Lab Guidance Document

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0
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IR-4 Laboratory Guidance Document

Introduction
This Guidance Document has been developed to provide consistency and to facilitate
communication among the IR-4 laboratories (LRDs), Regional Directors (management), Quality
Assurance (QA), and the IR-4 Study Directors (SD) and will serve as a resource for all facets of
IR-4. It indicates how IR-4 operates, by designating responsibilities and providing guidelines for
implementation of procedures, to assure that all studies conducted by IR-4 meet EPA GLP
regulations. Once this document is approved by the IR-4 PMC, it becomes an official policy
document for the conduct of studies in the IR-4 laboratories.

The main areas of attention in this document include personnel responsibilities in relation to IR-4
residue work, definitions and a significant section regarding lab operations with emphasis on
sample handling and storage; sample processing; analytical method validation; sample analysis
and extract storage; storage stability studies; communication with the study director; and the
Analytical Summary Report. This document will provide guidance for contract labs and will be
used as a training tool with regard to IR-4 analytical work.

Please note: paragraphs formatted with italics are taken directly from the Operational Handbook
of IR-4 Version 7.0

Committee members:
Daniel Kunkel, IR-4 Headquarters, Associate Director (Chair)
Debbie Carpenter, IR-4 Headquarters
Tom Hendricks, USDA-ARS, Tifton, GA
Matt Hengel, Western Regional Laboratory Coordinator, University of California
Wayne J iang, North Central Regional Laboratory Coordinator, Michigan State University
Christopher Lam, North Eastern Regional Laboratory Coordinator, Cornell
J im McFarland, Western Region QA Coordinator, University of California
Marion Miller, Western Region Director, University of California
J au Yoh, Southern Regional Laboratory Coordinator, University of Florida
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Table of Contents
Introduction ..................................................................................................................................... 2
Responsibilities ................................................................................................................................ 4
IR-4 Headquarters (HQ) Staff ............................................................................................................... 4
Regional Research Programs ............................................................................................................... 4
ARS Programs Research Personnel ...................................................................................................... 4
Definitions ....................................................................................................................................... 4
GLP Definitions ........................................................................................................................... 4
Archives ................................................................................................................................................. 4
Protocol ................................................................................................................................................. 4
Quality Assurance Unit ......................................................................................................................... 5
Sponsor ................................................................................................................................................. 5
Study ...................................................................................................................................................... 5
Study Director ....................................................................................................................................... 5
Testing Facility ..................................................................................................................................... 5
IR-4 Definitions ........................................................................................................................... 5
Laboratory Research Director .............................................................................................................. 5
Quality Assurance Coordinator (QAC) and Officers (QAO) ................................................................ 5
Regional Laboratory Coordinator ........................................................................................................ 6
References ....................................................................................................................................... 6
Lab Operations ................................................................................................................................ 6
Standard Operating Procedures ................................................................................................... 6
Standards and Solutions ............................................................................................................... 6
Obtaining Standards ................................................................................................................ 6
Characterization of Substances ............................................................................................... 6
Reagents and Solutions ........................................................................................................... 6
Sample Receipt, Processing and Storage ..................................................................................... 7
Sample Receipt ........................................................................................................................ 6
Sample Processing: ................................................................................................................. 7
Storage: ................................................................................................................................... 8
Working Method, Validation and Modifications: ....................................................................... 8
Other considerations ................................................................................................................ 8
Extraction ................................................................................................................................ 8
Clean-up steps ......................................................................................................................... 9
Detector ................................................................................................................................... 9
Working method approval and validation data ....................................................................... 9
Sample Analysis and Extracts ..................................................................................................... 9
Sample Analysis ...................................................................................................................... 9
Extracts .................................................................................................................................. 10
Storage Stability ........................................................................................................................ 10
Communication of Results with SD .......................................................................................... 10
Response Needed to Proceed: ............................................................................................... 10
Routine Results: .................................................................................................................... 10
ASR ................................................................................................................................................ 10
Training .......................................................................................................................................... 11
Contract/Company Labs ................................................................................................................ 11
Guideline Document review: ......................................................................................................... 11
Explaination of Attachements: ....................................................................................................... 11

Attachment 1: Sample processing Document.
Attachment 2: Sample Analytical Summary Report
Attachment 3: Checklist for Review of Analytical Summary Reports
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Responsibilities
IR-4 Headquarters (HQ) Staff coordinate the program among the regions and USDA-ARS, and
provide functions including:
1) GLP oversight including Study Director (SD) and Quality Assurance (QA).
2) Prepare research protocols.
3) Review, analyze, and archive raw data.
4) Prepare, review, and submit petitions to establish and maintain tolerances.
5) Interact with EPA and cooperating registrants.
6) Maintain a database to track projects.
7) Oversee Manufacturer and Contract Laboratories
The HQ office is administered by an Executive Director (Management Representative).

Regional Research Programs. Each Regional Program is administered by a Regional Director
who has overall responsibility for GLP compliance at the regional level. The
Regional Director has Regional Laboratory, Field and QA Coordinators who work with state
scientists within their region and provide them with research support.
1) Regional Laboratory Coordinator (RLC): Oversees and coordinates regional
laboratories and their satellite laboratories, and some contract laboratories, conduct
analyses to determine test substance residues on crop samples.
2) Regional QA Coordinator: Monitors the field and laboratory operations in each
region to assure that they are meeting GLP requirements.

ARS Programs Research Personnel The ARS Program is administered by an ARS
National IR-4 Director who has overall responsibility for GLP compliance at the ARS Facilities.
The ARS National IR-4 Director supports USDA-ARS residue laboratories and scientists
(Laboratory Research Directors) that conduct analyses and determine test substance residues on
crop samples. QA for these facilities is provided by other IR-4 QA and contract QA.

Definitions
GLP Definitions
Archives: All raw data developed by the IR-4 program will be archived as required under 40
CFR 160.190. Archivists will be designated by the Executive Director for IR-4 HQ and an index
of archived laboratory data from the RLCs will be sent periodically to the HQ Archivist.

Protocol: The regulations require an approved written protocol for each study. The SD is
responsible for the development of the protocol, which is prepared in accordance with the
information as outlined under 40 CFR 160.120. Protocols will contain both the field and
laboratory phases of each study and detail the proposed sites for the research. The regulations
require that the protocol be approved by the SD and sponsor by signing and dating. The Chair of
the PMC (sponsor) delegates approval of the protocols to the Executive Director or his/her
designee.

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Quality Assurance Unit: The QA unit will conduct facility inspections at all IR-4 test locations
and conduct critical phase inspections of each study at intervals adequate to ensure study
integrity. All QA audits from facility and critical phase inspections will be provided to the
appropriate SD and Management (IR-4 Executive Director) for review, appropriate response
and corrective action, and signature. Those reports that require action may be forwarded to the
Regional Directors as necessary. The HQ QA Manager will maintain a copy of the Master
Schedule for all IR-4 studies.

Sponsor: The sponsor is the person who initiates and provides financial or other support for a
study. The IR-4 Project Management Committee acts as sponsor for IR-4 studies and has
designated the Executive Director as sponsor for the purposes of GLP. The Executive Director
may delegate individuals to act as Sponsor Representative to sign the protocol, etc.

Study: An experiment conducted at the IR-4 Research Facilities (or contract facilities) to
determine the magnitude of the residue (test substance) in or on a given commodity to provide
the sponsor with residue chemistry data to support a pesticide tolerance.

Study Director: The SD represents the single point of study control, and is responsible for the
overall conduct of the study. The accountability provided by a single SD (who plans, oversees,
and controls the interpretation, analysis, documentation, and reporting of the results) is one of
the most important aspects of the GLP standards. For IR-4 studies, the SD oversees the research
of FRD and LRDs who are responsible for carrying out the field and analytical duties. The
RLCs, RFCs, and ARS National IR-4 Director assist the SDs in meeting their responsibilities.

Testing Facility: IR-4 HQ serves as the testing facility for the purposes of GLP. The Executive
Director will represent testing facility management, and the SDs and QAU will report to the
Executive Director.

IR-4 Definitions
Laboratory Research Director: A person with sufficient training and experience to be able to
conduct the laboratory analysis and appoint adequate personnel to assure this function will be
carried out for all studies. The LRD will report all deviations from the protocol or the SOPs to
the SD.

Quality Assurance Coordinator (QAC) and Officers (QAO): These persons, designated by the
Regional Director or Executive Director, report the findings of their audits to the SD, to the
Executive Director (Testing Facility Management) and to other research associated personnel.
The QAC/QAO will monitor studies, including facilities, equipment, personnel, methods,
practices, records and controls, for compliance with GLP. The QAU reviews the final report to
assure that it accurately reflects the raw data of the study and prepares and signs a Quality
Assurance Statement noting dates the inspections and findings were reported to the SD and SD
Management.

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Regional Laboratory Coordinator: This person assigns laboratory-testing sites within his/her
region for residue analyses conducted by the leader laboratory, its satellites, and private
contract laboratories.
References
Good science is key to successfully completing the analytical portion of any study. However, it
is just as important for SDs and LRDs to be aware of the impact of the following references.
These references provide a framework for all IR-4 study related work.

Operational Handbook of IR-4 (current version).
Good Laboratory Practice Standards, 40 CRF Part 160, August 17, 1989.
Food and Feed Crops of the United States, Markle et al. 1998.
OPPTS 860 Residue Chemistry Test Guidelines including:
OPPTS 860.1000, Background
OPPTS 860.1340, Residue Analytical Method
OPPTS 860.1380, Storage Stability Data
OPPTS 860.1500, Crop Field Trials
OPPTS 860.1520, Processed Food/Feed
Lab Operations
Standard Operating Procedures: Responsibility for the development of a comprehensive
set of SOPs that address the development, monitoring, and reporting of data from specific study
phases conducted at the research test site is the responsibility of each Laboratory Research
Director at that site. RLCs and the ARS National IR-4 Director provide guidance for and
approval of SOPs. This guidance document will take precedence over SOPs and they may
therefore need modification after this document is put in place.
Standards and Solutions
Obtaining Standards: IR-4s current policy is that all reference standards must be characterized
under GLP preferably before the analysis begins, but definitely before the study is completed
(signed by the study director). Due in part to the large number of registrants IR-4 works with,
obtaining GLP standards can be difficult. It is recommended that the LRD initiate discussions
with the cooperating registrant. If standards can not be acquired in sufficient time frame, then
the LRD is directed to contact the SD or Registrations Manager at IR-4 HQ to seek assistance in
obtaining standards. Purity value given on the Certificate of Analysis should be used for all
calculations of the standard concentration.

Characterization of Substances: Analytical Reference Standards; Documentation of the
characterization of the standards used in the analytical trial should be obtained by the
Laboratory Research Director and a copy sent to the SD along with the Analytical Summary
Report of the trial.

Reagents and Solutions: The GLP standards require all reagents and solutions in the laboratory
area to be labeled to indicate identity, titer or concentration, storage requirements, and
expiration date. This requirement can be difficult to accomplish when there is a mix of IR-4 and
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non-IR-4 personnel utilizing the laboratory and sharing the chemicals or when the chemical is
stable and has a long shelf life. The following is to be used as a guide for meeting the labeling
requirement:
1) Identity can be the common name(s), CAS number or chemical name of the reagent or
reagents in solution or mixture.
2) If the labeling of the original container provides the identity, concentration, storage
requirements (if any) and expiration date or shelf life, no additional information is
needed. If the labeling does not contain this information, than a supplemental label
containing the missing information should be permanently attached to the container
where it does not obscure other critical information.
3) All mixtures of chemicals prepared by laboratory personnel for use in IR-4 trials should
have labels with the information as shown in 2 above.
4) Expiration dates for stable chemicals should be determined by the Laboratory Research
Director following methods outlined in their SOPs.
5) Adequate precautions should be taken to avoid contamination of reagents and solutions
so that purity of their content is preserved.

Sample Receipt, Processing and Storage
Maintaining a representative sample and maintaining sample integrity are the important
considerations to keep in mind during sample receipt storage, processing, and extraction/analysis
(see Appendix 1.).

Sample Receipt: Samples are generally received from either ACDS or Fed Ex. For receipt of
samples from an overnight air express carrier such as FedEx, it is critical that the lab know a
shipment is in transit. If the shipment is not received as expected, laboratory personnel will
follow-up to track the shipment.

When samples are received, laboratory personnel check the condition of the samples; verify
receipt of the correct samples by checking sample identification and matrices against the
shipping papers. Unique laboratory numbers are assigned and recorded with cross reference to
field samples IDs. Shipping forms (8B) received with the samples may be used to record the
cross reference or custom forms may be used. At a minimum, custom forms must contain the
same information required on the FDB forms, and must show that protocol conditions have been
met (for example, acknowledging that forms 8B and 8C were shipped with the samples). The
SD, RFC, and FRD are notified when samples are received, and any problems with the shipment
are noted.
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Sample Processing: Information regarding sample preparation, size and providing homogeneous
representative samples see Attachment 1. Great care is taken in the field to collect samples from
all areas of the plot, so that the sample is representative of the whole field. When processing the
samples, the total sample must be processed, and thoroughly mixed. If this is not possible,
guidance from the Study Director and/or Registration Manager must be sought. Sample integrity
is generally maintained by processing samples with dry ice. The study analytical data must
document how representative samples were prepared.

Storage: Storage of samples is in accordance with the protocol requirements and SOPs. To
prepare for the loss of power or a freezer failure, consideration should be given to the availability
of backup freezers and dry ice, generators (power backup) and spare parts. Temperature
monitors, alarms, and established lines of notification are methods for providing the LRD with
information on the temperature of the storage areas. For a longer term power outage, samples
may need to be transported to another location to maintain sample integrity. These samples
represent a significant investment and their integrity should be safeguarded accordingly.

Working Method, Validation and Modifications: IR-4 methods are provided initially by
the cooperating registrant (reference method). Given the number of commodities IR-4 works
with, it is likely that each method will require some modification to work effectively. It should
be noted that once these methods are modified for other commodities, these methods become the
enforcement method for EPA. Significant changes to the initial working method may trigger an
independent method validation (OPPTS 860.1340), and thus are not encouraged unless needed to
develop an adequate method for the specific matrix. The LRD should discuss significant
changes with the SD and/or gatekeeper prior to making the change.

Other considerations: Approval for significant changes to the reference method must be
requested from the SD, HQ gatekeeper and registrant. Depending on the number of proposed
changes and familiarity with the method, the laboratory should keep in mind that such changes
will need to be dealt with well in advance of analysis, so that when the samples are received
analysis may proceed without delay.

Extraction: In most cases the extraction solvent and procedures must remain the same as the
reference method. Sample weights and extraction volume must stay proportional to the original
method. Exchange of equipment can be made only when the equipment is basically carrying out
the same function as noted in the method (for example tissuemizer and polytron). Other
substitutions (from tissuemizer to shaker tray) should be discussed with the registrant providing
the reference method and in consultation with the SD (and the gatekeeper
1
at HQ).

1
The role of the Gatekeeper (J ohannes Corley, Debbie Carpenter, the Registration Manager, or Bill Barney) is to
provide greater consistency from IR-4 HQ by utilizing personnel with greater chemistry experience.
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Clean-up steps: EPA has noted that as long as the extraction procedures are the same, clean-up
steps maybe removed. However, this should be done in consultation with the registrant
providing the reference method. It should also be noted that removing an excessive number of
steps may result in excessive wear and tear on the column and instrument. The impacts of
removing clean-up steps from the method, such as matrix enhancement effects, must be
evaluated as chromatography must be clean and sharp. Modifications should be discussed with
the SD, IR-4 gate keeper
2
as well as the registrant so they can share their experiences.
Chemist should also consider cost and time relating to removal of cited clean-up steps.

Detector: Using LC/MS/MS has generally become the norm and essentially all of the IR-4
laboratories have at least one instrument. It is likely that any new equipment purchases will be
directed toward using this technology. Therefore, replacing the detectors noted in the reference
method with LC/MS/MS should have minimal effect on the method while providing better
quantitation and confirmation.

Working method approval and validation data: Current minimum protocol requirements
indicate that the LRD will send the SD the working method and recovery data from the method
validation. If the recovery data are within 70 to 120% then weathered sample analysis may
proceed. However, it is expected that the SD take an active role in this process and acknowledge
that the method and data are acceptable. If the recovery data are not within the 70 to 120%
range, the SD must acknowledge that he/she is aware the data are out of range and if the data are
acceptable. If the validation recoveries are within the 70 120% range it is at the discretion of
the LRD to request SD approval prior to analysis of the field (weathered) samples in order to
note SD responsibility. If study director approval is needed or requested, the study director
should make every effort to respond within 2 working days. Recognizing that study directors
have other responsibilities including traveling, the lab will need to provide time for the study
director to respond in these situations. For urgent needs, or situations where the SD is not able to
respond within 2 working days, approval to proceed may be sought from the gatekeeper,
J ohannes Corley, Debbie Carpenter, the registration manager, or Bill Barney. However, the SD
must provide approval when he/she becomes available.
Sample Analysis and Extracts
Sample Analysis: IR-4 laboratories generally agree that double injections for each weathered
sample should be used. If there is a study with a large number of samples, the LRD may
consider doing single injections: however, it should be noted that double injections provide a
number of benefits such as instrument stability and detection of bad injections in real time,
allowing the chemist to respond to situations more quickly and efficiently.

Laboratory personnel should be mindful when unusual results are obtained and notify the SD
immediately. (Lab personnel may want to re-extract and re-analyze samples to confirm prior to
notification of SD). Examples of unusual situations are samples that have no residues compared
to other weathered (field samples) samples from treated plots, or decline samples where no

2
The role of the Gatekeeper (J ohannes Corley, Debbie Carpenter, the Registration Manager, or Bill Barney) is to
provide greater consistency from IR-4 HQ by utilizing personnel with greater chemistry experience.
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residues are detected, samples from untreated control plots with residues or if residues from
samples taken from the same treated plot have measurable residues and the values for each
sample vary by a factor of 5X or more.

Extracts: Registrants are advised to routinely include the storage of extracts, unless their
standard laboratory practice is to analyze extracts on the same day as they are obtained
(860.1380). Always run samples with concurrent recoveries to demonstrate extract stability.

Storage Stability: IR-4 does not carry out guideline storage stability studies as outlined in
860.1380. Our purpose is to show the samples are stable under the storage conditions used.
Currently, storage stability, with analysis of one time point is carried out for most studies. For
many compounds, the registrant may have adequate storage stability data available. IR-4 is
working with EPA to determine this. It is hoped that eventually, fewer storage stability analyses
will be required. IR-4 will transition to fewer storage stability studies per year for each lab
working with the compound, but analyze the data at two time points. The first time point will be
when the method is validated (3 samples) and another after an appropriate storage period
(another 3 samples). A minimum of nine samples will be spiked at 10x LLMV. If the samples
cover 90% of the storage time (from sample date to extraction date), this is sufficient. Note that
the protocol requires study director notification and approval if the sample storage period does
not cover 100% of the storage interval.

Communication of Results with SD:
Response Needed to Proceed:
There may be instances where the lab needs to communicate study related activities to the study
director, and a response is needed to proceed. One example includes out of range recoveries. If
the recovery data are not within the 70 to 120% range, the SD must acknowledge that he/she is
aware the data are out of range, accepts the recoveries, and that the analysis may proceed. If
study director approval is needed or requested, the study director should make every effort to
respond within 2 working days. Recognizing that study directors have other responsibilities
including traveling, the lab will need to provide time for the study director to respond in these
situations. For urgent needs, or situations where the SD is not able to respond within 2 working
days, approval to proceed may be sought from the gatekeeper, J ohannes Corley, Debbie
Carpenter, the registration manager, or Bill Barney. However, the SD must provide approval
when he/she becomes available.

Routine Results: The LRD (or designate) will provide routine updates to the SD (e.g. residue
analysis spreadsheet, residue result summaries) on a regular basis, along with background
information and assessment of the data. The lab will decide the frequency of updates, based on
their own operations. At a minimum, it is expected that the residue results will be shared with
the study director as soon as possible, once all samples for the study have been analyzed.
Acknowledgment of their receipt from the study director is expected.

Analytical Summary Report: a sample ASR is provided in Attachment 2.
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Training. This document will be used as a training tool for new Laboratory
Coordinators, IR-4 chemists, QA officers and Study Directors.

Contract/Company Labs. This document may also be used as a tool to provide guidance for
contract and company laboratories used by IR-4 for residue analysis.

Guideline Document review: Target review is for every three years. Please note that significant
material has been taken from the Operational Handbook of IR-4 and updates to that document
will affect this document as well.

Explanation of Attachments:

Attachment 1: Guidelines for the Preparation of Raw Agricultural Commodity Samples
For Residue Analysis
This instructional guideline has been prepared to aid in insuring uniformity and
consistency among IR-4 analytical facilities when preparing raw agricultural
commodities (RAC) for Magnitude of the Residue determinations. The attachment
provides information regarding sample preparation, size and providing homogeneous
representative samples. Great care is taken in the field to collect samples from all areas
of the plot, so that the sample is representative of the whole field and this guideline will
help to insure that samples remain representative when processed in the IR-4 laboratories.

Attachment 2: Sample Analytical Summary Report.
This example report is provided to illustrate a typical IR-4 Analytical Summary Report
and the critical elements that must be in a report. The tables etc have been updated to
help aid final report preparation. For electronic copies of this example please contact Dr.
Matt Hengel, Western Regional Laboratory Coordinator, University of California

Attachment 3: Checklist for Review of Analytical Summary Reports
This checklist (version 1.0, 05/12/08) is being provided as reference informaiton to assist
in the internal quality evaluation of analytical data. The checklist can be used to identify
and insure that appropriate information is included in the final reports submitted to EPA.
The checklist identifies items which must be brought to the study directors attention in
order for the study director to carry out his/her responsibilities under GLP.

LABORATORY SAMPLE PROCESSING GUIDANCE DOCUMENT (v.4, 12/01/08)
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GUIDELINES FOR THE PREPARATION OF RAW AGRICULTURAL COMMODITY
SAMPLES FOR RESIDUE ANALYSIS

PURPOSE:

This instructional guideline has been prepared to aid in insuring uniformity and
consistency among IR-4 analytical facilities when preparing raw agricultural
commodities (RAC) for Magnitude of the Residue determinations.

This guideline contains general directions for:
obtaining in a safe manner homogeneous RAC sub-samples with minimum risk of
residue cross-contamination (General Procedures section A)
processing guidelines for specific crop groupings with specific instructions on
inspecting and what portion of the RAC is to be prepared for residue
determination (Guidelines for Determining Portion of RAC to be Analyzed"
section B)
uniform sample preparation and comminuting procedures (i.e., pulverizing/
reduce to powder) for whole and sub-sampled RACs ("Guidelines for Sample
Preparation section C)

Definitions of Terms Used in this Guideline:
Raw Agricultural Commodity
Fresh fruits, whether or not they have been washed and colored or
otherwise treated in their unpeeled natural form; vegetables in their raw or
natural state, whether or not they have been stripped of their outer leaves,
waxed, prepared into fresh green salads, etc.; grains, nuts, eggs, raw milk,
meats, and similar agricultural produce. It does not include foods that have
been processed, fabricated, or manufactured by cooking, freezing,
dehydrating, or milling (40 CFR 180)

Sample
The amount of individual agricultural commodity units (e.g. specific
number of fruits or tubers, a set weight of grain, etc.) randomly selected
from a plot which may be composited for pesticide analysis (OPPTS
860.1500)
PROCEDURE:

A. General Guidelines
Persons given responsibility for processing agricultural crops (Processor) will be fully
trained in properly processing agricultural commodities and also in the safe use of
processing equipment and cryogenic materials. Proper ventilation is mandatory when
working with cryogenic materials such as liquid nitrogen and carbon dioxide. It is the
responsibility of the Processor to immediately notify her/his immediate supervisor and/or
the Laboratory Research Director or if unsafe working conditions exist.

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Processing equipment often operate at high revolutions to pulverize/powder the RAC.
This equipment can be hazardous and should be routinely checked for proper operation
before processing agricultural commodities.

The sample should not be brushed, stripped, trimmed, or washed except to the extent that
these are commercial practices before shipment or to the extent allowable (see 40 CFR
180) or the Pesticide Assessment Manual (PAM). Details for cleaning or trimming
specific crop types are outlined under "Guidelines for Determining Portion of RAC to
be Analyzed" section B and Appendix 1. In each case, the protocol and Study
Director will be consulted to clarify any potential problems prior to sample
processing.

The total sample should be processed whenever feasible. If the sample size is too large to
process, a representative sub-sample of each component part should be taken (e.g., 1/4 of
each cantaloupe from the original residue sample bag for maceration). Sub-sampling of
the component parts will be done in a manner to represent the residue distribution to be
found on all surfaces of the whole vegetative part. Details for specific crop types are
outlined under "Guidelines for Sample Preparation section C. If sub-sampling must
occur, due to large sample size or unit size, the Study Director will be consulted
prior to sample processing.

The order in which samples are processed should be chosen to minimize the potential for
residue cross-contamination. For each trial location, untreated samples should always be
processed first. Treated samples with the lowest application rate and the longest pre-
harvest interval (PHI) should follow. Samples with the highest application rate and the
shortest PHI should be processed last. In addition, crop fractions should also be
considered (e.g. nut meat fractions should be processed before hull fractions).

If cryogenic materials are required, the pulverized sample can quickly liquefy and
separate at room temperature soon after processing. All attempts should be made to
immediately transfer the sample to a properly labeled sampling bag and place in frozen
storage.

Processing equipment should be thoroughly washed and rinsed with distilled water and
acetone or methanol before attempting to process the next sample irrespective if the next
sample is a replicate from the same treatment location or a replicated control sample.

B. Guidelines for Determining Portion of RAC to be Analyzed
40 CFR 180 specifies that the sample taken should be of the whole raw agricultural
commodity (RAC) as it moves through interstate commerce. In certain cases, the portion
to be analyzed for a residue tolerance may not represent the whole RAC. Instructions on
what portion of the RAC should be analyzed are provided for nine individual food
commodities (e.g., bananas) and crop group commodities (e.g., root vegetables) in this
regulatory guideline. To fill this void, the FDA has provided additional guidance for
RACs that fall under a more complete crop groupings list (see 40 CFR 180.34 (f)). The
portion of the sample to be analyzed as described under PAM Volume 1 takes into
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account practical considerations of sample preparation. Appendix 1 on page 4 (Table
102-a: Portion of Raw Agricultural Commodity to be Analyzed for Pesticide Residues)
provides a compilation of EPA regulations and FDA directions to be followed for RAC
preparation. If sample processing procedures for a particular RAC are not specified under
the above crop grouping guidelines, or in the protocol, additional guidance from the
Laboratory Research Director and IR-4 Study Director approval will be sought before
preparing samples for residue determination.

C. Guidelines for Sample Preparation
The relatively small 2.5 to 100 gram laboratory sample taken from the whole RAC must
represent the entire treated or control sample. Often these samples are bulky or can be
comprised of a few large units or many smaller items. Whenever feasible, the total RAC
sample should be pulverized and a homogeneous 2.5 to 100 gram sample taken to assure
uniformity. Processing the entire sample may not always be feasible. Guidelines are
provided below to aid in preparing representative residue determination samples from
bulky, large unit and many small item RAC samples. In addition to the guidelines below,
Table 1 offers examples of current processing practices of several commodities by IR-
4/ARS facilities.

Bulky Samples: For more bulky samples [i.e., Alfalfa (green and dry), Barley, Field Corn
(silage, stover), Sweet Corn (forage, husks), Clover Grass, Mint (hay), Oats (forage,
fodder, or straw), Rice (plants), Rye, Sorghum (plants), Soybean (plants), Sugar Cane
(green and/or dry) Tobacco (green, cured), and Wheat (forage, fodder, or straw)],
acquiring the relatively small laboratory sample usually consists of two steps. First, the
crop is chopped into smaller size fractions using either a chopping knife or scissors or
through use of a large capacity chopper/mixer/grinder such as a spinning bowl or vertical
chopper (ie: Hobart HCM-450, 84142, 84145, 84146, VCM-25, or equivalent). The
chopped sample is then frozen to a brittle consistency using either liquid nitrogen (LN
2
)
or dry ice. This frozen material is then processed to a fine consistency using a sample
grinder (ie: Hobart 4822 or equivalent). Alternatively the samples may be first broken or
chopped or into smaller size fractions as described above and then thoroughly processed
with a cryogen (LN
2
or dry ice) in a spinning bowl chopper/mixer, spinning blade food
processor (ie: Robot-Coupe. RSI-6V or 10B) or other food grinder/chopper

Sub-sampling: Typically, sub-sampling of bulky or heavy units is performed in the field
as directed by the Protocol. However, when there are physical limitations for the
laboratory processing of the whole sample due to mass or sample size, sub-sampling of
the component parts must be done in a manner that assures the residue distribution is
representative of the whole vegetative part. Laboratory sub-sampling should only be
performed by GLP trained staff and in consultation with the Study Director and or
Registration Manager. If absolutely necessary, this practice must be limited to special
circumstances and be conducted by properly trained staff that understands the importance
of maintaining a fully representative sub-sample and the risks of possible residue/cross
contamination and/or deterioration of the crop matrix. Some examples of representative
sub-sampling in the laboratory include:
Page 4 of 14
Taking a well mixed portion of a large sample of very small items (berries, nuts,
grain, and immature vegetables). This may be necessary due to sample capacity of
processing/milling/grinding equipment (i.e., small Hobart/Robot-Coupe choppers,
Tekmar Analytical Mills and other similar chopping/grinding devices). For
example, a well-mixed 1 kg sub-sample from the 5 kg composited RAC sample
bag of coffee beans can be pulverized by the Tekmar Analytical Mill to produce a
representative sample.
For larger items when ca.12 units may comprise the entire composited RAC
(melons, pineapples, squash, see CODEX, reference 3 and PAM section 120c),
of each unit can be separated and composited to produce a representative sample
for processing.
In preparing a homogeneous tree fruit sample, where 6 fruits from each of 4 trees
is recommended (CODEX, reference 3), of each unit can be separated and
composited to produce a representative sample for processing.
When the processing or chopping of samples results in rapid degradation or loss
of residues during storage, a representative sub-sample shall be processed just
prior to analysis. The crop unit number, crop unit size, and the number of analyses
will determine the amount of sample to process with dry ice for each analysis.

If there is too much sample bulk to add the entire sample all at once and sub-sampling is
not an option, process a portion of the sample, add addl. sample and cryogen (if using),
process and repeat until the chopper is full. Bulk bag and repeat processing until the
entire sample is chopped. Combine all chopped matrix in the bulk bag, mix well and
remove sample for analysis/storage.

Page 5 of 14
Table 1.
Crop Group
(Subgroup)
Number and Name
Representative
Commodities
Pre-Processing
Preparation
1

Processing
2
Commodities
1. ROOT AND TUBER
VEGETABLES
Carrot, potato, radish, and
sugar beet.
While inside IR4 bag and frozen break
up with a mallet into approx. 2 inch
pieces and mix to combine. If greater
than 10 pounds cut each unit in half,
returning opposite half to sample bag.
Continue until all can fit in chopper. If
tops are included, cut with an electric
knife. A heavy knife and hammer are
useful if sample is too hard.
Robot Coupe, Grinder or
Hobart with cryogen
Arracacha; arrowroot; artichoke, Chinese; artichoke,
J erusalem; beet, garden; beet, sugar; burdock, edible;
canna, edible; carrot; cassava, bitter and sweet;
celeriac; chayote (root); chervil, turnip-rooted; chicory;
chufa; dasheen (taro); ginger; ginseng; horseradish;
leren; parsley, turnip-rooted; parsnip; potato; radish;
radish, oriental; rutabaga; salsify; salsify, black; salsify,
Spanish; skirret; sweet potato; tanier; turmeric; turnip;
yam bean; yam, true.
1A. Root vegetables
subgroup
Carrot, radish, and sugar beet While inside IR4 bag and frozen break
pieces and mix to combine. If tops
are included, cut with an electric knife.
A heavy knife and hammer are useful
if sample is too hard.
Hobart with cryogen
Beet, garden; beet, sugar, burdock, edible; carrot;
celeriac; chervil, turnip-rooted; chicory; ginseng;
horseradish; parsley, turnip-rooted; parsnip; radish;
radish, oriental; rutabaga; salsify; salsify, black; salsify,
Spanish; skirret; turnip
1B. Root vegetables (except
sugar beet) subgroup
Carrot and radish While inside IR4 bag and frozen break
pieces and mix to combine. If tops
are included, cut with an electric knife.
A heavy knife and hammer are useful
if sample is too hard.
Hobart with cryogen
Beet, garden; burdock, edible; carrot; celeriac; chervil,
turnip-rooted; chicory; ginseng; horseradish; parsley,
turnip-rooted; parsnip; radish; radish, oriental; rutabaga;
salsify; salsify, black; salsify, Spanish; skirret; turnip.
1C. Tuberous and corm
vegetables subgroup
Potato While inside IR4 bag and frozen break
pieces and mix to combine
Hobart with cryogen
J erusalem; canna, edible; cassava, bitter and sweet;
chayote (root); chufa; dasheen (taro); ginger; leren;
potato; sweet potato; tanier; turmeric; yam bean; yam,
true
1D. Tuberous and corm
vegetables (except potato)
subgroup
Sweet potato While inside IR4 bag and frozen break
Hobart with cryogen
J erusalem; canna, edible; cassava, bitter and sweet;
chayote (root); chufa; dasheen (taro); ginger; leren;
sweet potato; tanier; turmeric; yam bean; yam, true
2. LEAVES OF ROOT AND
TUBER VEGETABLES
(HUMAN FOOD OR
ANIMAL FEED)
Turnip and garden beet or
sugar beet
up with a mallet into approx. 2 inch or
smaller pieces or cut with electric knife
and then thoroughly mix to combine.
Hobart with cryogen. If too
much sample bulk to add
all at once, process in
batches until chopper is full
as described in footnote 2.
Beet, garden; beet, sugar; burdock, edible; carrot;
cassava, bitter and sweet; celeriac; chervil, turnip-
rooted; chicory; dasheen (taro); parsnip; radish; radish,
oriental (daikon); rutabaga; salsify, black; sweet potato;
tanier; turnip; yam, true

3. BULB VEGETABLES Onion, green; and onion, dry
bulb
pieces and mix to combine or cut in ~
1in pieces
Hobart with cryogen (LN2
or dry ice)
Garlic; garlic, great-headed; leek; onion, dry bulb and
green; onion, Welsh; shallot
1
and
2
see footnotes at bottom of page 11
Page 6 of 14
Table 1, cont.
Crop Group
(Subgroup)
Number and Name
Representative
Commodities
Pre-Processing
Preparation
1

Processing
2
Commodities
4. LEAFY VEGETABLES
(EXCEPT BRASSICA
VEGETABLES)
Celery, head lettuce, leaf
lettuce, and spinach
or dry ice). If too much
sample bulk to add all at
once, process in batches
until chopper is full as
described in footnote 2.
Amaranth (Chinese spinach); arugula (roquette);
cardoon; celery; celery, Chinese; celtuce; chervil;
chrysanthemum, edible-leaved; chrysanthemum,
garland; corn salad; cress, garden; cress, upland;
dandelion; dock (sorrel); endive (escarole); fennel,
Florence; lettuce, head and leaf; orach; parsley;
purslane, garden; purslane, winter; radicchio (red
chicory); rhubarb; spinach; spinach, New Zealand;
spinach, vine; Swiss chard
4A. Leafy greens subgroup Head lettuce and leaf lettuce,
and spinach
or dry ice).
Amaranth; arugula; chervil; chrysanthemum, edible-
leaved; chrysanthemum, garland; corn salad; cress,
garden; cress, upland; dandelion; dock; endive; lettuce;
orach; parsley; purslane, garden; purslane, winter;
radicchio; spinach; spinach, New Zealand; spinach,
vine
4B. Leaf petioles subgroup Celery While inside IR4 bag and frozen break
or dry ice).
Cardoon; celery; celery, Chinese; celtuce; fennel,
Florence; rhubarb; Swiss chard
5. BRASSICA (COLE)
LEAFY VEGETABLES
Broccoli or cauliflower;
cabbage; and mustard greens.
May need to quarter lengthwise, using
opposite pieces prior to mixing to
reduce bulk.
Broccoli; broccoli, Chinese (gai lon); broccoli raab
(rapini); Brussels sprouts; cabbage; cabbage, Chinese
(bok choy); cabbage, Chinese (napa); cabbage,
Chinese mustard(gai choy); cauliflower; cavalo broccolo;
collards; kale; kohlrabi; mizuna; mustard greens;
mustard spinach; rape greens
5A.Head & Stem Brassica
subgroup
Broccoli or cauliflower and
cabbage
pieces and mix to combine. May need
to quarter lengthwise, using opposite
pieces prior to mixing to reduce bulk.
or dry ice).
Broccoli; broccoli, Chinese; brussels sprouts; cabbage;
cabbage, Chinese (napa); cabbage, Chinese mustard;
cauliflower; cavalo broccolo; kohlrabi
5B.Leafy Brassica greens
subgroup
Mustard greens While inside IR4 bag and frozen break
pieces and mix to combine or cut with
an electric knife.
or dry ice).
Broccoli raab; cabbage, Chinese (bok choy); collards;
kale; mizuna; mustard greens; mustard spinach; rape
greens
Page 7 of 14
Table 1, cont.
Crop Group
(Subgroup)
Number and Name
Representative
Commodities
Pre-Processing
Preparation
1

Processing
2
Commodities
6. LEGUME VEGETABLES
(SUCCULENT OR DRIED)
Bean ( Phaseolus),(succulent
& dried),pea (Pisum)
(succulent & dried) and
soybean
Pre-processing not required. Robot Coupe, Grinder or
or dry ice)

For dried peas/beans -
grinder type processor,
coffee grinder or Robot
Coupe
Bean (Lupinus) (includes grain lupin, sweet lupin, white
lupin, and white sweet lupin); bean (Phaseolus)
(includes field bean, kidney bean, lima bean, navy bean,
pinto bean, runner bean, snap bean, tepary bean, wax
bean); bean (Vigna) (includes adzuki bean, asparagus
bean, blackeyed pea, catjang, Chinese longbean,
cowpea, crowder pea, moth bean, mung bean, rice
bean, southern pea, urd bean, yardlong bean); broad
bean (fava); chickpea (garbanzo); guar; jackbean;
lablab bean; lentil; pea (Pisum) (includes dwarf pea,
edible-podded pea, English pea, field pea, garden pea,
green pea, snowpea, sugar snap pea); pigeon pea;
soybean; soybean (immature seed); sword bean
6A.Edible-podded legume
vegetables subgroup
Any one succulent cultivar of
edible-podded bean
(Phaseolus) and any one
succulent cultivar of edible-
podded pea (Pisum)
Pre-processing not required Robot Coupe, Grinder or
or dry ice).

For dried peas/beans -
grinder type processor,
Coupe
Bean (Phaseolus) (includes runner bean, snap bean,
wax bean); bean (Vigna) (includes asparagus bean,
Chinese longbean, moth bean, yardlong bean);
jackbean; pea (Pisum) (includes dwarf pea, edible-
podded pea, snow pea, sugar snap pea); pigeon pea;
soybean (immature seed); sword bean
6B.Succulent shelled pea
and bean subgroup
Any succulent shelled cultivar
of bean (Phaseolus) and
garden pea (Pisum)
or dry ice)
Bean (Phaseolus) (includes lima bean, green; broad
bean, succulent); bean (Vigna) (includes blackeyed pea,
cowpea, southern pea); pea (Pisum) (includes English
pea, garden pea, green pea); pigeon pea
6C.Dried shelled pea and
bean (except soybean)
subgroup
Any one dried cultivar of bean
(Phaseolus) and any one dried
cultivar of pea (Pisum)
Pre-processing not required Grinder type processor,
Coupe with cryogen (LN2
or dry ice)
Dried cultivars of bean (Lupinus); bean (Phaseolus)
(includes field bean, kidney bean, lima bean (dry), navy
bean, pinto bean, tepary bean); bean (Vigna) (includes
adzuki bean, blackeyed pea, catjang, cowpea, crowder
pea, moth bean, mung bean, rice bean, southern pea,
urd bean); broad bean (dry); chickpea; guar; lablab
bean; lentil; pea (Pisum) (includes field pea); pigeon
pea

7. FOLIAGE OF LEGUME
VEGETABLES
Any cultivar of bean
(Phaseolus), field pea (Pisum)
and soybean
or dry ice)

Plant parts of any legume vegetable included in the
legume vegetables that will be used as animal feed.
7A.Foliage of legume
vegetables (except
soybeans) subgroup
Any cultivar of bean
(Phaseolus) and field pea
(Pisum)
or dry ice)

Plant parts of any legume vegetable (except soybeans)
included in the legume vegetables group that will be
used as animal feed.
Page 8 of 14
Table 1, cont.
Crop Group
(Subgroup)
Number and Name
Representative
Commodities
Pre-Processing
Preparation
1

Processing
2
Commodities
8. FRUITING VEGETABLES
(EXCEPT CUCURBITS)
Tomato, bell pepper, and one
cultivar of non-bell pepper
pieces and mix to combine or chop
with a knife.
or dry ice).
Eggplant; groundcherry (Physalis spp); pepino; pepper
(includes bell pepper, chili pepper, cooking pepper,
pimento, sweet pepper); tomatillo; tomato
9. CUCURBIT
VEGETABLES
Cucumber, muskmelon, and
summer squash

Chop entire fruit including seeds and
rind.
or dry ice).
Chayote (fruit); Chinese waxgourd (Chinese preserving
melon); citron melon; cucumber; gherkin; gourd, edible
(includes hyotan, cucuzza, hechima, Chinese okra);
Momordica spp (includes balsam apple, balsam pear,
bittermelon, Chinese cucumber); muskmelon (includes
cantaloupe); pumpkin; squash, summer; squash, winter
(includes butternut squash, calabaza, hubbard squash,
acorn squash, spaghetti squash); watermelon
9A.Melon subgroup Cantaloupe While inside IR4 bag and frozen break

rind.
or dry ice).
Citron melon; muskmelon; watermelon
9B. Squash/Cucumber
subgroup
One cultivar of summer squash
and cucumber

rind.
or dry ice).
Chayote (fruit); Chinese waxgourd; cucumber; gherkin;
gourd, edible; Momordica spp; pumpkin; squash,
summer;squash, winter
10. CITRUS FRUITS Sweet orange, lemon and
grapefruit
or dry ice).
Calamondin; citrus citron; citrus hybrids (includes
chironja, tangelo, tangor); grapefruit; kumquat; lemon;
lime; mandarin (tangerine); orange, sour; orange, sweet;
pummelo; Satsuma mandarin
11. POME FRUITS Apple and pear While inside IR4 bag and frozen break
up with a mallet into approx. 1 to 2
inch pieces and mix to combine. May
need to quarter lengthwise, using
opposite pieces prior to mixing to
reduce bulk.

peel.
or dry ice).
Apple; crabapple; loquat; mayhaw; pear; pear, oriental;
quince
Page 9 of 14
Table 1, cont.
Crop Group
(Subgroup)
Number and Name
Representative
Commodities
Pre-Processing
Preparation
1

Processing
2
Commodities
12. STONE FRUITS Sweet or tart cherry, peach,
and plum or fresh prune
Pre-processing not required. May
need to be pitted or cut into smaller
pieces.
or dry ice)
Apricot; cherry, sweet; cherry, tart; nectarine; peach;
plum; plum, Chickasaw; plum, Damson; plum,
J apanese; plumcot; prune (fresh)
13. BERRIES Any one blackberry or any one
raspberry; and blueberry
Pre-processing typically not required.
If larger than 1 to 2 in cut into smaller
pieces.
or dry ice).
A coffee grinder can be
used for small sample
sizes.
Blackberry (including bingleberry, boysenberry;
dewberry; lowberry, marionberry, olallieberry,
youngberry); blueberry; currant; elderberry; gooseberry;
huckleberry; loganberry; raspberry, black and red
13A.Caneberry (blackberry
and raspberry) subgroup
Any one blackberry or any one
raspberry
or dry ice).
Blackberry; loganberry; red and black raspberry;
cultivars and/or hybrids of these
13B. Bushberry subgroup Blueberry, highbush Pre-processing not required Robot Coupe, Grinder or
or dry ice).
Blueberry, highbush and lowbush; currant; elderberry;
gooseberry; huckleberry
14.TREE NUTS Almond and pecan Pre-processing typically not required.
Nut meat may need to be separated.
or dry ice).
sizes.
Almond; beech nut; Brazil nut; butternut; cashew;
chestnut; chinquapin; filbert (hazelnut); hickory nut;
macadamia nut; pecan; walnut, black and English
15. CEREAL GRAINS Corn (sweet and field), rice,
sorghum, and wheat
Pre-processing not required Wiley mill, coffee grinder or
Robot Coupe or with
cryogen (LN2 or dry ice).
Barley; buckwheat; corn; millet, pearl; millet, proso; oats;
popcorn; rice; rye; sorghum (milo); teosinte; triticale;
wheat; wild rice
16.FORAGE, FODDER AND
STRAW OF CEREAL
GRAINS
Corn, wheat, and any other
cereal grain crop
Use an electric knife if needed.
smaller Hobart with
cryogen (LN2 or dry ice)
Forage, fodder, and straw of all commodities included in
the cereal grains group
17.GRASS FORAGE,
FODDER, AND HAY
GROUP
Bermuda grass; bluegrass; and
bromegrass or fescue
Any grass, Gramineae family (either green or cured)
except sugarcane and those included in the cereal
grains group, that will be fed to or grazed by livestock,
all pasture and range grasses and grasses grown for
hay or silage
Page 10 of 14
Table 1, cont.
Crop Group
(Subgroup)
Number and Name
Representative
Commodities
Pre-Processing
Preparation
1

Processing
2
Commodities
18.NONGRASS ANIMAL
FEEDS (FORAGE,
FODDER, STRAW AND
HAY)
Alfalfa and clover (Trifolium) While inside IR4 bag and frozen break
or dry ice).
Alfalfa; bean, velvet; clover (Trifolium, Melilotus); kudzu;
lespedeza; lupin; sainfoin; trefoil; vetch; vetch, crown;
vetch, milk
19.HERBS AND SPICES Basil (fresh & dried); black
pepper; chive; hop cones; and
celery seed or dill seed
or dry ice).
For hops keep dry ice to a
minimum and do not leave
hops in chopper too long.
Allspice; angelica; anise; anise, star; annatto (seed);
balm; basil; borage; burnet; camomile; caper buds;
caraway; caraway, black; cardamom; cassia bark;
cassia buds; catnip; celery seed; chervil (dried); chive;
chive, Chinese; cinnamon; clary; clove buds; corainder
leaf (cilantro or Chinese parsley); coriander seed
(cilantro); costmary; culantro (leaf); culantro (seed);
cumin; curry (leaf); dill (dillweed); dill (seed); fennel
(common); fennel, Florence (seed); fenugreek; grains of
paradise, hop cones; horehound; hyssop; juniper berry;
lavender; lemongrass; lovage (leaf); lovage (seed);
mace; marigold, marjoram; mustard (seed); nasturtium;
nutmeg; parsley (dried); pennyroyal; pepper, black;
pepper, white; poppy (seed); rosemary; rue; saffron;
sage; savory, summer and winter; sweet bay; tansy;
tarragon; thyme; vanilla; wintergreen; woodruff;
wormwood
19A.Herb subgroup Basil (fresh & dried) and chive Pre-processing typically not required.
or dry ice).
sizes.
Angelica; balm; basil; borage; burnet; camomile; catnip;
chervil (dried); chive; chive, Chinese; clary; coriander
(leaf); costmary; culantro (leaf); curry (leaf); dillweed;
horehound; hyssop; lavender; lemongrass; lovage (leaf);
marigold; marjoram; nasturtium; parsley (dried);
pennyroyal; rosemary; rue; sage; savory, summer and
winter; sweet bay; tansy; tarragon; thyme; wintergreen;
woodruff; and wormwood
19B.Spice subgroup Black pepper; and celery seed
or dill seed
Pre-processing not required Wiley mill, coffee grinder or
Robot Coupe or with
cryogen (LN2 or dry ice).
Allspice; anise (seed); anise, star; annatto (seed); caper
(buds); caraway; caraway, black; cardamom; cassia
(bark); cassia (buds);celery (seed); cinnamon; clove
(buds); coriander (seed); culantro (seed); cumin; dill
(seed); fennel, common; fennel, Florence (seed);
fenugreek; grains of paradise; juniper (berry); lovage
(seed); mace; mustard (seed); nutmeg; pepper, black;
pepper, white; poppy (seed); saffron; and vanilla
TROPICAL FRUIT CROPS
Grapefruit
grapefruit, punimelo, and their
citrus hybrids (including
Uniq(Ugli) fruit)
or dry ice).
Corresponds to Codex Citrus Fruits Definitions
Page 11 of 14
Table 1, cont.
Crop Group
(Subgroup)
Number and Name
Representative
Commodities
Pre-Processing
Preparation
1

Processing
2
Commodities
Sugar Apple sugar apple, cherimoya,
atemoya, custard apple ilama,
soursop, biriba
or dry ice).
All crops in the Annonaceae; similar gross morphology;
inedible peel
Lychee lychee, longan, Spanish lime,
rambutan, pulasan
or dry ice).
All crops in the Sapindaceae; inedible peel
Papaya papaya, star apple, black
sapote, mango, sapodilla,
canistel, mamey sapote
or dry ice).
Make sure seeds are
chopped.
All crops have inedible peel; corresponds to Codex
classification
Avocado avocado, papaya, star apple,
black sapote, mango,
sapodilla, canistel, mamey
sapote
or dry ice).
All crops have inedible peel; corresponds to Codex
classification
Guava guava, feijoa, jaboticaba, wax
jambu, starfruit, passionfruit,
acerola
or dry ice).
Primarily edible peel; note/peel rarely contaminates
Passiflora spp. during juicing
Citrus Fruits add White sapote (Casimiroa),
and other cultivars and/or
hybrids of these
or dry ice).
White sapote is in the Rutaceae (citrus)

1.
Typical pre-processing tools include, but are not limited to: mallet, hammer, hatchet, cleaver, heavy knife, ginzu type knife, scissors, electric knife, and paper cutter. Caution must be taken
when attempting to break samples with mallets while in the IR-4 bags. The sample bag may break. A secondary bag may be used to contain the pieces. Be aware that there may be a
possibility of sample contamination with slivers of the bag/plastic lining. Alternatively, break-up of difficult frozen items using a heavy bladed knife, cleaver or heavy hammer/ mallets (2.5-
4lb) may be done on a chopping board lined with butcher paper with the edges folded up to contain sample pieces. Care must be exercised when using metal knives, choppers or
hammers that pieces do not cause personal injury in the event of breakage.
2.
Use of serrated S-blades will improve chopping efficiency of Robot Coupe Systems when processing fibrous and hard sample matrices including green coffee bean, roasted coffee beans,
and lychee whole fruit (with seed). Use of the Pulse or High speed (~3600 rpm) option for variable speed models is recommended for these difficult frozen matrices. A coffee grinder is
useful for dry seeded samples. If there is too much sample bulk to add the entire sample all at once and sub-sampling is not an option, process a portion of the sample, add addl. sample
and cryogen (if using), process and repeat until chopper is full. Bulk bag and repeat processing until entire sample is chopped. Combine all chopped matrix in bulk bag, mix well and
remove sample for analysis/storage.
Page 12 of 14
Appendix 1: FromPesticide Assessment Manual (PAM) Volume 1, 3
rd
Edition

Page 13 of 14
Appendix 1 (cont)

Page 14 of 14
References Cited:

1. 40 CFR 180.1 Tolerances And Exemptions From Tolerances For Pesticide Chemicals
In Food. Subpart A(j) Definitions and Interpretative Regulations

2. Codex Guidelines on Minimum Sample Sizes for Agricultural Commodities from
Supervised Field Trials for Residue Analysis, ALINORM 87/24A (1987)

3. Codex Alimentarius Volume 2 Pesticides Residues In Food Section 2 Codex
Classification Of Foods And Animal Feedstuffs. FAO, Rome 1993

4. Pesticide Assessment Manual (PAM) Volume 1, 3
rd
Edition, Section 102 and Section
203.

5. Residue Chemistry Test Guidelines OPPTS 860.1500 Crop Field Trials

ANALYTICAL SUMMARY REPORT
Acequinocyl: Magnitude of the Residue on Tomato

PR# 08356

Author(s)
Gregory L. Hall
Laboratory Research Director
Matt Hengel

Testing Facility
IR-4 Western Region Laboratory
Department of Environmental Toxicology
One Shields Ave.
University of California, Davis
Davis, CA 95616-5270

Laboratory ID# 08356.03-CAR02

Study Director
Van Starner
Sponsor
Interregional Research Project #4 (IR-4)
IR-4 Project Headquarters
500 College Road East, Suite 201 W
Princeton, New J ersey 08540

Field ID. Numbers
08356.03-TX08 08356.03-CA18 08356.03-CA19
08356.03-CA21 08356.03-CA22 08356-CA23
08356.03-NJ 06 08356.03-TXP04

Study Timetable
Study Initiation Date: 01/07/03
Experimental Termination Date: 04/13/04

Report Date
06/16/04
Page 1 of 154
GLP STANDARDS COMPLIANCE STATEMENT

PR#: 08356 Lab ID#: 08356.03-CAR02

The study reported herein for residues of Acequinocyl on Tomato was conducted and reported in
compliance with GLP Title 40, part 160 of the Code of Federal Regulations of the United States
of America. With the exception of the following:

The reference substance was characterized by Arvesta, and all the characterization
information is retained by them. No characterization of the reference substance was
performed by the IR-4 Western Region Laboratory.

Signature/Date Signature/Date
Matt Hengel Gregory L. Hall
Laboratory Research Director Analyst

IR-4 Western Region Laboratory IR-4 Western Region Laboratory
Department of Environmental Toxicology Department of Environmental Toxicology
One Shields Ave. One Shields Ave.
University of California, Davis University of California, Davis
Davis, CA 95616-5270 Davis, CA 95616-5270
Tel. No.: (530) 752-2402 Tel. No.: (530) 752-4742
IR-4 Western Region Laboratory, University of California, Davis Analytical Summary Report PR# 08356
2
QA STATEMENT
3
LABORATORY PERSONNEL

Name Designation

Marion Miller IR-4 Western Region Director

Matt Hengel Laboratory Research Director

Gregory L. Hall Principal Analyst
J o Engebretson Assistant to the Analyst
Riza Punongbayan Assistant to the Analyst

Bronson Hung Sample Control Officer
Chava Torres Asst. to Sample Control Officer

Bronson Hung Subsampling
Chava Torres Subsampling

Riza Punongbayan Report Preparation
4
TABLE OF CONTENTS
Page

GLP Compliance Statement..........................................................................................2

Quality Assurance Statement.........................................................................................3

Laboratory Personnel ....................................................................................................4

Table of Contents...........................................................................................................5

Archives (location of raw data).....................................................................................6

Summary
I. Objective/Introduction......................................................................................7
II. Sample Inventory/History................................................................................7
Table II.1 ......................................................................................................................8
III. Preparation of Storage Stability Study.............................................................9
Table III.1: Acequinocyl ..............................................................................................9
Table III.2: Acequinocyl-OH.......................................................................................9
IV. Standard Preparation........................................................................................9
V. Analytical Procedure.......................................................................................10
VI. Quantitation.....................................................................................................12
VII. Results and Discussion.................................................................................14
Table VII.1.1: Summary of Recoveries, Acequinocyl.....................................15
Table VII.1.2: Summary of Recoveries, Acequinocyl-OH..............................16
Table VII.2.1: Summary of Storage Stability, Acequinocyl ............................18
Table VII.2.2: Summary of Storage Stability, Acequinocyl-OH.....................18
Table VII.3.1: Residue Data Results, Acequinocyl and Acequinocyl-OH......19
Table VII.4: Summary of Residue Data from Crop Field Trial .................20

ATTACHMENTS
A. Index to Representative Chromatograms........................................................21
B. Data Summaries and Standard Curves............................................................75
C. Standard Use Forms.......................................................................................137
D. Certificates of Analysis..................................................................................151

All Chromatograms, Data Summaries, Standard Curves, Standard Use Form, and Certificates of Analysis
are True Copies reduced to 90% of the original size.

5
LOCATION OF RAW DATA

Original raw data, a certified copy of the signed protocol, amendments, correspondence logs and
all relevant information for the study titled: Acequinocyl: Magnitude of the Residue on
Tomato, PR#08356 along with a certified copy of the signed analytical summary report will be
maintained in the archives of the testing facility. The original copy of the analytical summary
report will be forwarded to the sponsor.

Portions of the field samples will be retained at the testing facility in a freezer at less than 5C
for at least 12 months after submission of the laboratory report. The long term storage stability
samples will be stored for at least 5 years at less than 5C. The study director will be consulted
before the field samples or the storage stability samples are discarded.

Laboratory Research Director: Matt Hengel

Testing Facility: IR-4 Western Region Laboratory
Department of Environmental Toxicology
One Shields Ave.
University of California, Davis
Davis, CA 95616-5270

6

IR-4 NATIONAL PESTICIDE CLEARANCE RESEARCH PROGRAM
ANALYTICAL SUMMARY REPORT PR#08356:
ACEQUINOCYL/TOMATO

I. Objective/Introduction

At the request of IR-4 Headquarters, the Western Region Leader Laboratory at University
of California Davis (UCD) has assayed tomato for residues of acequinocyl (EPA Reg.
No. Pending, CAS#57960-19-7) to provide data to support the establishment of a
pesticide tolerance. The method used in this study was derived from Determination of
Acequinocyl and Acequinocyl-OH in Almond Nutmeats and Almond Hulls, Analytical
Method#Meth-135, Original, October 19, 2001, Morse Laboratories, Inc. Steps where
the UCD working method significantly diverges from the registrants method are noted in
Section V. 1.0 Modifications to Referenced Analytical Method. The study followed
IR-4 National Pesticide Clearance Laboratory Phase Protocol PR#08356 as amended.
The validated method sensitivity is 0.01 ppm acequinocyl and 0.025 ppm for the matrices
of fruit, puree and paste.

II. Sample Inventory/History

Upon arrival at the laboratory, samples were opened, inspected, and checked against the
enclosed shipping form. Unique laboratory ID numbers were assigned as listed in Table
II.a. Samples were stored frozen. Raw Agricultural Commodity (RAC) samples were
subsampled by Hobart chopping with equal amounts of dry ice. After the entire sample
was chopped, a portion was placed in a labeled glass quart jar and surplus, if any, was put
back into the sample bag and both were stored frozen (generally <20C). Processed
sample cans (1 per treatment) were opened and their contents transferred to labeled glass
pint jars and stored frozen.

7
Table II.1: Sample Inventory

Field
Trial
Matrix Field Rep
No.
IR-4 Lab# Sampling Date
Lab Receipt
Date
Subsampling
Date
A 16059 02/19/03 02/26/03 02/26/03
B 16060 02/19/03 02/26/03 02/26/03
C 16061 02/19/03 02/26/03 02/27/03
TX08 Fruit
D 16062 02/19/03 02/26/03 02/27/03
A 16446 10/15/03 10/16/03 10/22/03
B 16447 10/15/03 10/16/03 10/22/03
C 16448 10/15/03 10/16/03 10/22/03
CA18 Fruit
D 16449 10/15/03 10/16/03 10/22/03
A 16136 06/11/03 07/16/03 07/24/03
B 16137 06/11/03 07/16/03 07/24/03
C 16138 06/11/03 07/16/03 07/25/03
CA19 Fruit
D 16139 06/11/03 07/16/03 07/25/03
A 16450 10/14/03 10/16/03 10/22/03
B 16451 10/14/03 10/16/03 10/22/03
C 16452 10/14/03 10/16/03 10/22/03
CA21 Fruit
D 16453 10/14/03 10/16/03 10/23/03
A 16162 07/23/03 08/08/03 08/19/03
B 16163 07/23/03 08/08/03 08/19/03
C 16164 07/23/03 08/08/03 08/25/03
CA22 Fruit
D 16165 07/23/03 08/08/03 08/25/03
A 16140 06/12/03 07/16/03 07/25/03
B 16141 06/12/03 07/16/03 07/25/03
C 16142 06/12/03 07/16/03 07/25/03
CA23 Fruit
D 16143 06/12/03 07/16/03 07/25/03
A 16079 04/14/03 06/11/03 06/16/03
B 16080 04/14/03 06/11/03 06/16/03
C 16081 04/14/03 06/11/03 06/19/03
D 16082 04/14/03 06/11/03 06/19/03
E 16083 04/15/03 06/11/03 06/18/03
F 16084 04/15/03 06/11/03 06/18/03
G 16085 04/17/03 06/11/03 06/17/03
H 16086 04/17/03 06/11/03 06/17/03
I 16087 04/21/03 06/11/03 06/16/03
NJ 06 Fruit
J 16088 04/21/03 06/11/03 06/17/03
Fruit F 16462 10/20/03 11/14/03 11/20/03
Paste G 16463 10/22/03 11/14/03 At Extraction
Puree H 16464 10/21/03 11/14/03 AtExtraction
Fruit J 16465 10/22/03 11/14/03 11/20/03
Paste K 16466 10/27/03 11/14/03 AtExtraction
TXP-04
(CA21)
Puree L 16467 10/24/03 11/14/03 AtExtraction

8
III. Preparation of Storage Stability Samples

Table III.1: Preparation of Storage Stability Samples, Acequinocyl
Field
Trial
Field
Rep
No.
Crop
Part
No.
Prepared
Sample
Size
(g)
Std #. Conc.
g/mL
L
Added
g
Added
Fort.
Level
ppm
Date
Fortified
TX08 A Fruit 6 40 286-1S1 20 200 5 0.1 02/27/03
TXP04 G Paste 6 10 286-2S1 50 200 5 0.1 11/18/03
TXP04 H Puree 6 10 286-2S1 50 200 5 0.1 11/18/03

Table III.2 Preparation of Storage Stability Samples, Acequinocyl-OH
Field
Trial
Field
Rep
No.
Crop
Part
No.
Prepared
Sample
Size
(g)
Std #. Conc.
g/mL
L
Added
g
Added
Fort.
Level
ppm
Date
Fortified
TX08 A Fruit 6 40 287-1S1 20 200 5 0.1 02/27/03
TXP04 G Paste 6 10 287-2S1 50 200 5 0.1 11/18/03
TXP04 H Puree 6 10 287-2S1 50 200 5 0.1 11/18/03

Note: All Samples were weighed into 8 oz. glass jars stored in the dark at generally <-20 C.

IV. Standard Preparation

Stock Solutions:
Typically, 10.0 mg (corrected for purity) of each analytical standard is accurately
weighed and quantitatively transferred to a separate 50 mL volumetric flask. Acequinocyl
is brought to volume with acetonitrile and Acequinocyl-OH is brought to volume with
acetone. The resulting concentration of each solution is 200 g/mL. These solutions are
to be stored in the dark at 1 to 8 C when not in use.

Fortification Standards:
Typically the following concentrations of Acequinocyl and Acequinocyl-OH are
prepared. Suitable mixtures may be prepared accordingly. All solutions are stored in the
dark at 1 to 8 C when not in use.

100 g/mL: Transfer 12.5 mL of 200 g/mL standard solution to a 25 mL volumetric
flask. Bring to volume in acetonitrile. Mix well.

10 g/mL: Transfer 2.5 mL of 100g/mL standard solution to a 25 mL volumetric flask.
Bring to volume with acetonitrile. Mix well.

1 g/mL: Transfer 250 L of 100 g/mL standard solution to a 25 mL volumetric flask.
Bring to volume in acetonitrile. Mix well.
9

HPLC (Calibration) Standard Solutions:
All standard solutions prepared in this section are to be stored in the dark at 1 to 8 C
when not in use. Typically the following concentrations of HPLC standard solution
mixtures are prepared:

0.5 g/mL: Transfer 1250 L of 10 g/mL standard solution to a 25 mL volumetric flask.
Bring to volume in acetone:acetonitrile:0.4% aqueous formic acid (2:2:1, v/v). Mix well.


0.10g/mL: Transfer 250 L of 10 g/mL standard solution to a 25 mL volumetric flask.


V. Analytical Procedure

1.0 Modifications to the Referenced Analytical Method:
For Tomato Fruit and Puree
1. Used 2x100mL hexane extract instead of 2x150mL. Because some aqueous was
present, solids were not rinsed into the filter at extraction.
2. Used TurboVap nitrogen evaporator instead of rotary evaporator.
3. Eliminated acetonitrile/hexane partition.
4. Increase Silica SPE to 1gm size to increase capacity, and adjusted column patterns to
give separate fractions for parent and metabolite.
5. Eliminated unnecessary GPC cleanup.
6. Substituted PFP Propyl for Phenyl-Hexyl HPLC column, and adjusted gradient
appropriately.

In addition for Tomato Paste only:
7. Use 5 gm Silica SPE to increase capacity, and adjust column patterns to give separate
fractions for parent and metabolite.

2.0 Extraction: (For tomato fruit, puree or paste)
2.1 Weigh 10.0 grams of the semi-frozen sample into a 250 mL aluminum wrapped
polyethylene centrifuge bottle. (Fortify at this point for recovery samples).
Note: All samples should be kept in at least a semi-frozen state until the addition of
the first extraction solvent and the extraction process begins. Acequinocyl and
Acequinocyl-OH are very sensitive to light. Glassware used during analysis should
be protected with aluminum foil or use amber/dark glassware. Specific glassware
includes any items in which there may be lengthy extract storage (>30 minutes)
10
such as: evaporation flasks, mixing cylinders, separatory funnels, test tubes. Cover
evaporator to exclude light when concentrating samples.
2.2 Add 20 grams of hexane-saturated anhydrous sodium sulfate.
2.3 Add 100 mL of hexane.
2.4 Blend using high-speed homogenizer at medium speed for 2 minutes, and allow
solids to settle for a few minutes.
2.5 Centrifuge for 15 minutes on maximum, after balancing with hexane.
2.6 Decant the hexane extract through a #541 filter paper (contained in a 100mm filter
funnel) into 250mL Turbovap tube.
2.7 Repeat with a second 100mL of hexane, centrifuge and combine.
2.8 Add 0.5 mL of 1% keeper to hexane extract.
2.9 Evaporate at approximately 30C to approximately 5mL on a Turbovap evaporator.

3.0 SPE Column for Tomato Fruit and Puree:
3.1 Run SPE, Varian Bond Elute-Si, 1 gm, Part #12256008 on each sample.
3.2 Condition SPE cartridge with 5 mL hexane.
3.3 Pass hexane extract ( 5mL) through the SPE cartridge.Note: 2-3 drops/sec. Stop
with solvent at top of frit.
3.4 Wash Turbovap tube two times with 5 mL of hexane, swirling to rinse walls of tube
and pass rinse through sample-laden cartridge.
3.5 Wash cartridge with 10mL hexane & discard.
3.6 Wash cartridge with 15mL hexane/ethyl ether (49:1, v/v) and discard.
3.7 Elute SPE with 10mL hexane/ethyl ether (48:2,v/v), collect (Acequinocyl).
3.8 Elute SPE with 15-20 mL hexane/ethyl acetate (9:1, v/v), collect (Acequinocyl-
OH)*
*Note: Volume of acequinocyl-OH eluate collected depends on matrix and SPE column lot variability.
Collect Acequinocyl-OH eluate in two tubes (a 10mL fraction & a 5-10mL fraction) that will be
combined later into one. Add 0.2 mL of 1% keeper solution to the first 10mL test tube and begin
to evaporate at approx 30C, adding the second 5-10mL cut, rinsing the tube and pooling the
cuts.

4.0 SPE Column for Tomato Paste:
4.1. Run Bond Elute-Si, 5gm (Varian Part #1225-6026).
4.2. Pre-wash Silica SPE column with 15 mL hexane.
Note: 2-3 drops/sec. Stop with solvent at top of frit.
4.3. Place sample on Si column, rinse and discard.
4.4. Wash Turbovap tube with 15 mL of hexane while swirling to rinse walls of tube.
Pass rinse through sample-laden cartridge and discard.
4.5. Rinse column with 40 mL hexane and discard.
4.6. Wash cartridge with 100 mL 98% hexane/ethyl ether (49:1, v/v) and discard.
4.7. Elute SPE with 60 mL 98% hexane/ethyl ether (49:1, v/v) and collect
(Acequinocyl). Note: Collect eluate in 40ml tube and pool the two collections in
Turbovap tube.
4.8. Elute SPE with 40 mL hexane/ethyl acetate (9:1, v/v) and collect (Acequinocyl-
OH). Note: Collect eluate in 40ml tube, transfer to a Turbovap tube and rinse with
hexane.
4.9. Add 0.4 mL keeper to each Turbovap tube.
Note: Cover sub-samples with aluminum foil until ready for analysis.
11
5.0 Sample Concentration:
5.1. Evaporate to dryness, with care, at approx 30C.
5.2. Redissolve residue in 0.4 mL acetonitrile. Add 0.4 mL of acetone and mix well.
5.3. Sonicate to insure that all residue is either dissolved or in suspension.
5.4. Finally add 0.2 mL of 0.4% aqueous formic acid and mix. The combination and
proportions of solvents used results in a mixture of acetone:acetonitrile:0.4%
aqueous formic acid (2:2:1, v/v). Mix well and submit to HPLC analysis. Final
concentration of HPLC-ready extract is 1.0 mL =10.0 g. Dilute with 2:2:1 (v/v) if
necessary.
5.5. If necessary, filter through 0.45 um filter with nylon and GF, into sample vial.
Cover sub-samples with aluminum foil until ready for analysis.
For Example:
(500mg injected =10gm/1ml x 50l), (5.0ng=0.10ng/l x 50l)
LOQ=0.010g/g=ng/mg=(5ng/500mg)
LOD=0.005g/g=(0.05ng/l x 50l)/(10gm/1mlx50l)

VI. Quantitation:

Calculations:
Prepare a four-point standard curve by injecting constant volumes of mixed standard
solutions. Use constant volume injections for sample extracts as well. Sample responses
not bracketed by the standard curve require dilution and re-injection. Inject a curve check
standard at least every 4-5 sample injections.

Calculations for instrumental analysis are conducted by Perkin-Elmer Analyst software
to create a standard curve based on linear regression. The regression functions are used to
calculate a best fit line (from a set of standard concentrations in g/mL versus peak
response) and to determine concentrations of the analyte found during sample analysis
from the calculated best fit line.

The equation used for the least squares fit is: y=mx +b, where y=peak response, x =
g/mL found for peak of interest, m=slope and b=y-intercept.
Control samples are fortified with known amounts of standard prior to extraction. Percent
recovery (if calculated by measuring the peak area) and ppm found are calculated as
shown:

pg/L determined X 50L injected =ppm found
mg crop *1000

ppm found x 100% =% Recovery
ppm expected

12
Example 1: Recovery Sample: 16079V.01R5

88.5 pg/L determined X 50L injected =0.0089 ppm found
500 mg crop *1000

0.0089 ppm found x 100% =89% Recovery
0.01 ppm expected

Example 2: Sample 16143

279 pg/L determined X 50L injected =0..0279 ppm found*
500 mg crop *1000

Instrument Parameters:

Instrumentation: HPLC-MS/MS

Autosampler: Perkin Elmer Series 200

Pumps: Perkin Elmer Series 200 micro pump

Data System: PE Sciex Analyst Software on a Dell computer running
Windows 2000. Data exported to Microsoft Excel.
Mobile Phase:
Conditions: Flow; 800l/min, Column temperature: Ambient, Injection size;
50l
HPLC Column: Restek Allure PFP Propyl (Catalog #9169553-700, 50 x 3.2mm
ID, 5 m particle size).
Gradient Table:
Step Total Time A (%) B (%)
0 0.0 45.0 55.0
1 0.5 45.0 55.0
2 2.0 12.5 87.5
3 14.0 12.5 87.5
4 14.1 45.0 55.0
5 16.0 45.0 55.0
A =0.1% formic acid in water B =0.1% formic acid in methanol.

Retention Time: Acequinocyl-OH 5.8-6.8 minutes, Acequinocyl 6.8-7.8 minutes
Note: Retention times between days can vary with conditioning of pre-column.

Detector: PE Sciex API 2000 tandem mass spectrometer
HPLC-MS/MS Interface: Heated Nebulizer
Heated Nebulizer Temperature: 450C
13
Curtain Gas Pressure: 50 psi Nitrogen
Collision Gas Pressure: 4 psi Nitrogen
Nebulizer Current: 2.0
Ion Source gas1: 50-85 psi Nitrogen
Ion Source gas2: 15 psi Nitrogen
Analyte Monitored: m/z => (i.e. Q1 Mass: 343 AMU, Q3 Mass: 189 AMU)
Mode: Positive Ion

VII. Results and Discussion:

Results are reported below as ppm acequinocyl or acequinocyl-OH. All out of range recoveries
were reported to the Study Director. Slight modifications to the SPE elution pattern were
required for various column lots and for fruit and puree analysis. The first set of storage samples
for acequinocyl-OH puree failed due to variations in solvents, or the SPE column, on 02/11/04.
Modifications to the elution pattern were made for the second stability set on 03/16/04 that are
reported. Additional stability study samples were prepared for acequinocyl-OH on puree for
long term storage. Tomato paste analysis required a larger SPE column and further
modifications to the elution pattern and quantities of solvents to remove matrix interference.
Summary of results are listed below:

Table VII.1.1: Summary of Recoveries, Acequinocyl

Matrix Spike
Level
ppm
Lab Sample ID Type of
Recovery
Acequinocyl
Found
ppm
Average
ppm
Recoveries
(%)
Average
Recovery
(%)
16079V.01R4 MV 0.0091 91
16079V.01R5 MV 0.0089 89
16079V.01R6 MV 0.0086 86
16079V.01R8 MV 0.0096 96
16079V.01R9 MV 0.0085 85
0.010
16080V.01R10 MV 0.0093
0.0090
93
904
16080V0.1R4 MV 0.0894 89
16080V0.1R5 MV 0.0848 85
16080V0.1R6 MV 0.0896 90
16059V.10R10 CR 0.0916 92
16059V.10R11 CR 0.0968 97
16059V.10R12 CR 0.0994 99
16140V.10R13 CR 0.0850 85
16140V.10R14 CR 0.0794 79
16140V.10R15 CR 0.0828 83
16462V.10R22 CR 0.0956 96
16462V.10R23 CR 0.0962 96
16462V.10R24 CR 0.0856 86
16059V.10R25 SSCR 0.0908 91

Fruit

0.10
16059V.10R26 SSCR 0.1012
0.0906
101

916

14
Matrix Spike
Level
ppm
Recovery
Acequinocyl
Found
ppm
Average
ppm
Recoveries
(%)
Average
Recovery
(%)
16080V1.0R4 MV 0.956 96
16080V1.0R4 MV 0.942 94
16080V1.0R4 MV 0.926 93
16466V1.0R19 CR 0.852 85
16466V1.0R20 CR 0.864 86
16466V1.0R21 CR 0.860 86
16079V1.0R22 CR 0.946 95
16079V1.0R23 CR 1.008 101
Fruit
(cont.)
1.0
16079V1.0R24 CR 0.990
0.9271
99

936

16463V0.01R7 MV 0.0096 96
16463V0.01R8 MV 0.0095 95
16463V0.01R9 MV 0.0088 88
16463V0.01R10 MV 0.0112 112
16463V0.01R11 MV 0.0109 109
0.01
16463V0.01R13 MV 0.0088
0.0098
88
9810
16463V0.10R7 MV 0.0844 84
16463V0.10R8 MV 0.0896 90
16463V0.10R9 MV 0.0828 83
16463V.10R10 CR 0.0870 87
16463V.10R11 CR 0.0888 89
16463V.10R12 CR 0.0916 92
16463V.10R13 SSCR 0.0908 91
16463V.10R14 SSCR 0.0802 80

0.10

16463V.10R15 SSCR 0.0888
0.0871

89

874
16463V1.0R7 MV 0.928 95
16463V1.0R8 MV 0.934 93
Paste

1.0
16463V1.0R9 MV 0.846
0.9027
85
915
16464V0.01R1 MV 0.0097 97
16464V0.01R2 MV 0.0113 113
16464V0.01R3 MV 0.0100 100
16464V0.01R4 MV 0.0102 102
16464V0.01R5 MV 0.0095 95
0.01
16464V0.01R6 MV 0.0100
0.0101
100
1016
16464V0.10R1 MV 0.0866 87
16464V0.10R2 MV 0.0896 90
16464V0.10R3 MV 0.0958 96
16464V.10R4 CR 0.0942 94
16464V.10R5 CR 0.0876 88
16464V.10R6 CR 0.0944 94
16464V.10R7 SSCR 0.0946 95
16464V.10R8 SSCR 0.0968 97
0.10
16464V.10R9 SSCR 0.0942
0.0926
94
934

Puree

1.0 16464V1.0R1 MV 0.848 0.858 85 861
15
Matrix Spike
Level
ppm
Recovery
Acequinocyl
Found
ppm
Average
ppm
Recoveries
(%)
Average
Recovery
(%)
16464V1.0R2 MV 0.854 85 Puree
(cont.) 16464V1.0R3 MV 0.872
(cont.)
87
(cont.)
MV=Method Validation, CR=Concurrent Recovery, SSCR=Storage Stability
Concurrent Recovery

Table VII.1.2: Summary of Recoveries for Fruit, Acequinocyl OH

Matrix Spike
Level
ppm
Recovery
Acequinocyl
-OH
Found
ppm
Average
ppm
Recoveries
%
Average
Recovery
%
16080V.025R1 MV 0.0232 93
16080V.025R2 MV 0.0234 93
16080V.025R3 MV 0.0228 91
16080V.025R4 MV 0.0231 92
16080V.025R5 MV 0.0213 85
0.025
16080V.025R6 MV 0.0272
0.0228
109
948
16080V0.1R4 MV 0.0900 90
16080V0.1R5 MV 0.0930 93
16080V0.1R6 MV 0.0906 91
16059V.10R10 CR 0.0776 78
16059V.10R11 CR 0.0626 63
16059V.10R12 CR 0.0748 75
16140V.10R13 CR 0.0616 62
16140V.10R14 CR 0.0586 59
16140V.10R15 CR 0.0630 63
16462V.10R22 CR 0.0814 81
16462V.10R23 CR 0.0762 76
16462V.10R24 CR 0.0868 87
16059V.10R25 SSCR 0.0762 76
0.10
16059V.10R26 SSCR 0.089
0.0772
89
7711
16080V1.0R4 MV 0.910 91
16080V1.0R5 MV 0.856 86
16080V1.0R6 MV 0.742 74
16446V1.0R19 CR 0.846 85
16446V1.0R20 CR 0.884 88
16446V1.0R21 CR 0.902 90
16079V1.0R22 CR 0.792 79
16079V1.0R23 CR 0.762 76
Fruit

1.0
16079V1.0R24 CR 0.800
0.8327
80
836
16463V0.025R7 MV 0.0293 117
16463V0.025R8 MV 0.0273 109
16463V0.025R9 MV 0.0212 85

Paste

0.025

16463V0.025R10 MV 0.0186

0.0239

75

9617

16
Matrix Spike
Level
ppm
Recovery
Acequinocyl
-OH
Found
ppm
Average
ppm
Recoveries
%
Average
Recovery
%
16463V0.025R11 MV 0.0207 83 0.025
(cont.) 16463V0.025R13 MV 0.0264
(cont.)
106
(cont.)
16463V0.10R7 MV 0.0718 72
16463V0.10R8 MV 0.0812 81
16463V0.10R9 MV 0.0762 76
16463V.10R10 CR 0.0716 72
16463V.10R11 CR 0.0782 78
16463V.10R12 CR 0.0704 70
16463V.10R13 SSCR 0.0780 78
16463V.10R14 SSCR 0.0744 74

0.10

16463V.10R15 SSCR 0.0768
0.0754

77
754
16463V1.0R7 MV 0.5360 54
16463V1.0R8 MV 0.5140 51
Paste
(cont.)
1.0
16463V1.0R9 MV 0.6160
0.5553
62
566
16464V0.025R1 MV 0.0219 87
16464V0.025R2 MV 0.0205 82
16464V0.025R3 MV 0.0180 72
16464V0.025R4 MV 0.0198 79
16464V0.025R5 MV 0.0220 88
0.025
16464V0.025R6 MV 0.0204
0.0204
82
826
16464V0.10R1 MV 0.0770 77
16464V0.10R2 MV 0.0834 83
16464V0.10R3 MV 0.0812 81
16464V0.10R4 CR 0.0644 64
16464V0.10R5 CR 0.0662 66
16464V0.10R6 CR 0.0698 70
16464V.10R10 SSCR 0.0992 99
16464V.10R11 SSCR 0.0994 99
0.10
16464V.10R12 SSCR 0.1112
0.0835
111
8316
16464V1.0R1 MV 0.672 67
16464V1.0R2 MV 0.670 67
Puree

1.0
16464V1.0R3 MV 0.638
0.66
64
662

MV=Method Validation, CR=Concurrent Recovery, SSCR=Storage Stability
Concurrent Recovery

17
Table VII.2.1: Summary of Storage Stability, Acequinocyl (Samples were stored at <-20C)
Matrix Lab Sample
ID
Storage
Stability
Recoveries
(%)
Date
Fortified
Date
Extracted
Maximum Actual
Samples Storage
Duration
(days)
Limit of
Demonstrated
Storage Stability
(days)
2

16059S.10R01 75
16059S.10R02 79 Fruit
16059S.10R03 85
02/27/03 01/30/04
285

337
16463S.10R01 61
16463S.10R02 69 Paste
16463S.10R03 74
11/18/03 04/12/04
156

146
16464S.10R01 76
16464S.10R02 76 Puree
16464S.10R03 75
11/18/03 02/11/04
93

119

Table VII.2.2: Summary of Storage Stability, Acequinocyl-OH (Samples were stored at <-20C)
Matrix Lab Sample
ID
Storage
Stability
Recoveries
(%)
Date
Fortified
Date
Extracted
Maximum Actual
Samples Storage
Duration
(days)
Limit of
Demonstrated
Storage Stability
(days)
2

16059S.10R07 56
16059S.10R08 58 Fruit
16059S.10R09 63
02/27/03 01/30/04 285 337
16463S.10R07 25
16463S.10R08 25 Paste
16463S.10R09 28
11/18/03 04/12/04 156 146
16464S.10R10 30
16464S.10R11 33 Puree
16464S.10R12 28
11/18/03 03/16/04 93 119

1
Calculated from harvest to extraction

2
Calculated from spiking to extraction
18
Table VII.3.1: Residue Data Results for Fruit, Acequinocyl and Acequinocyl-OH
Extraction Date Analysis Date

Residue Results (ppm) Trial
ID
Crop
Part
Field
Rep
No.
IR-4
Lab#
Sampling
Date
Acequinocyl Acequinocyl-
OH
OH
Storage
(Days)
Treated
Samples
Acequinocyl

Acequinocyl-
OH
A 16059 02/19/03 12/01/03
01/30/04
12/01/03
01/30/04
12/01/03
01/30/04
12/01/03
01/30/04
<0.01
<0.01
<0.025
<0.025
<0.025
B 16060 02/19/03 12/01/03 12/01/03 12/01/03 12/01/03 N/A* N/A
C 16061 02/19/03 12/01/03 12/01/03 12/01/03 12/01/03 285 0.10 <0.025
TX08 Fruit
D 16062 02/19/03 12/01/03 12/01/03 12/01/03 12/01/03 285 0.10 <0.025
A 16446 10/15/03 01/07/04 01/07/04 01/07/04 01/07/04 <0.01 <0.025
B 16447 10/15/03 01/07/04 01/07/04 01/07/04 01/07/04 N/A N/A
C 16448 10/15/03 01/07/04 01/07/04 01/07/04 01/07/04 84 0.13 <0.025
CA18 Fruit
D 16449 10/15/03 01/07/04 01/07/04 01/07/04 01/07/04 84 0.13 <0.025
A 16136 06/11/03 12/01/03 12/01/03 12/01/03 12/01/03 <0.01 <0.025
B 16137 06/11/03 12/01/03 12/01/03 12/01/03 12/01/03 N/A N/A
C 16138 06/11/03 12/01/03 12/01/03 12/01/03 12/01/03 173 0.04 <0.025
CA19 Fruit
D 16139 06/11/03 12/01/03 12/01/03 12/01/03 12/01/03 173 0.04 <0.025
A 16450 10/14/03 01/07/04 01/07/04 01/07/04 01/07/04 <0.01 <0.025
B 16451 10/14/03 01/07/04 01/07/04 01/07/04 01/07/04 N/A N/A
C 16452 10/14/03 01/07/04 01/07/04 01/07/04 01/07/04 85 0.20 <0.025
CA21 Fruit
D 16453 10/14/03 01/07/04 01/07/04 01/07/04 01/07/04 85 0.20 <0.025
A 16162 07/23/03 12/03/03 12/03/03 12/03/03 12/03/03 <0.01 <0.025
B 16163 07/23/03 12/03/03 12/03/03 12/03/03 12/03/03 N/A N/A
C 16164 07/23/03 12/03/03 12/03/03 12/03/03 12/03/03 133 0.05
CA22 Fruit
D 16165 07/23/03 12/03/03 12/03/03 12/03/03 12/03/03 133 0.04
A 16140 06/12/03 12/03/03 12/03/03 12/03/03 12/03/03 <0.01 <0.025
B 16141 06/12/03 12/03/03 12/03/03 12/03/03 12/03/03 N/A N/A
C 16142 06/12/03 12/03/03 12/03/03 12/03/03 12/03/03 174 0.03 <0.025
CA23 Fruit
D 16143 06/12/03 12/03/03 12/03/03 12/03/03 12/03/03 174 0.02 <0.025
A 16079 04/14/03 11/13/03
01/13/04
11/13/03
01/13/04
11/13/03
01/13/04
11/13/03
01/13/04
<0.01
<0.01
<0.025
<0.025
B 16080 04/14/03 11/17/03 11/17/03 11/17/03 11/17/03 <0.01 <0.025
C 16081 04/14/03 01/13/04 01/13/04 01/13/04 01/13/04 275 0.17 <0.025
D 16082 04/14/03 01/13/04 01/13/04 01/13/04 01/13/04 275 0.11 <0.025
E 16083 04/15/03 01/13/04 01/13/04 01/13/04 01/13/04 273 0.16 <0.025
F 16084 04/15/03 01/13/04 01/13/04 01/13/04 01/13/04 273 0.17 <0.025
NJ 06
Fruit
G 16085 04/17/03 01/13/04 01/13/04 01/13/04 01/13/04 271 0.11 <0.025
19
Extraction Date Analysis Date

Residue Results (ppm) Trial
ID
Crop
Part
Field
Rep
No.
IR-4
Lab#
Sampling
Date
OH
OH
Storage
(Days)
Treated
Samples
Acequinocyl

Acequinocyl-
OH
H 16086 04/17/03 01/13/04 01/13/04 01/13/04 01/13/04 271 0.16 <0.025
I 16087 04/21/03 01/13/04 01/13/04 01/13/04 01/13/04 267 0.12 <0.025
J 16088 04/21/03 01/13/04 01/13/04 01/13/04 01/13/04 267 0.12 <0.025
Fruit
F 16462 10/20/03 01/28/04
01/29/04
01/28/04
01/29/04
01/28/04
01/29/04
01/28/04
01/29/04
<0.01
<0.01
<0.025
<0.025
Paste
G 16463 10/22/03 03/24/04
03/29/04
03/31/04
04/12/04
03/24/04
03/29/04
03/31/04
04/13/04
03/24/04
03/29/04
03/31/04
04/12/04
03/24/04
03/29/04
04/01/04
04/13/04
<0.01
<0.01
<0.01
<0.01
<0.025
<0.025
<0.025
<0.025
Puree
H 16464 10/21/03 01/20/04
01/22/04
02/12/04
01/20/04
01/22/04
03/16/04
01/20/04
01/22/04
02/12/04
01/20/04
01/22/04
03/16/04
<0.01
<0.01
<0.01
<0.025
<0.025
<0.025
Fruit J 16465 10/22/03 01/28/04 01/28/04 01/28/04 01/28/04 100 0.14 <0.025
Paste K 16466 10/27/03 03/31/04 03/31/04 03/31/04 04/01/04 156 0.05 <0.025
TXP04
(CA21)
Puree L 16467 10/24/03 01/22/04 01/22/04 01/22/04 01/22/04 93 0.04 <0.025
*N/A=Not Analyzed

Table VII.4.1: Summary of Residue Data from Processing Crop Field Trial, Acequinocyl and Acequinocyl-OH
RAC Processed
Fraction
Total Application Rate
(lb a.i./A)
PHI
(days)
Analyte Residue Level (ppm) Processing Factor
1

Fruit Fruit 0.303 1 Acequinocyl 0.14 N/A
Fruit Paste 0.303 1 Acequinocyl 0.05 0.36
Fruit Puree 0.303 1 Acequinocyl 0.04 0.28
Fruit Fruit 0.303 1 Acequinocyl-OH <0.025 --
Fruit Paste 0.303 1 Acequinocyl-OH <0.025 --
Fruit Puree 0.303 1 Acequinocyl-OH <0.025 --
1
Factor determined by dividing the residue in Paste and Puree by residue in Fruit in the same trial.
20
IR-4 Western Region Laboratory, University of California, Davis Analytical Summary Report PR#08356

ATTACHMENT A: REPRESENTATIVE CHROMATOGRAMS
INDEX TO REPRESENTATIVE CHROMATOGRAMS

Chromatogram(s) Page Nos.

A. Standard curves:
Acequinocyl ...........................................................................................22
Acequinocyl-OH....................................................................................26

B. Miscellaneous Controls:
Acequinocyl
Tomato fruit..................................................................................30
Puree.............................................................................................31
Paste..............................................................................................34
Acequinocyl-OH
Tomato fruit..................................................................................37
Puree.............................................................................................38
Paste..............................................................................................41
C. Fortified Recoveries:
Acequinocyl
Tomato fruit..................................................................................44
Puree.............................................................................................47
Paste..............................................................................................50
Acequinocyl-OH
Tomato fruit..................................................................................53
Puree.............................................................................................56
Paste..............................................................................................59

D. Treated Samples:
Acequinocyl
Tomato fruit..................................................................................62
Puree.............................................................................................67
Paste..............................................................................................68
Acequinocyl-OH
Tomato fruit..................................................................................69
Puree.............................................................................................74

Each chromatogram represents a 50 L injection.

(Chromatograms, Data Summaries, and Standard Curves are for demonstration
purposes only, not all of the required chromatograms are included)
21
22
23
24
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5
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75
APPENDIX B: DATA SUMMARIES AND STANDARD CURVES
76
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N
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D
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%

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135
136
137
APPENDIX C: STANDARD USE FORMS
138
139
140
141
142
143
144
145
146
147
148
149
150
151
APPENDIX D: CERTIFICATES OF ANALYSIS
152
153
154

Version 1.0 05/12/08
Page 1 of 5
Checklist for Review of Analytical Summary Reports

PR #: Active Ingredient/Crop:

Yes No NA Notes
1)Sample Preparation
1.1 For each sample, was the full sample ground, and
mixed thoroughly?

2)Instrument Condition
2.1 For GC/MS, are tune files or other appropriate
documentation available for each run, to show that the
instrument was in good working order at the time the run
was made?

If 2.1 is no, or if the analyst has concerns regarding
the instrument condition, the LRD must be consulted. Was
the LRD consulted?

2.2 For other detectors, was the instrument in good
working order for each run? The answer to this question
will rely on the analysts professional judgment and will
include an evaluation of appropriate data obtained
throughout the study, for example, the standard curve, the
peak retention times, the area counts of the standards and
the signal to noise ratio. Note what data was considered.

the instrument condition, the LRD must be consulted. Was
the LRD consulted?

Version 1.0 05/12/08
Page 2 of 5

Yes No NA Notes
3)Analysis
3.1 Is the peak of interest distinct on each chromatogram?
(No shoulder peaks on the peak of interest and no
interfering peaks.)

3.2 Is the S/N ratio adequate? For example, when viewing
the chromatograms for the standards through the course of
the study, are there any runs where the S/N ratio has
dropped significantly? A low S/N ratio is a concern. The
answer to this question will rely on the analysts
professional judgment.

a change in S/N ratio during a study, the LRD must be
consulted. Was the LRD consulted?

3.31 Are recoveries during method validation comparable
to the recoveries in the reference method? (When the
average recoveries are compared, the difference is <20%.
Spot check the data, detailed calculations are not needed)

3.32 Are concurrent recoveries during analysis
comparable to those seen in method validation? (When the
average recoveries are compared the difference is <15%.
Spot check the data, detailed calculations are not needed)

3.4 Did the r-squared value remain consistent during
method validation and analysis of samples (Range <0.02)?

If 3.4 is no, what is the range of the r-squared
values? Provide an explanation.

3.5 Are there manual integrations?
If 3.5 is yes, were any standards manually
integrated?

If 3.5 is yes, is a reason provided in the ASR?

Version 1.0 05/12/08
Page 3 of 5

Yes No NA Notes
3.61 Were duplicate injections used for concurrent
fortifications?

If 3.61 is yes, were duplicate injections within 30%
of each other?

3.62 Were duplicate injections used for unknowns?
If 3.62 is yes, were duplicate injections within 30%
of each other?

4)Results
4.1 Are control values non-detectable?
If 4.1 is no, are they <20% of the highest residue
value? (860.1340, p.2)

If 4.1 is no, is this noted in the ASR and the
pertinent chromatograms included?

4.21 Are method validation recoveries and concurrent
recoveries consistently >100%? (860.1340, p.2)

4.22 For method validation and concurrent recoveries, is
the CV (defined as the standard deviation/average) <20%?
(860.1340, p.3)

Version 1.0 05/12/08
Page 4 of 5

Yes No NA Notes
5)Analytical Summary Report
5.1 Were any samples re-extracted and rerun?
If 5.1 is yes, was the LRD consulted? The LRD
needs to approve samples needing re-extraction due to
judgment calls, for example, samples with unexpectedly
high or low residue results, or a need for manual
integration.)

If 5.1 is yes, was the study director notified? (If the
situation is covered in an SOP i.e. samples needing
dilution, reanalysis due to a power failure or the vial
location was incorrect for injection, document in the study
file. The study director does not need to be notified). If
answer to this question is no, please note why.

If 5.1 is yes, is there information explaining the
situation and its resolution in the ASR?

5.2 Is there documentation in the raw data regarding any
unexpected circumstances during the run?

If 5.2 is yes, was the LRD consulted? The LRD
needs to approve judgment calls, for example, samples
with unexpectedly high or low residue results, or a need for
manual integration.)

If 5.2 is yes, was the study director notified? (If the
situation is covered in an SOP i.e. reanalysis due to a
power failure or the vial location was incorrect for
injection, the study director does not need to be notified).
If answer to this question is no, please note why.

If 5.2 is yes, is there information explaining the
situation and its resolution in the ASR?

If 5.2 is yes, are chromatograms of samples with
unusual or inconsistent results included in the ASR?
(860.1000, p.18)

Version 1.0 05/12/08
Page 5 of 5

Yes No NA Notes
5.3 Are standard curves and peak heights/areas for all
standards available in the ASR for each run? (860.1000, p.
18)

If 5.3 is no, please attach a copy of any missing
standard curves with peak heights/areas, so the study
director may add them as an appendix to the final report.

5.4 Are the dates the test compounds (standard solutions)
were prepared included in the ASR? (860.1500, #3, p. 37)

If 5.4 is no, please include this information with
this checklist (A copy of the standards prep form(s) is
fine).

5.5Are all residue values reported in the ASR bracketed by
the standard curve?

If 5.5 is no, was the study director contacted?

Analytical Summary Report Reviewed by:

Signature Date

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