifier ions must be within +/- 20% of the target. If not, consult a supervisor or repeat the assay or sample. QC results are maintained in the Toxicology Laboratory. Instrument Operating Procedure: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebook. Instrument Methods: Refer to the Instrument Methods Notebook. References: Rachel R. McCusker, Helen Paget-Wilkes, Chris W. Chronister, and Bruce A. Goldberger. Analysis of Gamma-Hydroxybutyrate (GHB) in Urine by Gas Chromatography-Mass Spectrometry. J. Anal. Toxicol. 23: 301-305 (1999).
GAMMA-HYDROXYBUTYRATE TRAINING NOTES
1. If GHB is detected in urine (sexual assault cases primarily) and further blood testing is required, the blood samples should be extracted with a curve and QC samples made in blank blood. Different matrices have varying effects on the area counts. 2. You may prepare the eluant while samples are drying in step 5. 3. Care should be taken in drying down samples in step 12; samples must be completely dry, but do not over dry. 4. The Bel-Art pipette pump (green) may be used to start the fluids through the column.
LEAD BY GRAPHITE FURNACE ATOMIC ABSORPTION Principle: Lead is a ubiquitous element in the environment and is routinely present in the human body. It is particularly to toxic in children where it impairs normal development of the central nervous system. In this procedure, lead is mixed with modifier and analyzed using graphite furnace atomic absorption. Atomic absorption is an analytical process in which an element is volatilized in a sample chamber. An element specific lamp emits a constant intensity of light of known wavelength through the sample chamber. Lead atoms absorb light at 282.3 nm, and the amount of light absorbed is proportional to the concentration of lead in the sample. Equipment: Varian SpectrAA-400Z with Zeeman background correction equipped with sample dispenser Sample cups, 2 mL, for the sample dispenser Forked platform graphite tube Micropipettors ranging from 25L to 900L Volumetric flasks Pipets Bulk Reagents: Ammonium Dihydrogen Phosphate ( NH4H2PO4 ) suitable grade for trace metal analysis. Nitric Acid (HNO3), concentrated Trace metal grade Lead Stock Standard 1000g/mL (Pb) suitable grade for Atomic Absorption use for trace metals Triton X-100 External control such as Contox Reference Material, Biorad Level III Whole Blood Metals or other suitable control follow reconstitution and storage instructions provided by the manufacturer Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eyewash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary.
Lead Version 2.1
Nitric acid is corrosive. Avoid contact with skin, eyes, and mucous membranes. Avoid inhaling vapors. Lead may cause damage to various organ systems. Avoid contact with the skin, eyes, and mucous membranes. Avoid ingestion or inhalation. Preparation of Reagents, Calibrators, Controls, and Standards: 2% HNO3 (Make-up solution) In a 200 mL volumetric flask, add approximately 75 mL deionized water and 4 mL of concentrated nitric acid. Mix carefully and bring to volume with deionized water. In a 100 mL volumetric flask place 10 ml Triton X-100 into approximately 70 mL deionized water. Stir for ~ 1 hour. The solution may need to be slightly warmed or sonicated for complete dissolution. Bring to volume with deionized water. Expires One year after preparation date. Note: This solution is also used for CO analysis. In a 100 mL volumetric flask, dissolve 20g of NH4H2PO4 in ~75 mL deionized water. Bring to volume with deionized water. Expires Six months from prep date. In a 100 mL volumetric, add ~ 30 mL of deionized water, 5 mL of 10% Triton X-100, 1 mL of 20 % NH4H2PO4, and 200 L concentrated nitric acid. Mix. Bring to volume with deionized water. Expires 3 weeks from prep date. Into a 100 mL volumetric, pipet 1.0 mL stock Pb standard and 2.0 mL concentrated nitric acid into ~75mL deionized water. Mix. Bring to volume with deionized water. Expires 1 week from prep date Into a 50 mL volumetric flask, pipet 2 mL of 10 ug/mL Intermediate Pb Standard and bring to volume with 2% HNO3. Expires - Make fresh daily (24 hr expiration) Into a 50 mL volumetric flask, pipet 1mL of 10 ug/mL Intermediate Pb Standard and bring to volume with 2% HNO3. Expires - Make fresh daily (24 hr expiration) Prepare in 2mL auto sample cup. Add 900L of Lead Working Modifier and 100L of 2% HNO3. Expires - Make fresh daily (24 hr expiration)
10% Triton X-100 (v/v)
20% NH4H2PO4 (w/v)
Lead Working Modifier
10g/mL Intermediate Pb Standard
400 g/L Pb High Calibration Std* *( PbHC Std A ) 200 g/L Pb High Calibration Std* *( PbHC Std B ) Negative Control
Lead Version 2.1
Positive Control, 300ug/L
Prepare in 2mL auto sample cup. Add 900 uL of Lead Working Modifier and 75uL of PbHC Std A + 25uL 2% HNO3 Expires Make fresh daily (24 hour expiration)
Sample Requirements: Acceptable specimens: blood, serum/plasma, urine Sample Collection and Preservation: It is recommended but not required that blood specimens are preserved with sodium fluoride (gray top) and that postmortem specimens are collected from femoral vessels. Liquids are usually refrigerated during storage. Solid samples in plastic cups are usually frozen for storage. Glass containers often break if frozen. Sample Preparation: Dilutions and homogenates must be so noted in the case file. Analytical Procedure: 1. Preparation of Calibrators: In each 2 mL auto sample cup place 900L of lead working modifier and 50g/L (ppb) lead calibrator - 25L of PbHC std B (200g/L) + 75L of 2% HNO3 100g/L (ppb) lead calibrator - 25L of PbHC std A + 75L of 2% HNO3 200g/L (ppb) lead calibrator - 50L of PbHC std A + 50L of 2% HNO3 300g/L (ppb) lead calibrator - 75L of PbHC std A + 25L of 2% HNO3 400g/L (ppb) lead calibrator - 100L of PbHC std A Gently stir with pipet tip to mix, or invert gently with parafilm covering top. Make fresh daily. Preparation of Samples: In each 2 mL auto sample cup place 900 uL of lead working modifier and 100 uL of sample. Gently stir with the sample tip, or inverted with a cover of parafilm to ensure good mixing. Preparation of Controls: See table above. Place all auto sample cups in auto sampling rack in appropriate labeled slot. Load sampler with the prepared auto sampling cups as follows: Vial # Solution Concentration in ug/dL 41 0 ppb (ug/L) 0 42 50 ppb (ug/L) 5 43 100 ppb (ug/L) 10 44 200 ppb (ug/L) 20 45 300 ppb (ug/L) 30 46 400 ppb (ug/L) 40
2. 3. 4. 5.
Lead Version 2.1
52 1 2 3 4 5
2% Nitric Acid Negative Control Positive Control External Control Sample Sample, etc. 0 30
Quality Control: A negative control, a positive control, and an external control such as the Contox must be run at the beginning and end of every set. In general, a negative control and positive control should be run every 5-6 samples. Control values should be +/- 15% of the target for the positive control, and the negative control should be representative of the calibration blank. The external control should match manufacturers range or be within +/- 15% of target. Instrument Operating Procedures: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebook. Instrument Parameters: Program name Instrument Mode Calibration Mode Measurement Mode Lamp Current (mA) Slit Width (nm) Slit Height Wavelength (nm) Sampler Introduction Time Constant (sec) Replicates Background Correction Lead in Blood Template Absorbance Concentration Peak Area 5.0 0.5 Normal 283.3 Sampler Premix 0.05 3 BC On
Lead Version 2.1
Step No. 1 2 3 4 5 6 7 8 9
Temp C 150 250 600 600 850 850 2300 2300 2500
Time (sec) 20.0 20.0 10.0 5.0 10.0 1.0 1.5 2.0 6.0
Gas Flow Gas Type (l/min) 3.0 Normal 3.0 Normal 3.0 Normal 3.0 Normal 3.0 Normal 0.0 Normal 0.0 Normal 0.0 Normal 3.0 Normal
Read Command No No No No No No Yes Yes No
Qualitative Identification: Elemental lead is known to absorb light at 283.3 nm. Calculations: 1. Units are reported from the instrument and entered into the final report in g/dL to be consistent with units commonly used in medicine. a. To convert from ug/L or ppb to ug/dL divide by 10. 2. Use only two decimal places by truncating the last place (no rounding). 3. If other dilutions were made, reported result represents the concentration of lead in the diluted sample. To calculate the concentration in the original sample, multiply by the dilution factor. References: Varian Applications Notes: Determination of Lead in Blood by GFAAS-Deuterium and Zeeman Background Correction
Lead Version 2.1
LITHIUM BY FLAME ATOMIC ABSORPTION Principle: Lithium (Li) salts are used to treat and control bipolar psychiatric disorders. Lithium has a low therapeutic index, i.e. there is only a small difference in therapeutic and toxic concentrations. In this procedure, lithium is analyzed by flame atomic absorption spectrophotometry. Flame atomic absorption is an analytical process in which an element is volatilized in a flame. An element specific lamp emits a constant intensity of light of known wavelength through the sample chamber. Lithium atoms absorb light at 670.8 nm, and the amount of light absorbed is proportional to the concentration of lithium in the sample. Equipment: Varian SpectrAA-220FS Sample Preparation System SPS5 Test tubes, 25 mL, for autosampler Centrifuge tubes for standards rack Micropipettors ranging from 50L to 900L Volumetric flasks Pipets External Control such as BioRad Therapeutic Drug Monitoring Control, Level 2 (approximately 1.5mEq/L) Reagents: Lithium Stock Standard 1000g/mL (Li) suitable grade for Atomic Absorption use for trace metals. Potassium Chloride, KCl External Standard such as BioRad Therapeutic Drug Monitoring Control, Level 2 follow reconstitution and storage instructions provided by the manufacturer. Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eyewash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary.
Lithium Version 2.1
Preparation of Reagents, Calibrators, Controls, and Standards: 100g/mL Intermediate Li Standard A Into a 10 mL volumetric, pipet 1mL stock lithium standard and bring to volume with deionized water. Expires 1 week from prep date In a 200 mL volumetric flask, place 0.8g of KCl and bring to volume with deionized water. Add 1mL of blank blood to 4mL of deionized water + 5mL of KCl in a 25mL test tube. Invert gently to mix. Expires - Make fresh daily (24 hr expiration) Add 1mL of blank blood to 4mL of deionized water + 5mL of KCl + 140L of Li Standard A in a 25mL test tube. Invert gently to mix. Expires - Make fresh daily (24 hr expiration) Add 1mL of BioRad Level 2 and 4mL of deionized water + 5mL of KCl in a 25mL test tube. Invert gently to mix. Expires - Make fresh daily (24 hr expiration)
4000 g/mL KCl Negative Control
Positive Control, 2 mEq/L
BioRad External Control
Sample Requirements: Acceptable specimens: blood, serum/plasma, urine, gastric Sample Collection and Preservation: It is recommended but not required that blood specimens are preserved with sodium fluoride (gray top) and that postmortem specimens are collected from femoral vessels. Liquids are usually refrigerated during storage. Solid samples in plastic cups are usually frozen for storage. Glass containers often break if frozen. Sample Preparation: Dilutions and homogenates must be so noted in the case file. Analytical Procedure: 1. Preparation of Calibrators a. To a 25 mL test tube, add one of the following sets: i. 0 mEq/L Li standard: 2mL of blank blood + 10 mL of KCl solution ii. 0.25 mEq/L Li standard: 35L of Li Standard A (100g/mL) + 2mL of blank blood + 10 mL of KCl solution iii. 0.5 mEq/L Li standard: 70L of Li Standard A (100g/mL) + 2mL of blank blood + 10 mL of KCl solution
Dallas County Institute of Forensic Sciences Toxicology Laboratory 2 Lithium Version 2.1
2.
3. 4. 5.
iv. 1.0 mEq/L Li standard: 140L of Li Standard A + 2mL of blank blood + 10 mL of KCl solution v. 2.0 mEq/L Li standard: 280L of Li Standard A + 2mL of blank blood + 10 mL of KCl solution vi. 4.0 mEq/L Li standard: 560L of Li Standard A + 2mL of blank blood + 10 mL of KCl solution b. Add deionized water to each tube in sufficient volume to bring total volume to 20 mL. c. Cap and invert gently to mix. d. Make fresh daily. Preparation of Samples a. To a 25 mL test tube add i. 1mL of sample (whole blood, serum, gastric) ii. 5mL of KCl solution iii. 4mL of deionized water iv. Cap and gently invert to mix.. v. NOTE: Total volume for samples is 10mL. Preparation of Controls a. See table above. If samples contain obvious particulate matter, they should be centrifuged or allowed to settle before being aspirated. DO NOT aspirate particulate matter into the nebulizer. The autosampler unit may be used and loaded as follows, or alterternativley, manual introduction of sample may be used. SPS-5 Rack Type A (Standards Rack 1-12) Slot# Solution 1 0 mEq /L 2 0.25 mEq /L 3 0.5 mEq /L 4 1.0 mEq /L 5 2.0 mEq /L 6 4.0 mEq /L SPS-5 Rack Type 25 (Sample Rack 1-60) 1 Negative Control 2 Positive Control 3 BioRad QC 4 Sample 5 Sample 6 Sample etc.
Quality Control: A negative control, a positive control, and an external control such as the BioRad must be run at the beginning and end of every set. In general, a negative control and positive control should be run every 5-6 samples.
Dallas County Institute of Forensic Sciences Toxicology Laboratory 3 Lithium Version 2.1
Control values should be +/- 15% of the target for the positive control, and the negative control should be representative of the calibration blank. The external control should match manufacturers range or be within +/- 15% of target. Instrument Operating Procedures: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebook. Instrument Parameters: Program name Instrument Mode Calibration Mode Lamp Current (mA) Slit Width (nm) Slit Height Wavelength (nm) Flame Air/ Delay Replicates Background Correction Air Flow Acetylene Qualitative Identification: Elemental lithium is known to absorb light at 670.8 nm. Calculations: 1. Units are as reported from the instrument in mEq/L. 2. Report only two decimal places by truncating the last place (no rounding). 3. If other dilutions were made, reported result represents the concentration of lithium in the diluted sample. To calculate the concentration in the original sample, multiply by the dilution factor. 4. Relationship between ug/mL, mmol/L, and mEq/L a. For lithium, mmol/L = mEq/L b. To convert from mg/L to mmol/L, divide ug/mL by the molecular weight of lithium (6.94 mg/mmol). i. Example 14 mg/L x mg/6.94 mmol = 2 mmol/L or 2 mEq/L c. To convert from mmol/L to mg/L multiply mmol/L by the molecular weight of lithium (6.94 mg/mmol). i. Example 2mmol/L x 6.94 mmol/mg = 14 mg/L Lithium in Blood Template Absorbance Concentration 5.0 1.0 Normal 670.8 Acetylene 10 sec 3 BC Off 13.5 2.00
Lithium Version 2.1
Interpretation of Results: Lithium is usually administered orally in the form of lithium carbonate or lithium citrate. Common doses are 0.4 0.6 grams three times a day. Therapeutic serum concentrations are 0.5 2.0 mEq/L. Toxic serum concentrations are regarded as >2.0 mEq/L. The lower limit of quantitation for this assay is 0.25 mEq/L. References: Stafford DT and Saharovici F. Serum Lithium Determinations Using Flameless Atomic Absorption Spectroscopy. Spectrochimica Acta 29B: 277-281, 1074. Varian Applications Note: Determination of Lithium, Zinc, and Copper in Blood Serum by Flame Microsampling.
Lithium Version 2.1
MERCURY BY VGA ATOMIC ABSORPTION Principle: Mercury is a nonessential trace element and is routinely present in all human tissues. In this procedure, samples are acid digested to break down organic constituents. Mercury is volatilized from the digestate using a cold vapor generation technique and analyzed by atomic absorption spectrometer using a vapor generation accessory (VGA). Atomic absorption is an analytical process in which an element is volatilized in a sample chamber. An element specific lamp emits a constant intensity of light of known wavelength through the sample chamber. Mercury atoms absorb light at 253.7 nm, and the amount of light absorbed is proportional to the concentration of arsenic in the sample. Equipment: Varian SpectrAA-220FS Sample Preparation System SPS5 Test tubes, 25 mL for autosampler Centrifuge tubes for standards rack Micropipettors ranging from 50L to 900L Volumetric flasks and beakers Pipets External Control such as BioRad Urine Metals Control, Level 2 Water Bath with temp range capability of 40-85 degrees Reagents: Nitric Acid (HNO3), concentrated Trace metal grade Sulfuric Acid (HSO4), concentrated Trace metal grade Hydrochloric Acid (HCl), concentrated Trace metal grade Perchloric Acid (H2ClO4), concentrated Trace metal grade Mercury Stock Standard 1000g/mL (Hg) suitable grade for Atomic Absorption use for trace metals. External Standard such as BioRad Urine Metals Control, Level 2 follow reconstitution and storage instructions provided by the manufacturer. Stannous Chloride (Sn2Cl) trace metal grade or equivalent Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eyewash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary.
Mercury Version 2.0
Sulfuric, nitric, perchloric, and hydrochloric acids are corrosive. Avoid contact with skin, eyes, and mucous membranes. Avoid inhaling vapors. Stannus chloride may cause irritation to the respiratory tract, eyes, skin and digestive tract. Use caution if pregnant. Preparation of Reagents, Calibrators, Controls, and Standards: Into a 100 mL volumetric, pipet 500L Mercury Stock Standard. Bring to volume with deionized H20. Expires 1 week from preparation date In a hood with eye protection, place 500mL of concentrated nitric into a glass-stoppered container. Add 200mL of concentrated perchloric acid. Add 100mL of concentrated nitric acid. Carefully swirl to mix. Allow to cool before use In a hood, place 25g Sn2Cl, 25mL of deionized H20, and 20 mL of HCl into a 100 mL volumetric flask. Swirl. Bring to volume with deionized H20. Expires Make fresh for each set. Add 2.0 mL of blank blood to a 50mL conical tube. Negative Control To a 50 mL conical tube, add 2.0 mL of blank blood and 40L or 60L respectively of Hg Standard A and vortex to mix. Place 2.0 mL of Urine Metal Control Level 2 into a 50mL conical tube.
5g/mL Intermediate Hg Standard A
5:2:1 Nitric : Perchloric: Sulfuric Acid (digestion acid)
25% Stannous Chloride (reductant)
Positive Control, 100 ug/L or 150 ug/L
External Control such as BioRad Urine Metal Control Level 2
Sample Requirements: Acceptable specimens: blood, serum/plasma, urine, gastric, hair, tissues, etc. Sample Collection and Preservation: It is recommended but not required that blood specimens are preserved with sodium fluoride (gray top) and that postmortem specimens are collected from femoral vessels. Liquids are usually refrigerated during storage. Solid samples in plastic cups are usually frozen for storage. Glass containers often break if frozen.
Mercury Version 2.0
To collect hair, select a pencil size bundle of hair on the head. Tie at intervals down the hair bundle. Pull from the scalp (postmortem) or cut close to the scalp. Mark the head end of the bundle. Measure the length of the bundle. The hair bundle is cut into sections at known distances from the head. Sections may be analyzed separately to assist in determining time of exposure. Sample Preparation: Other samples: Place a known volume or weight of other samples into the digestion tube. Mince if appropriate to aid acid digestion.
Dilutions and homogenates must be so noted in the case file. Analytical Procedure: Use acid washed glassware: Clean glassware in 30% HNO3 1. 2. Fill water bath with deionized water and bring to 40 degrees C; this takes about 1 hour. Preparation of Calibrators: Place the following in a 50 mL conical tube: 0 g/L Mercury 40 g/L Mercury 100 g/L Mercury 150 g/L Mercury 200 g/L Mercury 3. 2.0mL of blank blood 16L of Hg Standard A (10g/mL)+ 2.0mL of blank blood 40L of Hg Standard A + 2.0mL of blank blood 60L of Hg Standard A + 2.0mL of blank blood 80L of Hg Standard A + 2.0mL of blank blood
Preparation of Samples: Place 2.0 mL of each sample into individual 50mL conical tubes for digestion. Use 2 g of tissue. Preparation of Controls: See table above. Add 5mL of digestion acid (5:2:1) to each of the test tubes. (Allowing samples to sit in acid solution overnight is recommended to avoid foaming and assist in digestion.) Place samples in 40 deg C water bath for 1 hr. Increase temperature to 85-90 degrees C. When the temperature reaches 85-90 degrees C, begin timing the digestion for at least 1 hour. When properly digested, the sample should be straw colored. Remove from water bath and allow to cool. Dilute to 50mL with deionized water. The autosampler unit may be used and loaded as follows, or manual introduction of sample may be used. SPS-5 Rack Type A (Standards Rack 1-12)
4. 5.
6. 7.
8.
9. 10.
Mercury Version 2.0
Slot# 1 2 3 4 5
Solution 0 g/L 40 g/L 100 g/L 150 g/L 200 g/L
SPS-5 Rack Type 25 (Sample Rack 1-60) 1 2 3 4 5 Negative QC Positive QC BioRad QC Sample Sample etc.
Quality Control: A negative control, a positive control, and an external control such as the BioRad must be run at the beginning and end of every set. In general, a negative control and positive control should be run every 5-6 samples. Control values should be +/- 15% of the target for the positive control, and the negative control should be representative of the calibration blank. The external control should match manufacturers range or be within +/- 15% of target. Instrument Operating Procedures: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebook. Instrument Parameters: Program name Instrument Mode Calibration Mode Lamp Current (mA) Slit Width (nm) Slit Height Wavelength (nm) Flame Delay Replicates Background Correction Air Flow
Mercury in Blood Template Absorbance Concentration 4.0 0.5 Normal 253.7 Air/ Acetylene 70 sec 3 BC On 13.5
Mercury Version 2.0
Acetylene Qualitative Identification:
2.00
Elemental mercury is known to absorb light at 253.7nm. Calculations: Data is truncated to two decimal places. Results are reported from the instrument in ug/L. Liquid samples are usually reported in ug/L. To calculate the concentration of a solid sample, multiply ug/1000ml (ug/L) reported by the instrument by 2.0 ml to obtain ug. Divide by the weight of the sample in Kg to obtain ug/Kg. If other dilutions were made, reported result represents the concentration of mercury in the diluted sample. To calculate the concentration in the original sample, multiply by the dilution factor. References: Varian Applications Note: Determination of Mercury in Blood and Urine by Cold Vapor AAS using the VGA-77. Varian Applications Note: Determination of Mercury in Fish Tissue, a Rapid, Automated Technique for Routine Analysis. Interpretation: The following is provided as a general guide for interpretation; interpretation of individual case data requires case specific evaluation.
Normal concentrations: Muscle 0.036-0.039 g/g Blood 0.009-0.011 g/g Kidney 1.26-1.31 g/g Liver 0.27-0.037 g/g
Mercury Version 2.0
METHOCARBAMOL BY GC/MS Principle of Assay: Methocarbamol is an older drug that is occasionally prescribed as a sedative or muscle relaxant and is detected in the acid/neutral screen. Guaifenesin is a pharmacologically-active, plasma metabolite that is formed by the hydrolysis of the methocarbamol amide group. Acid and neutral drugs and the internal standard, barbital, are extracted from biological specimens at a weak acid pH (potassium dihydrogen phosphate) using toluene-ether as the extracting solvent. After evaporating the extracting solvent, the residue is partitioned between acetonitrile and hexane. As a result of this partitioning, fat and other lipophilic matrix components stay in the hexane, and the drug of interest is concentrated in the acetonitrile. The extract is screened and quantitated by GC/MS.
Equipment: Agilent 5973 gas chromatograph/mass spectrometer, autosampler, and computer with capillary column such as: DB-1, 30m x 0.25 mm x 0.25 um film thickness Screw top culture tubes, 16mm x 125 mm, screw cap with Teflon liner Screw top conical tubes, 5 mL screw cap with Teflon liner Eppendorf pipet: 100 uL, 50 uL Repipet dispenser/diluter: 0.5, 1.0, 5.0 mL volumes Rotator Centrifuge Autosampler vials, 32 mm x 11 mm, Teflon lined seals and micro-volume glass inserts Vortex mixer Hamilton syringes Pasteur pipettes Serological pipettes, 5ml Reagents: Toluene, reagent grade Anhydrous diethylether, reagent grade Acetonitrile, reagent grade Hexane, reagent grade Potassium dihydrogen phosphate Barbital, free acid Instructions for Reagent Preparation:
Dallas County Institute of Forensic Sciences Laboratory 1 Methocarbamol Toxicology Version 2.0
Toluene-ether (1:1): In a hood, combine equal volumes of toluene and diethylether. Potassium dihydrogen phosphate, saturated: Add 270 grams potassium dihydrogen phosphate to 900 mL water in a beaker containing a stirring bar. Warm on a hot plate while stirring until the potassium dihydrogen phosphate goes into solution. Cool before use. Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Ether is very volatile and very flammable. Do not use near a source of electrical spark or open flame. Caution: vapors may pool and travel down a lab bench or floor to an ignition source. Toluene-ether should be made in a hood. Avoid repeated contact with the skin, eyes, and mucous membranes. Other solvents are also flammable and should be used in a hood or well ventilated area. Avoid repeated contact with the skin and contact with eyes or mucous membranes. Stocks, Standards, Controls, Calibrators: 0.2 mg/mL Barbital Internal Standard: Transfer 50 mg barbital free acid to 250 mL volumetric flask and dilute to volume with deionized water. Make fresh every 6 months. 1.0 mg/ml Drug Stock Standard: Transfer 10 mg free drug to a 10 mL volumetric flask and dilute to volume with methanol. If the drug is in the form of a salt or contains waters of hydration, the 10 mg must be adjusted accordingly to add 10 mg of the free base. The calculation to convert an amount of free acid to the salt is as follows: mg of salt = mg of free drug x molecular weight of salt / molecular weight free drug Methocarbamol Working Standard 0.2 mg/ml (200mg/L): Using a volumetric pipette, transfer 2.0 mL of a 1.0 mg/ml stock solution to a 10 mL volumetric flask and dilute to volume with methanol. Methocarbamol QC 0.1 mg/ml (100mg/L): Using a volumetric pipette, transfer 1.0 mL of a 1.0 mg/ml stock to a 10 mL volumetric flask and dilute to volume with methanol.
Dallas County Institute of Forensic Sciences Laboratory
Methocarbamol Toxicology Version 2.0
Negative control: Extract 1 mL of blank blood containing only the internal standard. All stock solutions and controls are kept in the refrigerator. Allow to come to room temperature before using. Sample Requirements: Acceptable specimens: blood, serum/plasma, urine, bile, gastric, vitreous, tissue homogenates, pharmaceutical preparations Sample Collection and Preservation: It is recommended but not required that blood specimens are preserved with sodium fluoride (gray top); however, most acid and neutral drugs are not as susceptible to in vitro enzymatic degradation as alkaline drugs. Femoral blood is preferable; however, acid and neutral drugs are typically less susceptible to postmortem redistribution than alkaline drugs. Liquid specimens are usually refrigerated during storage; solid specimens in plastic cups are usually frozen during storage. Sample Preparation: Tissues: Homogenize 5 grams tissue in 15.0 mL deionized water. Use 1 mL of the homogenate for analysis Gastric: Measure the total volume of gastric submitted before sampling; record in the case file. If gastric is a homogenous, pipettable liquid, it may be used as is in the analysis. If gastric is non-homogenous and/or thick, homogenize as tissue. For initial analysis, dilute gastric 1 to 100 with water. A significant quantity of drugs will overload the column. If dilution is negative, an undiluted or lesser diluted sample may be run. Other samples: Use 1.0 mL of sample in the analysis. High samples: Gastric, blood, and other specimens may require dilution. In this case, extract less than 1 mL and make up the volume with deionized water Non-standard dilutions and homogenates must be so noted in the case file. Analytical Procedure: 1. Set up one 15 mL screw cap tube for each calibrator, internal QC negative control, and sample. 2. Using a serological pipette, add 1.0 mL saturated potassium dihydrogen phosphate to each screw top culture tube. 3. Instructions for Preparation of Calibration Standards:
Calibrator (1 mg/L): Using a Hamilton syringe, transfer 5 ul working standard (0.2 mg/ml) to a 15 ml screw top culture tube containing 1.0 ml saturated potassium dihydrogen phosphate. Vortex. Calibrator (10 mg/L): Using a 50 ul Eppendorf pipet, transfer 50 ul working standard (0.2 mg/ml) to a 15 ml screw top culture tube containing 1.0 ml saturated potassium dihydrogen phosphate. Vortex. Calibrator (20 mg/L): Using a 100 ul Eppendorf pipet, transfer 100 ul working standard (0.2 mg/ml) to a 15 ml screw top culture tube containing 1.0 ml saturated potassium dihydrogen phosphate. Vortex. Calibrator (40 mg/L): Using a 100 ul Eppendorf pipet, transfer 200 ul working standard (0.2 mg/ml) to a 15 ml screw top culture tube containing 1.0 ml saturated potassium dihydrogen phosphate. Vortex. 4. For the QC sample, add 100 uL Methocarbamol QC with an Eppendorf pipette to a 15 ml screw top culture tube containing 1.0 ml saturated potassium dihydrogen phosphate. Vortex. 5. To each tube, add 100 uL of the 0.2 mg/mL barbital internal standard solution using an Eppendorf pipet. Vortex. 6. Using a wide bore glass pipette, add 1.0 mL blank blood to each Calibrator, the Negative Control, and the Internal QC sample. Vortex. 7. Using a wide bore glass pipette, add 1.0 mL sample to each sample tube. Vortex. 8. Add 5.0 mL toluene/diethylether to each tube using repipet dispenser. 9. Rotate for 10 minutes at medium speed. 10. Centrifuge for 3 minutes. 11. Using a Pasteur pipette, transfer the toluene/diethylether layer (top) to a 5 mL conical tube. 12. Take to residue in a hood using air and hot water bath (approximately 55 oC). Remove from water bath as soon as the specimen reaches dryness.* 13. To each tube, add 100 uL acetonitrile using pipet and 500 uL hexane using repipet dispenser. 14. Vortex for 1 minute. 15. Transfer acetonitrile layer (bottom) and some of hexane layer (top) to an autosampler vial with insert. Centrifuge on low for 1 minute. 16. Inject 1.0 uL on the GC/MS using AN method. 17. Data is recalculated using the Methocarbamol method after data analysis. *Note: If an oily residue remains after the extract is taken to dryness in step 10, perform the following procedure: Add 200 uL acetonitrile and 1.0 mL hexane. Vortex for one minute; centrifuge. Discard the top (hexane) layer. Take the bottom (acetonitrile) layer to dryness. Continue with step 11. If this procedure is used, it must be noted in the case file.
Quantitation: This GC/MS method (Methocarbamol) can be used to quantitate most acid/neutral drugs by generating a four-point calibration curve for each drug. GC/MS results are reported in mg/L. Tissues: The calculated result represents the concentration of drug in the homogenate. To calculate the concentration of drug in the tissue, multiply by the dilution factor. If the standard tissue homogenate is used (1 part tissue + 3 parts water = 1:4 dilution), multiply the homogenate concentration by 4 to obtain the concentration of drug in the tissue. Results are expressed in mg/Kg. Other Dilutions: The calculated result represents the concentration of drug in the diluted sample. To calculate the concentration of drug in the original sample, multiply by the dilution factor. For example if 0.5 mL of gastric + 0.5 mL of water was analyzed instead of 1 mL gastric, multiply the calculated concentration by 2 to determine the concentration of drug in the undiluted specimen,. If the original specimen was a solid and weighed, units are mg/Kg. If the original specimen was a pipettable liquid, units are mg/L. Gastric: Gastric is reported as mg/ total volume of gastric. A notation must be made in the case file regarding how all dilutions were made. Quality Control: A quality control sample will be included with each batch of samples for quantitation. QC sample results should be +/- 20% of the target concentration. QC results outside this range must be reviewed by a supervisor who will determine whether results may be reported or repeated. QC results are maintained in the Methocarbamol QC file. These files are located in the Toxicology Laboratory. Instrument Operating Procedures: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebooks. Instrument Methods: Refer to the Instrument Methods Notebooks. References:
E. H. Foerster, J. Dempsey, and J.C. Garriott. A gas chromatographic screening procedure for acid and neutral drugs in blood. J. Anal. Toxicol. 3: 87-91 (1979)
METHOCARBAMOL - TRAINING NOTES 1. Diethylether is extremely volatile and extremely flammable. Use caution to prevent solvent or vapors from reaching ignition source. 2. Some specimens particularly in decomposed cases have an oily residue left after evaporation of toluene/ether. Use the alternate method described in the procedure to handle this type of sample. Use of the alternate method should be noted in the case file. 3. Once the analytical process is begun, it should be followed to conclusion in a timely manner. The extraction process should not be left mid-stream for extended periods of time because sample degradation may occur with some chemicals at specific stages of the extraction process. 4. The wash bottles for the autosamplers should be filled with hexane washed acetonitrile to ensure solvent compatibility with the sample. 5. Do not report concentrations below the lowest standard unless approved by supervisor.
OPIATES BY GC/MS Principle of Assay: Hydromorphone, morphine, and 6-monoacetylmorphine are amphoteric and have both acidic and basic functional groups. Extraction of these substances from an aqueous matrix into an organic phase requires that the pH must be above the pKa for the acidic OH group and below the pKa for the amine function. Therefore, there is a narrow pH range required for extraction of these substances; this is why they cannot be analyzed using the alkaline drug screen procedure. This method also allows quantitation of codeine and hydrocodone which may also be analyzed using the alkaline drug screen. Heroin has a very short half life and is rapidly metabolized to 6-monoacetylmorphine and further to morphine. Presence of 6-monoacetylmorphine indicates heroin use. Plasma esterases cause degradation of heroin and 6-monoacetyl morphine in the body after death and in vitro. Because urine and vitreous are somewhat protected from the action of plasma esterases, these specimens are analyzed in addition to blood when available when heroin use is suspected. Morphine, hydrocodone, codeine, 6-monoacetylmorphine and hydromorphone and their corresponding deuterated metabolites are extracted using liquid-liquid extraction. The sample is made weakly basic (about pH 8.5), and the compounds of interest are extracted into ethyl acetate/isobutanol (9:1) solvent. After extraction into acid and back-extraction into organic, the organic phase is evaporated to residue. The hydroxyl groups found on the compounds of interest are derivatized using BSTFA-1%TMCS and analyzed using a GC/MS with selected ion monitoring (SIM). Equipment: Hewlett-Packard model 6890 series II gas chromatograph Hewlett-Packard model 5973 mass selective detector Hewlett-Packard model 7673 autosampler Capillary column: such as HP-1 (cross-linked methyl silicone gum phase) 12 m x 0.20 mm x 0.33 um film thickness. Culture tubes, 15 ml screw cap with Teflon liner Conical tubes, 5 ml, screw cap with Teflon liner Eppendorf pipets: 50 and 100 ul volumes Hamilton syringe: 10 uL volume Rotator Centrifuge Oven, approximately 1000C Vortex mixer Vacuum aspirator Autosampler vials, 32 x 11 mm, Teflon lines seals and glass inserts
Dallas County Institute of Forensic Sciences Toxicology Laboratory 1 Opiates by GC/MS Version 2.1
Water bath\evaporator Reagents: Sodium bicarbonate Sodium carbonate Ethyl acetate Isobutanol Hydrochloric acid, concentrated BSTFA with 1% TMCS (N,O-bis-(trimethylsily)trifluoroacetamide/Trimethylchlorosilane Carbonate Mix (8:3): Mix 80 grams of sodium bicarbonate and 30 grams sodium carbonate and pulverize with a mortar and pestle. Store at room temperature. 0.2N Hydrochloric acid: Add approximately 500 ml deionized water to a one liter volumetric flask. Add 16.6 ml concentrated hydrochloric acid. Mix and allow to cool. Dilute to volume with deionized water. Store at room temperature. Ethyl acetate/isobutanol (9:1): Add 900 ml of ethyl acetate to a one liter flask. Add 100 ml isobutanol. Mix and store at room temperature. Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Hydrochloric acid is corrosive. Avoid contact with skin, eyes, and mucous membranes. Ethyl acetate and isobutanol are volatile solvents. Avoid contact with skin; do not breathe vapors. BSTFA with 1% TMCS is corrosive. Avoid contact with skin, eyes, and mucous membranes. Stocks, Standards, Controls, Calibrators: D3-Stock Internal Standards (100ug/ml each of deuterated morphine, 6-monoacetylmorphine, codeine, hydromorphone, and hydrocodone): Prepared by manufacturer. Store in freezer. D3-Working Internal Standard Mixture (4ug/ml): Using a volumetric pipet, transfer 1.0 ml of each of the following Stock Internal Standards to a 25 mL volumetric flask and dilute to volume with acetonitrile; store in refrigerator: codeine D3 (Stock: 100 ug/ml in methanol) hydrocodone D3 (Stock: 100 ug/ml in methanol)
hydromorphone D3 (Stock: 100 ug/ml in methanol) morphine D3 (Stock: 100 ug/ml in methanol) 6-monoacetylmorphine D3 (Stock: 100 ug/ml in acetonitrile) Drug Stock Standard (1.0 mg/ml): Prepared by manufacturer. Store in refrigerator. Drug Working Mix Standard (10 ug/ml): Using a 50 ul Eppendorf pipet, transfer 50 ul each of the following drug stock standards (1.0 mg/ml) to a 5.0 ml volumetric flask and dilute to volume with acetonitrile; store in refrigerator: codeine morphine 6-monoacetylmorphine hydromorphone hydrococone Drug external QC standard (10 ug/ml): Using a 50 ul Eppendorf pipet, transfer 50 ul each of the codeine, morphine, 6-monoacetylmorphine, hydromorphone, and hydrocodone stock standards (1.0 mg/ml) to a 5.0 ml volumetric flask and dilute to volume with acetonitrile. (The vials containing the drug standards used as a check samples are located in the cooler and are labeled Alltech Drug Opiate Stds). Store in refrigerator. Sample Requirements: Acceptable specimens: blood, serum/plasma, urine, bile, vitreous, gastric, tissue homogenates, pharmaceutical preparations, etc. For Medical Examiner samples, blood is usually analyzed as the primary specimen. If morphine and/or 6-monoacetylmorphine are present in the blood or if the Medical Examiner suspects heroin use, the urine (or vitreous if urine is not available) will also be tested for opiates unless the analyst determines that based on history urine opiates are not applicable. Sample Collection and Preservation: It is recommended but not required that blood specimens are preserved with sodium fluoride (gray top) and that postmortem specimens are collected from femoral vessels. Liquids are usually refrigerated during storage. Solid samples in plastic cups are usually frozen for storage. Glass containers often break if frozen. Sample Preparation: Tissues: Homogenize 5 grams tissue in 15.0 mL deionized water. Use 2 mL of homogenate in the analysis.
Opiates by GC/MS Version 2.1
Gastric:
If gastric is a homogenous, pipettable liquid, it may be used as is in the analysis. If gastric is non-homogenous and/or thick, homogenize as tissue. For initial analysis, dilute gastric 1 to 100 with water. A significant quantity of most drugs will still overload the column. If dilution is negative, an undiluted or lesser diluted sample may be run. Other samples Use 2.0 mL of sample in the analysis. High samples: Gastric, blood, and other specimens may require dilution. In this case, extract less than 2 mL and make up the volume with deionized water Non-standard dilutions and homogenates must be so noted in the case file. Analytical Procedure: Instructions for Preparation of Calibration Standards: Calibrator (20 ug/L): Using a 10.0 ul Hamilton syringe, transfer 4.0 ul working mix standard (10 ug/ml) to a 15 ml screw top culture tube containing 1.0 ml deionized water. Vortex and add 2.0 ml blank blood or appropriate matrix. Calibrator (250 ug/L): Using a 50 ul Eppendorf pipet, transfer 50 ul working mix standard (10 ug/ml) to a 15 ml screw top culture tube containing 1.0 ml deionized water. Vortex and add 2.0 ml blank blood or appropriate matrix. Calibrator (500 ug/L): Using a 100 ul Eppendorf pipet, transfer 100 ul working mix standard (10 ug/ml) to a 15 ml screw top culture tube containing 1.0 ml deionized water. Vortex and add 2.0 ml blank blood or appropriate matrix. Calibrator (1000 ug/L): Using a 100 ul Eppendorf pipet, transfer 200 ul working mix standard (10 ug/ml) to a 15 ml screw top culture tube containing 1.0 ml deionized water. Vortex and add 2.0 ml blank blood or appropriate matrix. Instructions for Preparation of Controls: 250 ug/L Quality Control Sample: Using a 50 ul Eppendorf pipet, transfer 50 ul of Alltech Drug check standard (10 ug/ml) to a 15 ml screw top culture tube containing 1.0 ml deionized water. Vortex and add 2.0 ml blank blood or appropriate matrix. Negative Control: Transfer 2.0 ml blank blood or appropriate matrix to a 15 ml screw top culture tube containing 1.0 mL of deionized water. Instructions for Preparation of Samples:
Place 1 ml deionized water and 2 ml of each specimen in a 15 ml screw cap vial. Note: for urine, use 2 ml deionized water and 1 ml of urine. (See training notes for discussion of hydrolysis.) 1. 2. 3. 4. 5. 6. With a Eppendorf pipet, add 50 ul Working Internal Standard Mixture (10 ug/ml) to all tubes (calibrators, controls and samples). Vortex to mix. Note: Samples should be vortexed soon after Internal Standard Mix is added, for example in groups of six samples. Add approximately 0.3-0.4 grams (3 scoops) carbonate mix to each tube. Vortex to mix. Add 8.0 ml ethyl acetate/isobutanol (9:1) and rotate for 10 minutes. Centrifuge for approximately five minutes. Transfer the organic layer (top) to a clean 15 ml screw top tube; the organic layer from blood may be poured but must be transferred by pipette for urine and vitreous. Add 2.0 ml 0.2N hydrochloric acid and rotate for 5 minutes. Centrifuge for approximately two minutes. Aspirate the organic layer (top). To the aqueous layer, add 0.2-0.3 grams (2 scoops) carbonate mix, and vortex to mix. Add 6.0 ml ethyl acetate/isobutanol (9:1) and rotate for five minutes. Centrifuge for about two minutes. Transfer the organic layer (top) to a 5.0 ml screw cap conical tube and evaporate to dryness. Heat residue in oven at 1050C for five minutes. Using a Eppendorf pipet, add 100 ul BSTFA-1%TMCS to each tube. Cap tube and vortex approximately one minute. Heat in oven for an additional 5 minutes at 700C. Cool and transfer to an autosampler vial, cap and run on GC/MS using method file Opiate.M.
7. 8. 9. 10. 11. 12. 13. 14. 15.
Calculations: The GC/MS reports the results in ug/L which must then be converted to mg/L by the chemist performing the assay. To convert ug/L to mg/L, divide by 1000. Other Dilutions: The calculated result represents the concentration of drug in the diluted sample. To calculate the concentration of drug in the original sample, multiply by the dilution factor. For example if 1 mL of urine + 2 mL of water was analyzed instead of 2.0 mL of sample, multiply the calculated concentration by 2/1 or 2 to determine the concentration of drug in the undiluted specimen. If the original specimen was a solid and weighed, units are mg/Kg. If the original specimen was a pipettable liquid, units are mg/L. Methods of dilution must be noted in the case file. Quality Control: A 250 ug/L external control (Alltech) is included in each run. QC results meet manufacturers
specifications or within +/- 20% of target. QC data is maintained in the Toxicology Laboratory. The sample results for codeine and hydrocodone are also compared with the results from the alkaline screen if performed and should be very similar. The lower limit of quantitation for the assay is 0.02 mg/L for each analyte. Linearity of the assay has been verified from 0.020 1.000 mg/L for all analytes. Ion qualifiers must match within +/- 20%; otherwise, the sample must be reviewed with a supervisor or repeated. Instrument Operating Procedure: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebook. Instrument Methods: Refer to the Instrument Methods Notebook. Reporting Guidelines: Because of the short half life and postmortem degradation of heroin and 6- MAM, blood plus urine or vitreous are typically analyzed when morphine is detected or heroin use is suspected. The following provides a guide for reporting results of testing on these multiple specimens. Medical Examiner Cases: If heroin use is suspected analyze blood and urine for opiates; run vitreous as a second specimen if urine is not available. If morphine is present in the blood and there is no history suggesting medical treatment with morphine, analyze urine (or vitreous) as a second specimen. H-Cases Sexual assault cases: Positive opiates in the urine are reported as detected. Opiates are quantitated in blood when that specimen is available. Other H-cases: Treat as ME cases. DWI Cases Review with the Toxicology Chemist III Codeine: Report codeine in blood when it is present at a concentration of 0.02 mg/mL or greater. Dilute blood specimens exceeding the upper curve limit and rerun. Do not report codeine if <0.02 mg/L. A high codeine may have morphine as a small metabolite; there is no need to run urine or vitreous opiates in this case or to report these results. (Do not confuse this with a high
morphine and a small codeine metabolite in which the urine or vitreous results would be reported). Hydrocodone: Report hydrocodone in blood when it is present at a concentration of 0.02 mg/mL or greater. Dilute blood specimens exceeding the upper curve limit and rerun. Do not report hydrocodone <0.02 mg/L. In most cases, hydrocodone alone is not reported in urine or vitreous when a blood result is reported.
Hydromorphone: Each case is evaluated independently. If hydromorphone is <0.02 mg/L re-extract with 4.0 ml of blood. Morphine: Morphine is typically not reported when it is present at a concentration < 0.02 mg/mL. o However, report morphine as < 0.02 mg/mL when 6-MAM is present in any specimen tested in a case, the qualifiers for morphine are met, and the morphine concentration is below 0.02 mg/mL. Dilute blood specimens exceeding the upper curve limit and rerun. If urine or vitreous is run, report all positive opiate results. If morphine in urine exceeds the upper curve limit you may report it as greater than the highest standard if the qualifiers are in and the peak shape is reasonable. Otherwise dilute appropriately and rerun. 6-Monoacetylmorphine (6-MAM): 6-MAM is always reported if the qualifiers are met. Report 6-mam as <0.02 mg/L if the qualifiers are met and the concentration is below 0.02 mg/L. Dilute blood specimens exceeding the upper curve limit and rerun. If urine or vitreous are run report all opiate results. If 6-MAM in urine exceeds the upper curve limit, it may be reported as greater than the highest standard if the qualifiers are in and the peak shape is reasonable. Otherwise dilute appropriately and rerun. References: Bexar County Medical Examiners Office, Cocaine and Opiates Procedure
OPIATES BY GC/MS TRAINING NOTES 1. Silyl derivatives are used to improve chromatographic properties of acids, alcohols, thiols, amines, and other functional groups containing reactive hydrogens. BSTFA forms silyl derivatives of opiates; see attached schematic. TMCS is present to enhance reactivity. Hydrocodone and hydromorphone can exist as both keto and enol forms resulting in monosilyl and di-silyl derivatives. Under standard conditions, the relative proportion of the two derivatives should be constant. 2. BSTFA is unstable in the presence of moisture. It is important that sources of moisture be removed prior to derivatization . It is also important to protect the bulk derivatization reagent from moisture during storage; for this reason, BSTFA is usually purchased and used in single use containers which are not reused. 3. Morphine and 6-monoacetylmorpine are present in both the free form and more extensively conjugated with glucuronide. It is standard practice in this Laboratory to report the free concentrations because it is the free fraction that can distribute across membranes. However on occasion, it may be useful to analyze the total drug present. In this case the specimen is hydrolyzed first as follows: Prepare specimens, calibrators, and controls. Add 50 ul Working Internal Standard Mixture (10 ug/ml) per standard procedure. Add 1 ml concentrated HCl and pressure cook for one hour. Allow the digested sample to cool. Adjust pH to 9.0-9.5 with 40% sodium hydroxide or 25% hydrochloric acid. Continue with step #4 of the procedure. 4. The conjugated amount of drug may be calculated by subtracting the free fraction from the total. In older literature, it is often the total drug that is reported; therefore, care must be taken when comparing results to reported literature concentrations. 5. In aqueous solution, heroin and 6-monoacetylmorphine hydrolyze to morphine. This degradation occurs in the body after death and in vitro. Collection of specimens in gray top tubes containing sodium fluoride under refrigeration retards but does not eliminate the degradation process. Opiate analyses should be performed on blood collected in gray top tubes if available. 6. Specimens are typically screened by ELISA, and positives confirmed by this method. 7. Rapid iv use of heroin may cause death before drug distributes into urine. Therefore, blood is the specimen of choice for screening tests such as ELISA. 8. In decomposed samples, hydrocodone and hydromorphone do not derivatize well. For this reason, it is also important to use fresh blank blood from the freezer when setting up the calibration curve. 9. Use method OPSCAN.M to set SIM windows.
SALICYLATE QUANTITATION BY FLUORESENCE SPECTROPHOTOMETRY Principle of Assay: Aspirin (acetylsalicylic acid) is rapidly metabolized to salicylate; therefore, aspirin concentrations in blood are low. Thus, it is customary to measure salicylate concentrations as an indication of aspirin use. Salicylate is detected and identified by GC/MS in the acid/neutral screen; it must be quantitated using this fluorescence assay. In this assay, salicylate is extracted into toluene-ether at an acidic pH. Salicylate is back extracted into borate buffer and quantitated using fluorescence spectrophotometry. Equipment: Fluorescence spectrophotometer such as Perkin-Elmer Fluorospectrophotomer model LS-5 Rotator 25ul Eppendorf pipet Vacuum aspirator Culture tubes, 15 mL, screw cap with Teflon liner SMI pipette Reagents: Toluene, reagent grade Diethylether, anhydrous Sodium borate Salicylic acid Hydrochloric acid, concentrated Toluene-Ether (1:1): Under a vent hood, combine 2 liters toluene and 2 liters diethylether. 5% Borate Buffer: Transfer 10.0 grams sodium borate to a 200 mL volumetric flask and dilute to volume with deionized water. 3 N Hydrochloric Acid: Add approximately 500mL deionized water to a one liter volumetric flask. Add 249mL of concentrated hydrochloric acid. Mix and allow to cool. Dilute to volume with deionized water. Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or
Dallas County Institute of Forensic Sciences Toxicology Laboratory Salicylates Version 2.0
eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Ether is very volatile and very flammable. Do not use near a source of electrical spark or open flame. Caution: Vapors may pool and travel down a lab bench or floor to an ignition source. Toluene-ether should be made in a hood. Avoid repeated contact with the skin and contact with eyes and mucous membranes. Hydrochloric acid is corrosive. Avoid contact with skin, eyes, or mucous membranes. Stocks, Standards, Controls, Calibrators: 1.0 mg/mL Salicylic Acid Stock Standard: Transfer 25.0 mg salicylic acid to a 25 mL volumetric flask and dilute to volume with deionized water. 50.0 mg/L Salicylic Acid Working Standard: Transfer 0.5 mL of 1.0 mg/mL Salicylic Acid Standard to a 10 mL volumetric flask and dilute to volume with deionized water. 100.0 mg/L Salicylic Acid Working Standard: Transfer 1.0 mL of 1.0 mg/mL Salicylic Acid Standard to a 10 mL volumetric flask and dilute to volume with deionized water. 200.0 mg/L Salicylic Acid Working Standard: Transfer 2.0 mL of 1.0 mg/mL Salicylic Acid Standard to a 10 mL volumetric flask and dilute to volume with deionized water. 300.0 mg/L Salicylic Acid Working Standard: Transfer 3.0 mL of 1.0 mg/mL Salicylic Acid Standard to a 10 mL volumetric flask and dilute to volume with deionized water. 200 mg/L Salicylic Acid QC Sample: 1.0 mg/mL salicylic acid stock standard make the above listed stock standard using salicylic acid standard from another vendor or lot as applicable. Transfer 2.0 mL of 1.0 mg/mL QC salicylic acid stock to a 10 mL volumetric flask and dilute to volume with deionized water. Sample Requirements: Acceptable specimens: blood, serum/plasma, urine, bile, gastric, vitreous, tissue homogenates, etc. Sample Collection and Preservation: Liquid specimens (blood, urine, etc.) should be refrigerated during storage; solid specimens (gastric, tissues, etc.) in plastic cups should be frozen during storage.
Sample Preparation: Tissues: Gastric: Homogenize 5 grams tissue in 15.0 mL deionized water. Use 25 uL of the homogenate for analysis For initial analysis dilute 1 part gastric with 99 parts water (1 to 100 dilution). Any significant quantity of a drug will be detected. If diluted sample is negative, an undiluted or less diluted sample may be run. Non-homogenous gastric should be homogenized prior to use. Make dilutions as necessary.
Other samples:
Analytical Procedure: 1. 2. 3. 4. 5. 6. To each 15 mL screw top culture tube, add 25 uL standard, QC, or sample. For the blank, add 25 ul of deionized water to the culture tube. To each tube, add 2 mL of 3 N hydrochloric acid and 10 mL toluene-ether. Cap and rotate for 10 minutes. Centrifuge for about 2 minutes. Transfer 1 mL toluene-ether (upper layer) to another labeled 15 mL screw top culture tube using a 1 mL volumetric pipette. (Do not discard the remaining toluene-ether layer because a dilution may be needed). 7. Add 4 mL 5% sodium borate buffer to each tube and rotate 10 minutes. 8. Centrifuge for about 2 minutes 9. Aspirate the toluene-ether layer (upper layer). 10. Aerate the borate buffer layer to remove traces of the organic phase by bubbling with compressed air. 11. Use the blank to zero the instrument at 400 nm. See attached Instrument Operation Procedures. 12. Record the fluorescence of samples, controls, and standards at 400 nm. 13. Enter data into the Excel spreadsheet as noted below. 14. Place a copy of the results in each case file; place a coy of the Excel spreadsheet in the salicylate QC binder. Note: exit the computer program without saving changes. Quality Control: Original calibration data is kept on file on the Toxicology Laboratory. The reportable range is 50 300 mg/L salicylate. Higher samples must be reported >300 mg/L or diluted. Calculations: Fluorescence of unknown X (Concentration of standard) = salicylic acid concentration (mg/l)
Fluorescence of standard The results are entered into Microsoft Excel QC/AA computer under the program for salicylates and the calculations are done by the computer program based on a standard linear regression. Instrument Operating Procedure: The instrument operating procedure is a part of this analytical procedure. Instrument Methods: Refer to the attached instrument operation procedure.
Salicylates Version 2.0
FLUORESCENCE SPECTROPHOTOMETER OPERATION Principle of Operation: Salicylate molecules absorb radiant energy at a particular wavelength (excitation wavelength) and are excited to a higher electronic state. This excitation causes the molecules to emit radiation at a different wavelength (emission wavelength) characteristic of salicylate. Emitted radiation (fluorescence) is measured at right angles to the excitation light path. Intensity of fluorescence is proportional to concentration and allows quantitation based on known standards. Instrument Parameters: Excitation Emission scan range Recorder speed Response Fixed scale Scan speed Quantitate at 400 nm 310 nm 350 to 450 nm 10 nm/cm 4 1 60 nm/min
Recorder Parameters: Chart speed Range 60 nm/min 1000 mV
Setting up the Instrument: Initial set up: 1. Turn on the power to the fluorescence spectrophotometer (back lower right). Allow instrument to warm up during extraction procedure. On the spectrophotometer: 2. Press [RESP]. Should read 4. If not, press [4],[RESP]. 3. Press [FXD SCL]. Should read 1; if not press [1], FXD SCL]. 4. Press [EX] 5. Press [3],[1],[0], [GO TO ] on left side. 6. Press [EM] 7. Press [3],[5],[0], [LOW ]. 8. Press [4],[5],[0], [HIGH ]. 9. Press [GO TO lambda] on the right. ( goes back to low A350")
Routine operation - Running a sample or standard: 10. Lift light shield and place cuvette, with sample, into cuvette holder (position #1). Close lid. 11. Allow luminescence to reach maximum. 12. Press [4],[0],[0], [GO TO ] on right side and take a reading, and record. Instrument shut down: 13. Clean cuvette. 14. Turn off fluorescence spectrophotometer.
SALICYLATES BY FLUORESENCE TRAINING NOTES 1. Sample concentrations greater than 300 mg/L may be reported as >300 mg/L or diluted. 2. When making the toluene-ether solution, check the expiration date on the ether container. Expired ether may cause erroneous results. 3. Check cuvettes for bubbles when adding sample. They may interfere with analysis. 4. Traces of toluene-ether solution left in borate buffer after aspiration (Procedure Step 8) must be removed by purging borate buffer with air prior to further analysis. 5. Other clinical salicylate procedures will not work with whole blood due to the interference of the heme group in red blood cells.
VALPROIC ACID QUANTITATION Principle of Assay: Valproic acid is an anticonvulsant drug that is detected in the acid/neutral screen. Because valproic acid is volatile, some of the drug may be lost during the evaporation step in the acid/neutral procedure. Therefore when valproic acid is detected in the acid/neural screen, it is quantitated using this method. In this analysis, the specimen is made acidic and valproic acid is extracted into chloroform. Quantitation is performed using gas chromatography/mass spectrometry. Initial identification of valproic acid in a sample is performed as a part of the standard acid/neutral procedure. Equipment: Hewlett-Packard GCD GC/MS Hewlett-Packard model 7673 autosampler Capillary columns : such as DB-5 (cross-linked methyl silicone gum phase) 12 m x 0.20 mm x 0.33 um film thicknness Culture tubes, 15 ml, screw cap with Teflon liner Conical tubes, 5 ml, screw cap with Teflon liner Eppendorf pipet - 100ul 5 ml serological pipets 1 ml serological pipets Vortex mixer Centrifuge Autosampler vials, 32mm x 11mm, Teflon lined seals and glass inserts Hamilton syringes 500 uL pipet Reagents: Hydrochloric acid, concentrated Chloroform 3N Hydrochloric Acid: Fill a 500 ml volumetric flask approximately half full with deionized water. Add 125 ml concentrated hydrochloric acid. Mix carefully and allow to cool if necessary. Fill to the mark with deionized water. Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary.
Dallas County Institute of Forensic Sciences Toxicology Laboratory 1 Valproic Acid Version 2.1
Hydrochloric acid is corrosive. Avoid contact with skin, eyes, and mucous membranes. Chloroform is a chlorinated solvent and is considered a human carcinogen. Use in a vent hood or in a well-ventilated area. Avoid contact with skin, eyes, and mucous membranes. Stocks, Standards, Controls, and Calibrators: Stock Standards: 1.0 mg/ml Thymol Internal Standard: Weigh 10 mg thymol and transfer to a 10 ml volumetric flask. Dilute to volume with methanol. 10 mg/ml Valproic Acid Stock Standard: Transfer 100 ul valproic acid (2-propylpentanoic acid 100% solution) to a 10 ml volumetric flask. Dilute to volume with methanol. 10 mg/ml Valproic Acid Stock Quality Conrol Standard: Transfer 100 ul valproic acid (2propylpentanoic acid 100% solution) to a 10 ml volumetric flask. Dilute to volume with methanol. Calibration Curve Standards: 10 mg/L Valproic Acid Calibrator: In a 15 ml screw top tube, add 5ul valproic acid stock standard (10 mg/ml) to 1 mL deionized water. Add 4 ml blank blood and rotate for 5 minutes. Use 500 ul for analysis. 30 mg/L Valproic Acid Calibrator: In a 15 ml screw top tube, add 15ul valproic acid stock standard (10 mg/ml) to 1 mL deionized water. Add 4 ml blank blood and rotate for 5 minutes. . Use 500 ul for analysis. 70 mg/L Valproic Acid Calibrator: In a 15 ml screw top tube, add 35ul valproic acid stock standard (10 mg/ml) to 1 mL deionized water. Add 4 ml blank blood and rotate for 5 minutes. . Use 500 ul for analysis. 100 mg/L Valproic Acid Calibrator: In a 15 ml screw top tube, add 50ul valproic acid stock standard (10 mg/ml) to 1 mL deionized water. Add 4 ml blank blood and rotate for 5 minutes. . Use 500 ul for analysis. Quality Control Standards: 50 mg/L Valproic Acid QC: In a 15 ml screw top tube, add 25ul valproic acid stock quality control standard (10 mg/ml) to 1 mL deionized water. Add 4 ml blank blood and rotate for 5 minutes. . Use 500 ul for analysis. External Control (such as Biorad): Reconstitute and store per manufacturers directions. Use 500 uL for analysis. Negative Control: Use 500 uL blank blood for analysis.
Valproic Acid Version 2.1
Sample Requirements: Acceptable specimens: blood, serum/plasma, urine, bile, gastric, vitreous, tissue homogenates, pharmaceutical preparations Sample Collection and Preservation: It is recommended but not required that blood specimens are preserved with sodium fluoride (gray top); however, most acid and neutral drugs are not as susceptible to in vitro enzymatic degradation as alkaline drugs. Femoral blood is preferable; however, acid and neutral drugs are typically less susceptible to postmortem redistribution than alkaline drugs. Liquid specimens (blood, urine, etc.) should be refrigerated during storage; solid specimens (gastric, tissues, etc.) in plastic cups should be frozen during storage. Sample Preparation: Tissues: Homogenize 5 grams tissue in 15.0 mL deionized water. Use 500 uL of the homogenate for analysis Gastric: For initial analysis dilute 1 part gastric with 99 parts water (1 to 100 dilution). Any significant quantity of a drug will be detected. If diluted sample is negative, an undiluted or less diluted sample may be run. Non-homogenous gastric should be homogenized prior to use. Other samples: Use 500 uL in the analysis. Non-standard dilutions must be noted in the case file. Analytical Procedure: 1. Place 500 uL of each sample and standard in a 5 mL conical tube. 2. Add 100 uL of thymol internal standard. Vortex. 3. While vortexing the conical tube, add 100 uL 3N HCl. Note: HCl must be added while vortexing or the blood in the tube will clot and not mix well. 4. Add 1 mL chloroform. Vortex. 5. Cap then centrifuge on high for about 5 minutes. 6. Transfer chloroform layer (bottom) to an autosampler vial for analysis. 7. Use VALPROQ.M method on GCD. Samples are run in full scan mode and quantitated using SIM. Quality Control: Controls should match manufacturers range or fall within +/- 20% of target concentration. If not, consult a supervisor or repeat the assay or sample. QC results are maintained in the Toxicology Laboratory. Qualifier ions should be within +/- 20% of target.
Dallas County Institute of Forensic Sciences Toxicology Laboratory 3 Valproic Acid Version 2.1
Instrument Operating Procedure: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebook. Instrument Methods: Refer to the Instrument Methods Notebook.
VALPROIC ACID QUANTIATION TRAINING NOTES 1. Valproic acid is identified by the acid/neutral drug screen procedure. It is quantitated using this procedure because it is volatile and loss may occur during evaporation in the acid/neutral procedure. 2. A wood applicator stick may be used for mixing when adding the HCl and chloroform.
VOLATILES BY HEADSPACE GAS CHROMATOGRAPHY Principle of Assay: The volatiles method allows for identification and quantitation of several volatile chemicals in biological specimens and aqueous solutions using headspace gas chromatography. Substances placed in a closed vial thermostatted at constant temperature will partition into the gaseous phase above the sample based upon the partition coefficient of the substance. Under constant conditions, equilibrium will be reached between the sample phase and gas phase. At this point the concentration of substance in the headspace or gas phase is proportional to the concentration of substance in the sample, and the amount of substance in the sample may be calculated. In this procedure, the sample and an aqueous internal standard solution are combined and thoroughly mixed in a headspace vial. The vial is sealed and placed in the headspace autosampler. The sample is heated for a pre-set time to establish equilibrium of volatile analytes between the liquid and vapor phases. A portion of the headspace is sampled and introduced into the gas chromatograph. Identification of the analyte(s) present in the sample is based on retention time on two different types of columns; quantitation is based on peak areas relative to the internal standard. Toluene is the volatile most commonly encountered in this laboratory; therefore, its analysis is described in more detail. Equipment: HP 5890 Series II and a Varian model 3800 gas chromatographs equipped with flame ionization detectors and two capillary columns such as: RTX BAC-1 and RTX BAC-2 (0.53 mm ID x 30 meter) Varian Genesis headspace autosampler Computer equipped with the Varian Star Workstation software package Headspace vial, septa and seals Repipet dispenser/diluter Micropipettor - 250ul Reagents: n-Propanol, reagent grade Appropriate standard listed below Detectable Analytes: Toluene Diethyl ether Methylene chloride
Volatiles by Headspace Version 2.0
Methyl ethyl ketone Hexane Ethyl acetate Carbon tetrachloride 1,1,1-Trichloroethylene Chlorobutane Benzene Ethylene dichloride Chloroform Trichloroethylene Paraldehyde
This method is also suitable for analysis of other volatiles. If an unknown compound is suspected, the specimen may be run by mass spectrometry to determine the compound(s) of interest. Once the compound(s) has been determined, the appropriate standards can be diluted and run. Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Stocks, Standards, Controls, Calibrators: 0.012% n-Propanol (internal standard solution): Transfer 0.72 grams of n-propanol to a 6 liter volumetric flask and dilute to volume with distilled water. 17.3 mg/L Toluene standard: Transfer 20 ul toluene to a 1000 ml volumetric flask and dilute to volume with deionized water. Other standards: Calculate the concentration of the standard by taking a known volume of pure standard and diluting it to 1000 mL in deionized water. Multiply the density of the standard (g/ml or mg/ul) by the volume of standard (in microliters) to get the standard concentration in mg/L. The concentration may be calculated using the formula: Volume std * Density std / Final Volume std = Concentration std
For example, the toluene standard prepared above is calculated as follows: 20 ul * 0.866 mg/ul / 1 L = 17.3 mg/L GC response and resultant assay sensitivity varies among volatile compounds. Therefore measurable standard concentrations will vary by compound. Consult a supervisor for assistance. Compounds such as halothane, isoflurane, desflurane, and other agents used in surgical procedures are often reported as detected only and not quantitated. Consult a supervisor for assistance. Sample Requirements: The following samples are suitable for analysis: antemortem blood, serum/plasma, postmortem blood, vitreous, bile, gastric, aqueous samples, tissue homogenates, etc. Urine is also an appropriate specimen; however, some volatiles are only slightly soluble in water may require a lesser dilution of urine with internal standard solution during the assay procedure (for example 1 part sample : 1 part internal standard solution). Sample Collection and Preservation: It is recommended that specimens are collected in volatile vials or analyses performed on unopened tubes of sample. Specimens should be stored refrigerated and should be analyzed as soon as possible upon receipt. Analytical Procedure: Liquid samples: 1. Using the Repipet dispenser, place 4.0 mL of n-propanol internal standard solution in headspace vial. 2. Add 250 ul of sample (standard, sample), using the micropipettor. 3. Wipe off the tip of the micropipettor and rinse the plunger and tube in water at least 5 times to clean. 4. Seal the vial with a septum and seal ring. 5. Mix the contents with a gentle swirling motion. 6. Analyze on the headspace GC using proper instrument parameters. 7. Calculations are performed manually as noted in the Quantitation section of this method; results are reported in milligrams per liter. Tissue samples: 1. Place 1.0 gram of tissue in the homogenizer with 16 milliliter of internal standard solution. 2. Place 4.25 mL of homogenate in the headspace vial using a wide bore serological pipette.
Dallas County Institute of Forensic Sciences Toxicology Laboratory Volatiles by Headspace Version 2.0
3. 4. 5. 6.
Seal the vial with a septum and seal ring. Mix the contents with a gentle swirling motion. Analyze on the headspace GC using proper instrument parameters. Calculations are performed manually as noted in the Quantitation section of this method; results are reported in milligrams per g tissue.
Qualitative Identification: The presence of an analyte is determined by matching retention times on two columns of different polarity and matching to retention times of known standards. If an unknown or unexpected peak is present in the alcohols and acetone analysis, a GC/MS will be run to determine substance identity; refer to the Volatiles by GC/MS procedure. Quantitation: Run a standard of known concentration and a sample. Obtain area counts for the internal standard and standard from the standard run. Also obtain area counts for the internal standard and compound(s) of interest in the unknown run. Calculate the concentration using the formula: Concentration unknown = Concentration Std * R unknown / R Std Where: R unknown = Area unk / Area Internal Std unk R Std = Area Internal Std std / Area std Quality Control: A standard should be run for each suspected volatile at a concentration similar to that of the sample. Quality control results are kept in the Toxicology Laboratory. Instrument Operating Procedure: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebook. Instrument Methods: Refer to the Instrument Methods Notebook.
References: Genesis Headspace Autosampler Operator's Manual Foerster, EH and Garriott, JC. Analysis of Volatile Compounds in Biological Samples. JAT 5:241 244, 1981.
VOLATILES BY HEADSPACE GAS CHROMATOGRAPHY TRAINING NOTES After the sample is added to the aqueous internal standard, mix by using a swirling motion. This will give more consistent results due to a more homogeneous solution. When adding internal standard solution to the headspace vial with the Repipet dispenser, do not allow the initial drop of solution that may be ejected when the plunger is pulled upward to go into the headspace vial. When adding internal standard solution to the headspace vial with the Repipet dispenser, do not allow the initial drop of solution that may be ejected when the plunger is pulled upward to drop on the outside of the vial. It has been observed that wet vials may stick to the auto sampler and not drop into the instrument resulting in no injection. Make sure that the top is crimped correctly. If the aluminum ring is not properly crimped, it may result in the needle hitting the ring and not properly piercing the septum. There is a substantial peak just beyond the internal standard that is a common component of tissue homogenates and should not be considered as a compound of interest in this procedure. Volatile samples are often only slightly soluble in water. It is recommended that when making the standard solution the standard is added to the volumetric and then the volumetric is first brought to approximately full with deionized water. The flask is mixed well and then brought to volume with deioinized water and mixed again. See the Alcohols and Acetone procedure for additional information about headspace analysis.
Instrument Parameters Volatiles by Headspace Gas Chromatography Instrument Parameters: Column Temperature Detector Temperature Injector Temperature Thermostatting Platen Temperature Sample Loop Transfer Line Thermostatting Time Helium (carrier gas) Flow Hydrogen Flow Air Flow Run time Method on Headspace Method on the Varian Workstation Column Pressure 90oC 150oC 150oC 60oC 110oC 150oC 15 minutes 10 mL/min 40 mL/min 320 mL/min 5 min 2 TolueneHP.mth 7 psi
VOLATILES SCREEN BY GC/MS Principle of Assay: This analysis allows qualitative identification of volatile compounds in blood and other specimens including unidentified liquids or solids using purge and trap gas chromatography/mass spectrometry. A sample of headspace from a closed container containing a suspected volatile substance is transferred to the purge and trap concentrator where it is then concentrated and desorbed onto a GC/MS for positive identification. Equipment: Hewlett-Packard model 5890 Series II Plus gas chromatograph with capillary column such as HP-5MS capillary column (5% phenyl methyl siloxane) 30m x 0.25mm x 0.25um film Hewlett-Packard 5972 Series Mass Selective Detector Hewlett-Packard 7695 Purge and Trap Concentrator Hewlett-Packard model 6890 autosampler Gastight Hamilton syringe, 5ml Luer-lock needle, in. Lumbar needle , 6 in. Hotwater Bath , 60 C. Liquid Nitrogen Reagents: None Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Use care when inserting needles into the stoppers since there is the danger of a sharps injury. Stocks, Standards, Controls, Calibrators: All standards are kept in the freezer. Standards are removed as fast as possible and returned to the freezer.
Volatiles by GC/MS Version 2.0
Sample Requirements: Acceptable samples include but are not limited to blood, plasma/serum, urine, bile, vitreous, gastric, tissue homogenates, liquids, etc. Sample Collection and Preservation: It is recommended that samples are collected in headspace vials, however, samples in unopened collection tubes may also be used. Samples should be refrigerated during storage and analyzed as soon as possible. Analytical Procedure: At least one blank room air sample should be run before the sample in order to check the system. Also, if possible a blank sample in the same type container should be analyzed to check for interference from the stoppers, etc. 1. 2. The closed vial or stoppered test tube containing the sample is placed in a beaker of water in water bath (approximately 60 deg C) for about 5 minutes. A 6 inch lumbar needle is inserted through the rubber top of the vial and lowered about halfway down the tube. If the sample is very viscous or solid, the needle is placed just above the sample. The plunger is removed from the lumbar syringe. A inch needle is attached to a 5 ml gas-tight syringe and inserted just through the rubber stopper of the vial and into the headspace above the sample. Lower the lumbar needle into the sample. Using the gas-tight syringe, collect 5 ml of headspace air from the vial and transfer the sample to the fritted sparger of the purge and trap concentrator. As you begin collecting headspace from the vial, you should notice air bubbling through the sample. The headspace sample is then purged for 11 minutes and trapped on a Tenax column located in the purge and trap concentrator. While the sample is being collected, prepare the computer for data acquisition by using the following schematic flowchart for running the GC/MS: GC/MS Instrument #1 Method Load TOXVOL.M MSTop Sequence Load TOXVOL.S OK Sequence Edit Sample Log Table (Highlight and correct Data File and Sample Name)
Dallas County Institute of Forensic Sciences Toxicology Laboratory Volatiles by GC/MS Version 2.0
3. 4.
5. 6.
OK Sequence Run (Enter todays date in Data File Directory, correct Sequence Comment) OK Sequence Save TOXVOL.S OK Overwrite? OK Sequence Run 7. Turn on liquid nitrogen for cryogenic GC operation. When the purge and trap is 10.5 min into the purging phase, hit Run Sequence on computer. This will initiate the acquisition and start the mass spectrometer after the GC has cooled down. Identification of GC/MS peaks is made by searching the Wiley 275k library or other available libraries.
8.
Qualitative Identification: Identification of volatiles is made using GC/MS. Air blanks and system blanks should be run to identify any interference from volatiles in rubber stoppers, lab air, etc. Carbon dioxide is usually detected at approximately 1.5 min. when analyzing blood samples. This is a normal product and also serves as verification that the analysis proceeded properly. Instrument Operating Procedure: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebook. Instrument Methods: Refer to the Instrument Methods Notebook. References: Volatile Organic Compounds By Gas Chromatography/Mass Spectrometry (GC/MS), U.S. EPA, Test Methods For Evaluating Solid Waste (SW-846), Method 8260B.
VOLATILES BY GC/MS TRAINING NOTES 1. Interference It is important to verify if there is interferences from sample containers such as vacutainer stoppers or the lab environment. Specimen blanks and lab air blanks should be run. All major peaks should be identified using the Wiley computer library or other library. A hard copy of the Total Ion Chromatogram (TIC) and mass spectra from major peaks should be kept with the case. A hard copy of the TIC of blanks run should also be kept. Occasionally, an air peak can be observed between 1 and 2 minutes on the air blank chromatogram. If you notice this, tighten the nut at the GC/MS interface and re-run the air blank. Remember to close the valve on the nitrogen cylinder when finished with the analysis.
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QUANTITATION OF GASES BY MULTIPLE HEADSPACE GAS CHROMATOGRAPHY Principle of Assay: The concentration of a gas or very volatile liquid in a liquid or solid sample can be determined using the technique of multiple headspace analysis. When a sample containing a volatile is placed in a closed sample vial and heated to a constant temperature, the volatile distributes between the sample phase and gas (headspace) phase based upon the partition coefficient of the volatile substance; under constant temperature conditions, an equilibrium will be achieved between the sample phase and the gas phase. After an aliquot of the headspace is removed for GC analysis and the sample vial is again allowed to come to equilibrium condition, the relative amount of volatile distributed between the sample and gas phase will be the same as in the sample container prior to removal of the headspace; however, the absolute amount of substance available for distribution will be reduced by the amount removed in the first headspace sample. If an infinite number of headspace samples are taken, all of the volatile will be removed from the sample, and the sum of all of the GC areas of all injections will correspond to the amount of the volatile in the sample placed into the vial. The quantity of volatile in the sample can be calculated by comparing the total area of the volatile in the sample to the total area of volatile in a known standard. A semi-log plot of area versus injection number is a straight line. Therefore as a practical matter, two headspace analyses per specimen are sufficient for quantitation of the unknown. In this assay, the sample is placed into a headspace vial. The vial is sealed, weighed, and placed in the headspace autosampler. The sample is heated for a preset time to establish equilibrium between the sample and vapor phases. A portion of the headspace is vented to the gas chromatograph for analysis. The sample vial is again placed in the headspace autosampler and analyzed. A known amount of standard is placed into a headspace vial and treated in the same manner. After the analysis the sample headspace vial is emptied, cleaned, dried, and weighed again. The difference in weight is the weight of the sample analyzed. Quantitation is performed using an excel spreadsheet. Identification of the volatile(s) present in the sample is based on retention times on two different types of columns and/or GC/MS identification. Equipment: HP 5890 Series II and a Varian model 3800 gas chromatographs equipped with flame ionization detectors and two capillary columns such as RTX BAC-1 and RTXBAC-2 (0.53 mm ID x 30 meters) Varian Genesis headspace autosampler Computer equipped with the Varian Star Workstation software package Headspace vial, septa and seals Gas tight syringe or other equipment to prepare gas standards (see standards preparation below)
Multiple Headspace Analysis Version 2.0
Reagents: Select and prepare the applicable calibration gas 1,1,1-trichloroethane calibration gas chlorodifluoromethane calibration gas (freon 22) dichlorodifluoromethane calibration gas (freon 12) ethyl chloride calibration gas isobutane calibration gas methane calibration gas n-butane calibration gas propane calibration gas difluoroethane calibration gas ethane calibration gas nitrous oxide calibration gas isoflurane calibration gas desflurane calibration gas halothane calibration gas
This is not a complete list of compounds suitable for analysis by this procedure; many gases and very volatile liquids are appropriate for analysis. If an unknown compound is suspected, the specimen may be run by mass spectrometry to determine the compound(s) of interest. Once the unknown compound has been identified, the appropriate standard can be diluted and run. Safety Precautions: The most common type of chemical or biological exposure in this type of laboratory is a splash to the skin or eye. Skin, mucous membranes, or eyes which have been splashed with commonly used chemicals or biologicals should be thoroughly washed for at least 15 minutes in cool tap water or eye wash station. Refer to appropriate MSDSs for additional chemical information. Report the incident immediately to a supervisor. Seek medical attention as necessary. Calibration gases are in pressurized containers that should be handled accordingly: do not puncture or overheat can. Stocks, Standards, Controls, Calibrators: Standards should be prepared according to GC response of the compound of interest and the expected or suspected concentration of compound in the sample. Response will vary according to the compound of interest. Several standard preparations may be necessary before determining the appropriate concentration. Prepare standards by mixing a known volume of air with a known volume of calibrator gas using positive pressure and two syringes with a three way stopcock connector. Pull a known volume
of air into a syringe. Attach the three way stopcock to the syringe with a pushing and twisting motion. Put a known volume of calibrator gas into the second syringe. Attach the second syringe to the three way stopcock and turn the handle so that the third port is closed. Note that the handle is always over the closed port. This will allow for the two syringes to be connected in such a way that the air from the most full syringe can be pushed into the least full syringe. Mixing will take place when the full syringe is pushed into the empty syringe several times. Once the mixing is done 1 ml of the gas is injected with a syringe into a sealed headspace vial and analyzed.
The volume of air and the volume of calibrator gas will depend upon the dilution that is to be made. This determination needs to be made by the chemist and a supervisor based upon the expected response of the compound of interest. The above information should be recorded for later use in the volatiles.xls calculation spreadsheet.
Sample Requirements, Collection, and Preservation: A wide variety of solid, liquid, and gas samples are appropriate for analysis by this techinique including but not limited to antemortem blood, postmortem blood, urine, vitreous, bile, gastric, lung, aqueous solutions, gases, other tissues, etc. Three separate samples of different volume or size should be collected in headspace vials as soon as possible and sealed. (Sample collection is normally performed by Medical Examiner staff at autopsy.) For a liquid sample, the headspace vials should be filled to approximately 3 mm, 5 mm, and 1 cm. For tissue, the headspace vial should be filled at three different levels of approximately , 1/3, and of the head space vial. Dilutions of gas canisters should be prepared as described for standards. Storage of specimen: Gas canisters - store at room temperature Biological samples and liquids - refrigerate Analysis should be performed as soon as possible after sample receipt.
Analytical Procedure: 1. Qualitative analysis by GC/MS should be performed first to identify the unknown volatile. GC/MS may be performed on one of the headspace vials or on the headspace from an unopened blood tube. (Note: GC/MS identification cannot be performed on substances with molecular weight of 16 amu or below.) Weigh each headspace vial prior to initial analysis and record the weight. Analyze on the headspace GC, using preset parameters. Analyze the vial a second time by
3 Multiple Headspace Analysis Version 2.0
2. 3.
4. 5. 6.
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headspace GC. Determine the approximate concentration of the compound(s) present and prepare standards for quantitation; seek supervisory assistance as needed. Add 1 ml of standard mixture to a sealed headspace vial. Analyze the vial using the preset parameters specified for the analysis of the unknown. Analyze the vial a second time. After the analysis is complete, empty the contents of the sample vial, rinse, and dry it completely. Weigh the empty vial plus rubber stopper and crimp cap. The difference in the weight of the full vial and the empty vial is the weight of the sample. Calculations are performed using the spreadsheet volatiles.xls in Microsoft excel. This can be accessed through the desktop icon on the QC/AA computer.
Qualitative Identification: Identification of volatiles present in the sample is made using GC/MS where appropriate; refer to the Volatiles by GC/MS procedure. Quantitation: Calculations are performed using the spreadsheet volatiles.xls in Microsoft excel. This can be accessed through the desktop icon on the QC/AA computer. A discussion of the calculations follows: 1. Determine the total area of volatile in the sample and standard vials: A total = A12 / A1 A2 ,where A1 = Area of the first injection of the sample or standard and A2 = Area of the second injection of the sample or standard Perform this calculation for the sample and for the standard. 2. Determine the amount of standard placed in the vial: m = ml of standard placed into the standard vial p = ppm of standard placed into the standard vial mw = molecular weight of the standard 22.4 L = volume of one mole of gas at STP p / 1,000,000 parts air x mw x 1 mole / 22.4 L x 1 L / 1000 ml x 1000 mg/ 1 L x 1000 ug/ 1 ml x m = ug of standard in vial Ex: If 1 ml of a 1000 ppm butane standard was placed in the standard vial: 1000 parts butane/1,000,000 parts air x 58.12 g butane/mole x 1 mole/22.4 L x 1L/1000ml x 1000 mg/1 L x 1000 ug/ 1 ml x 1 ml = 2.59 ug butane in the standard vial.
3.
Calculate the amount of volatile in the sample:

total
ug standard in the standard vial/A unknown 4.
standard = ug volatile in the volatile vial/A
total
Calculate the amount (grams) of sample placed into the sample vial: weight of sample vial with sample weight of sample vial without sample = weight of
sample For a liquid: multiply the weight of the sample by the density (g/ml) to determine the volume of sample in the sample vial; the density of blood is 1.06 g/ml. 5. Calculate concentration of volatile in sample: Solid: ug volatile in the volatile vial / weight of sample (g) = ug/g or mg/kg Liquids: ug volatile in the volatile vial / volume of the sample (ml) = ug/ml or ml/L Quality Control: Each batch will contain at least one standard of each of the suspected compounds in a concentration approximate to the suspected concentration of the unknown. QC results are kept in the Toxicology Laboratory. Instrument Operating Procedure: The instrument operating procedure may be found near the instrument; refer to instrument manual and/or instrument procedure notebook. Instrument Methods: Refer to the Instrument Methods Notebook. References: Genesis Headspace Autosampler Operator's Manual Ettre LS, Kolb B, and Hurt SG. Techniques of Headspace Gas Chromatography, American Laboratory, pp 76-83, October, 1983. Kolb B. Multiple Headspace Extraction A procedure for eliminating the influence of the sample matrix in quantiative headspace gas chromatography. Chromatographia 15:587-594, 1982.
Zhu J and Chai X. Automation in Determining Solute Concentration and Henrys Constant by Multiple Headspace Extraction Gas Chromatography. American Lab News Edition. August, 1999.
Procedure Review and Approval Form
VOLATILES BY MULTIPLE HEADSPACE GAS CHROMATOGRAPHY TRAINING NOTES Refer to the Alcohols and Acetone method, the Volatiles method, and selected references in the training portion of this manual. It is important that the septum is properly crimped to prevent loss of volatiles.
Multiple Headspace Analysis 6/02
INSTRUMENT PARAMETERS MULTIPLE HEADSPACE ANALYSIS Instrument Parameters: Instrument Parameters: Column Temperature Detector Temperature Thermostating Platen Temperature Injector Temperature Sample Loop Transfer Line Thermostatting Time Helium (carrier gas) Flow Hydrogen Flow Air Flow Run Time Method on Headspace Method on Varian Workstation Column Pressure 35C 150C 40C 150C 110C 150C 15 minutes 10 mL/min 40 mL/min 320 mL/min 10 minutes 3 VolatileHP.mth 7 psi
Multiple Headspace Analysis 6/02

SWIFS Toxicology Laboratory Procedure Manual v2.3 (03.13.2009) 267 Pages

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Dallas County Southwestern Institute of Forensic Sciences

TOXICOLOGY LABORATORY PROCEDURE MANUAL, Version 2.3

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Changes from Previous Version Version 2.0

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Dallas County Institute of Forensic Sciences Toxicology Procedure Manual

Corrections and Revisions Version 2.3

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Statement of Purpose Version 2.0

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Scope of Services Version 2.0

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Evidence Management Version 2.0

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Evidence Handling Procedure Version 2.0

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Evidence Handling Procedure Version 2.0

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Evidence Handling Procedure Version 2.0

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Evidence Handling Procedure Version 2.0

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Evidence Handling Procedure Version 2.0

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Dallas County Institute of Forensic Sciences Toxicology Laboratory

Evidence Handling Procedure Version 2.0

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Dallas County Institute of Forensic Sciences Toxicology Laboratory

Evidence Handling Procedure Version 2.0

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Dallas County Institute of Forensic Sciences Toxicology Laboratory

Evidence Handling Procedure Version 2.0

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Dallas County Institute of Forensic Sciences Toxicology Laboratory

Evidence Handling Procedure Version 2.0

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Dallas County Institute of Forensic Sciences Toxicology Laboratory

Evidence Handling Procedure Version 2.0

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Dallas County Institute of Forensic Sciences Toxicology Laboratory

Evidence Handling Procedure Version 2.0

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Dallas County Institute of Forensic Sciences Toxicology Laboratory

Evidence Handling Procedure Version 2.0

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