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29
Numerous methods have been proposed for the determination of catecholamines, serotonin, and their metabolites in biological uids. In this section, the following methods are presented: Method 29-1. Determination of Plasma Free Catecholamines by HPLC/EC Method 29-2. Determination of Plasma Free Metanephrines by Liquid Chromatography With Electrochemical Detection Method 29-3. Determination of Plasma Free Metanephrine and Normetanephrine by LC-MS/MS Method 29-4. Determination of Urinary Free Catecholamines by HPLC/EC Method 29-5. Determination of Urinary Free Catecholamines by LC-MS/MS Method 29-6. Determination of Urinary
Metanephrine and Normetanephrine by HPLC/EC Method 29-7. Determination of Urinary Metanephrine and Normetanephrine by LC-MS/MS Method 29-8. Determination of Urinary Vanillylmandelic Acid by HPLC/EC Method 29-9. Determination of Urinary Vanillylmandelic Acid by LC-MS/MS Method 29-10. Determination of Urinary Homovanillic Acid (3-Methoxy-4Hydroxyphenylacetic Acid) by HPLC/EC Method 29-11. Determination of Urinary Homovanillic Acid by LC-MS/MS Method 29-12. Determination of Urinary 5Hydroxyindoleacetic Acid by Quantitative HPLC Method 29-13. Determination of Urinary 5-Hydroxyindole-3-Acetic Acid by LC-MS/MS
Copyright 2006, 1999, 1994, 1986 Elsevier Inc. All rights reserved.
Appendix 29
The current resulting from this reaction is converted to a voltage signal and monitored as a function of time. At a constant temperature and ow rate, this oxidation current is directly proportional to the concentration of the analyte. Catecholamine reference materials are used to calibrate the system on the basis of peak heights and retention times. To calculate sample concentrations, peak height ratios relative to the internal standard (dihydroxybenzylamine) for unknowns are compared with those of the calibrations. Specimen Collection and Storage Different methods of blood collection have been reported; variations among them include the choice of anticoagulant, the addition of antioxidants, and various sample processing techniques. Standardization of posture is essential because plasma catecholamine levels increase twofold to threefold when a supine subject assumes an upright position. Most procedures recommend that morning specimens be drawn from subjects who have been resting quietly for 30 minutes in a recumbent position after insertion of a venous catheter. In addition, subjects should refrain from eating, using tobacco, or drinking coffee or tea for at least 4 hours before venipuncture. If at all possible, specimens should be obtained when patients are drug free. Most antihypertensive drugs (other than clonidine) and many other drugs can produce false-positive results. Use of these drugs should be discontinued 3 to 7 days before obtaining the sample. If hypertension must be treated before measuring catecholamines, then
Copyright 2006, 1999, 1994, 1986 Elsevier Inc. All rights reserved.
Appendix 29
11.
12.
13.
14.
15.
16.
17.
Instrumentation A commercially available HPLC system equipped with a 15 cm 4.6-mm (I.D.) cation-exchange silica column and an electrochemical detector is suitable. Quality Control Two controls are included in every run. For example, Bio-Rad Lyphochek Endocrine Control, Levels 1 and 2 (Bio-Rad Clinical Diagnostics Group, Hercules, Calif.) are reconstituted each day with distilled water and processed for immediate use. Controls are placed in an ice bath until ready to use. Procedure Preanalysis Check 1. Establish operating conditions of the chromatographic and detection systems. Suggested conditions are as follows: Flow rate: 1.1 mL/min Potential: 500 mV Sensitivity: 5 nA/V Chart speed: 1 cm/min 2. Inject 100 mL of the catecholamine prerun calibrator at least twice. 3. Inspect the chromatograms using the following guidelines to monitor performance of the chromatography system: a. Retention times are not different for the two injections. (Note: If retention times differ, reinject the calibrator as needed to conrm that the system is at equilibrium.)
Sample Analysis 4. Remove patient samples (heparinized plasma) and catecholamine-free plasma from frozen storage and thaw in a 37 C water bath. Mix well. Centrifuge the patient samples for 5 minutes. Place all samples in an ice bath until ready to use. 5. Prepare control samples as noted under Controls. 6. Pipette 4.0 mL of each patient sample and control into labeled 96 16.8-mm extraction tubes. 7. Pipette 20, 40, and 80 mL of the catecholamine combination calibrator into labeled tubes containing 4.0 mL of the catecholamine-free plasma. 8. To all tubes add 80 mL of the working internal standard solution (50 mg/L). 9. Using a measuring spoon, transfer two level spoonfuls of alumina (about 60 to 80 mg) to each tube. 10. Add 2 mL Tris buffer to each tube. 11. Immediately cap each tube and shake vigorously for 10 minutes using the mechanical shaker. 12. Centrifuge the tubes for 2 minutes at 3000 rpm. 13. Without disturbing the alumina pellet, remove the supernatant from each tube using a vacuum aspirator connected to a vacuum trap. 14. Using a squirt bottle, add about 1 mL of pH 7 adjusted water to each tube. Vortex mix the tubes for 15 seconds. (Note: To ensure thorough washing of the alumina, no portion of the alumina pellet should remain on the bottom of the tube during mixing.) 15. Centrifuge the tubes for 2 minutes at 3000 rpm. 16. Carefully remove as much supernatant liquid as possible without disturbing the alumina pellet. (Note: This step is critically important!) 17. Wash the alumina again by repeating steps 14, 15, and 16. 18. Add 400mL of HClO4, 0.1 mol/L, to each tube. Vortex mix for 30 seconds. Allow to stand for 5 minutes and then vortex mix for 30 seconds. (Note: Thorough mixing is important to ensure good recovery of the catecholamines.) 19. Centrifuge each tube for 2 minutes at 3000 rpm. (Note: Do not allow the supernatant extract to remain in contact with the alumina for more than 30 minutes. Otherwise, degradation of the catecholamines can result.) 20. Using a glass Pasteur pipette, carefully transfer the supernatant to a storage vial. At this time, samples may be stored at 4 C for up to 24 hours before injection into the HPLC system. (Note: Extracts should not be injected until the baseline has been established. Check regularly for baseline shifts, and rezero the output of the detector as necessary.) 21. Introduce 100mL of each standard, patient specimen, and control into the HPLC column. (Note: The remaining extract can be used for dilutions as required.) Chromatogram Figure A29-1 shows a representative chromatogram of catecholamines extracted from plasma control.
Continued
Copyright 2006, 1999, 1994, 1986 Elsevier Inc. All rights reserved.
Appendix 29
Figure A29-1 Representative chromatogram of catecholamines extracted from a plasma control sample. Epinephrine (A), dopamine (B), and 3,4-dihydroxybenzylamine (D) retention are identied on the chromatogram.
Calculations 1. Check sample identication of each chromatogram to ensure proper labeling. 2. Identify catecholamine and internal standard peaks by relative retention times. 3. For manual integration, use a ruler to draw a line under all peaks. Identify the baseline points (deepest valleys) and draw lines that connect these baseline points. (Note: Disregard small valleys between partially fused peaks.) 4. For each sample, measure the peak heights to the nearest 0.1 cm. Record results on the appropriate worksheet as the example shows below:
Calibrating Compound Epinephrine Norepinephrine Dopamine Internal standard Retention Time, min 5.4 6.7 7.4 8.2 Peak Heights, cm 9.0 6.1 1.8 4.3
(Note: The analyst should verify that the internal standard (IS) peak height for each patient sample or control is >75% of the IS peak height for the extracted calibrator.) 5. Calculate ratio of peak heights (peak height of catecholamine/ peak height of internal standard) for each patient sample, control, and calibrator. 6. Establish a calibration curve by plotting the peak height ratio of each catecholamine calibrator on ordinate versus concentration on abscissa (250, 500, and 1000 pg/mL).
Reference Modied from Koch DD, Polzin GL. Effect of sample preparation and liquid chromatography column choice on selectivity and precision of plasma catecholamine determination. J Chromatogr 1987;386:19-24.
Copyright 2006, 1999, 1994, 1986 Elsevier Inc. All rights reserved.
Appendix 29
METHOD 29-2 Determination of Plasma Free Metanephrines by Liquid Chromatography With Electrochemical Detection
Submitted by Graeme Eisenhofer, Ph.D. Clinical Neurocardiology Section, National Institutes of Health, Bethesda, Maryland. Principle This method allows measurement of picomolar plasma concentrations of normetanephrine, the O-methylated metabolite of norepinephrine; metanephrine, the metabolite of epinephrine; and methoxytyramine, the metabolite of dopamine. An initial extraction procedure serves to isolate and concentrate the low picomolar concentrations of free O-methylated amine metabolites into a puried low volume form appropriate for injection onto the HPLC apparatus. In this procedure, samples of plasma together with a known amount of internal standard and a small amount of dilute acetic acid are applied to extraction columns containing a cation exchange resin. The extraction columns are rst activated by washing with methanolic potassium hydroxide to bring up the negative charges on the column matrix. The positively charged O-methylated amine metabolites are thereby selectively adsorbed onto the activated cation exchange resin. The extraction columns are then washed with dilute solutions of acetic acid, ammonium phosphate, and water. The metabolites are eluted from extraction columns using ammoniacal methanol, which is then dried down and the residue reconstituted in a minimum volume of dilute acetic acid ready for measurement by liquid chromatography with coulometric detection. Measurement by HPLC involves separation of the O-methylated amines using a C18 reversed-phase HPLC column followed by their ordered post column detection by coulometry. Mobile phase is pumped through the autosampler and HPLC column using a solvent delivery system that provides a continuous, pulse-free ow of mobile phase through the system. Separation on the HPLC column is facilitated by hydrophobic and ionic interactions between components of the C18 analytical column stationary phase, mobile phase, and O-methylated amines. The mobile phase provides an adjustable vehicle for passage of the analytes of interest through the analytical column to the coulometric detector. Adjustments to the composition of the mobile phase, specically the pH and concentrations of octane sulfonate and acetonitrile, allow ne-tuning of the separation of analytes of interest on the analytical column. The negatively charged octane sulfonate ion-pairing reagent binds to positively charged O-methylated amines. In turn, interactions of the hydrophobic carbon chain of the octane sulfonate molecules with the hydrophobic C18 analytical column matrix lead to retention of the positively changed analytes of interest on the HPLC column. Thus by varying the octane sulfonate concentration, the retention of positively charged analytes of interest may be altered in relation to other uncharged or changed species or potentially interfering compounds. Subtle changes in the pH of the mobile phase (i.e., between pH 3.15 and 3.40) provide a further mechanism for altering the charge, and thus the retention on the analytical column of O-methylated amines or contaminating substances. Variations in the concentration of acetonitrile organic phase provide a further means to adjust the retention of O-methylated amines and potentially interfering substances by modifying the overall extent of hydrophobic interactions. Further changes to retention can be facilitated by alterations to the column temperature. Changes to the composition of the mobile phase may be made when there is need for adjustment of retention times of O-methylated amines and interfering compounds. Mobile phase eluting from the outlet of the analytical column and containing the O-methylated amines then passes through a triple electrode coulometric system, consisting of a conditioning cell and an analytical cell connected to the Coulochem detector. The functions of the electrochemical detection system are the detection and quantication of the O-methylated amines after their separation and elution on the analytical column. The rst electrode in the conditioning cell is set to an oxidizing potential to screen out irreversibly oxidized contaminating species that may otherwise interfere with subsequent detection by a reducing potential at the third analytical electrode. The hydroxyl groups on the amine metabolites are normally in the reduced state, but the compounds are reversibly oxidized and reduced so that measurement by reduction at the analytical cell is possible after the compounds are oxidized at the rst electrode in the conditioning cell. Outputs from the third electrochemical cell at a reducing potential are generated as chromatographic recordings suitable for subsequent analysis. Sample concentrations are calculated from peak heights of the amine metabolites relative to those of calibrators and corrected according to the procedural recovery of an internal standard. Specimen Collection and Storage Patients should be in the supine position for at least 20 minutes before and during collection of blood samples. To avoid the acute effects of the stress of venipuncture, it is preferable to use an indwelling IV placed in a vein at least 20 minutes before the blood sample is drawn. Ten 1-milliliter samples of blood should be drawn into a evacuated collection tubes containing heparin in any form or EDTA as an anticoagulant. The O-methylated amine metabolites are not stable in whole blood at room temperature. Therefore samples of blood should be placed on ice and centrifuged (preferably at 4 C) to separate the plasma within 2 hours of collection. Aliquots of plasma should be stored at -70 C or colder until analysis. Samples requiring shipping should be kept frozen using suitable amounts of dry ice packaged in styrofoam containers. The O-methylated metabolites in such samples are stable for up to 5 years. Analytical Interferences Acetaminophen produces known interference with assays of plasma free metanephrines. The patient should therefore not have taken acetaminophen in any form (e.g., Tylenol, Excedrin, and combined cold medications) for at least 5 days before the blood sample is drawn. Apart from acetaminophen there are no other established causes of direct analytical interference from common over the counter or prescribed medications (see below). However, occasional co-chromatographing or interfering peaks do occur from unknown substances in plasma of either endogenous or exogenous origin. These interferences can be particularly prevalent after meals or in patients with renal insufciency. Therefore to minimize the appearance of chromatographic interferences from dietary substances, samples of blood should be obtained after an overnight fast (water and decaffeinated soft drinks are permissible).
Continued
Copyright 2006, 1999, 1994, 1986 Elsevier Inc. All rights reserved.
Appendix 29
METHOD 29-2 Determination of Plasma Free Metanephrines by Liquid Chromatography With Electrochemical Detectioncontd
Reagents 1. Calibrators: -normetanephrine HCl, -metanephrine HCl, -methoxytyramine HCl, 4-hydroxy-3-methoxybenzylamine HCl (all analytical grade); 4-hydroxy-3-ethoxyphenylethanolamine, special NIH synthesis. Store refrigerated. Expiration3 years. 2. Prepare all calibrators to contain 1 mg/mL stock calibrator (free base) solutions in 0.2 mol/L acetic acid then dilute each of them to separate 100 ng/mL (normetanephrine, metanephrine, and methoxytyramine) or 200 ng/mL (internal standards) working standard solutions in 0.2 mol/L acetic acid. Store as aliquots in 1.5 mL Eppendorf tubes at -70 C to -80 C. Expiration2 years. 3. Mobile phase: a. Acetonitrile, HPLC grade (no xed source). Store at room temperature. Expiration3 years. b. 1-Octane sulfonic acid, ultrapure grade (Sigma O 0133). Store at room temperature. Expiration3 years. c. Phosphoric acid, HPLC grade (no xed source). Store at room temperature. Expiration3 years. d. Sodium phosphate monobasic (NaH2PO4 H2O), analytical grade (no xed source). Store at room temperature. Expiration3 years. e. Ethylenediamine-tetraacetic acid disodium salt: Dihydrate (EDTA), analytical grade (Sigma ED2SS) Store at room temperature. Expiration3 years. f. Mobile phase stock solution: Add to a clean 1 L bottle 138 g of sodium phosphate monobasic, 1.28 g of octane sulfonate, and 100 mg EDTA. Dilute to 1 L with deionized water and stir thoroughly to dissolve. Store at room temperature. Expiration6 months. g. Mobile phase working solution: Add to a clean 1 L bottle, 100 mL of mobile phase stock solution and 50 to 90 mL of HPLC grade acetonitrile (75 mL is often most appropriate for a new columnsee note below). Dilute to 950 mL with milliQ water and adjust pH with stirring to an appropriate point between pH 3.15 and pH 3.45 with HPLC grade phosphoric acid (a pH of 3.25 is a most appropriate point to start with a new columnsee note below). Dilute to a nal volume of 1 L with milli-Q water and Millipore lter through a 0.22 mm type of GV (Millipore) lter under vacuum. Decant to a clean bottle and pump through HPLC system. Expiration7 days or sooner depending on background current. 4. Ion exchange extraction reagents: a. Bond Elut LRC Accucat Cation Exchange columns with 200 mg packing (Varian P/N 1228-2005, Analytichem International, Harbor City, Calif.) Store at room temperature. Expiration 1 year. b. One percent KOH in methanol: Dissolve 5.0 g KOH (MW 56) in 500 mL of methanol or mix 89 mL 1 mol/L methanolic potassium hydroxide solution with 411 mL methanol. Store at room temperature. Expiration6 months. c. Ten mmol/L acetic acid in methanol: Add 570 mL glacial acetic acid to 1000 mL of deionized H2O (10 mmol/L acetic acid). Mix together 900 mL 10 mmol/L acetic acid with 100 mL methanol. Store at room temperature. Expiration6 months. d. Ammonium phosphate 10 mmol/L, pH 8.5: Dissolve 1.15 g monobasic ammonium phosphate (MW 115.03) in 800 mL deionized H2O. Adjust the pH to 8.5 with 5% ammonium hydroxide (5 mL ammonium hydroxide in 95 mL H2O). Adjust volume to 1 L with deionized H2O and add 0.05 g EDTA. Store at room temperature. Expiration6 months. e. Ten percent ammonium hydroxide/methanol solution (1 : 3): Add together 90 mL deionized H2O, 10 mL concentrated ammonium hydroxide solution, and 300 mL HPLC grade methanol. Made fresh on day of use. Expiration1 day. f. Acetic acid, 0.2 mol/L: Dilute 12 mL of glacial acid with milliQ water to 1 L. Store at room temperature. Expiration1 year. HPLC Instrumentation 1. Isocratic solvent delivery system (HPLC pump): Should be relatively pulse free (e.g., Waters model 515, Waters Chromatography, Milford, Mass.) 2. Autosampler: Should be refrigerated and capable of delivering at least 90% of a 100 mL sample onto the analytical column (Waters 717 plus autosampler is recommended, Waters Chromatography, Milford, Mass.). 3. Analytical column: 5-mm particle size 4.6 250 mm C18 reversedphase column (LUNA, P/N 00G4252-EO, Phenomenex, Torrance, Calif.). 4. Guard column (P/N KJO4282, Phenomenex, Torrance, Calif.) C18 inserts changed before each run. 5. Column temperature control unit: Required for maintaining constant column temperature (column cooler 250, Cera Inc., Baldwin Park, Calif.). 6. Coulometric detector: (Model 5100A or Model 5200A Coulochem electrochemical detector, Environmental Sciences Associates Inc., Chelmsford, Mass.). 7. Multiple electrode system (Model 5021 conditioning cell and Model 5011 analytical cell, Environmental Sciences Associates Inc., Chelmsford, Mass.). 8. Data acquisition hardware and software system. Extraction Equipment 1. Vacuum manifold for placement of extraction columns and diversion of solutions to waste or to collection tubes (Vac-Elut SPS 24, Analytichem International, Harbor City, Calif.). 2. Vacuum concentrator for drying down and concentration of ammoniacal methanol extracts in glass culture tubes. Should include centrifugation to avoid loss of sample from glass culture tubes (SVC200H Speed Vac Concentrator and RVT 4104 Refrigerated Vapor Trap, both from Thermo Savant, Holbrook, N.Y.). Quality Control Each extraction and HPLC run should include a water blank, a QC sample (normal QC) and 1 spiked QC sample (high QC) at the beginning of the run. This should be followed by the samples to be assayed. A second set of QC samples should be included in the middle and at the end of the run depending on the expected duration of the run. Procedures Extraction Procedure 1. Remove plasma samples and calibrators from freezer and thaw at room temperature. Spin thawed plasma at 3000 g for 10
Copyright 2006, 1999, 1994, 1986 Elsevier Inc. All rights reserved.
Appendix 29
METHOD 29-2 Determination of Plasma Free Metanephrines by Liquid Chromatography With Electrochemical Detectioncontd
minutes to pellet brin and other particulate matter (Note: It is essential that samples are free of particulate matter for their efcient passage through extraction columns). Number extraction columns, 13 100 mm culture tubes, and sample sheet according to the sequence of samples to be extracted and run on the HPLC. Wash extraction columns with 5 mL NH4OH : methanol under light vacuum (1 to 5 mmHg). Do not allow columns to dry out. Activate columns with 2 mL KOH : methanol under reduced vacuum (1 to 5 mmHg). Again do not allow columns to dry out. Wash columns with 2 mL deionized H2O under reduced vacuum (1 to 5 mmHg). Again do not allow columns to dry out. Add to columns 1 mL deionized H2O, 70 mL of 0.2 mol/L acetic acid, then the water (for blank) or plasma sample (2 mL) followed by 20 mL of internal standard solution. Pass the whole solution slowly through the column under reduced pressure (Elution time should be between 2 and 5 minutes). Wash columns with 2 mL acetic acid : methanol under reduced vacuum and dry the column out by increasing the vacuum to -20 mmHg for 2 minutes. Wash columns with 2 mL ammonium phosphate solution under reduced vacuum. Wash columns with 2 mL deionized H2O under reduced vacuum. Elute all H2O. Rotate the vacuum manifold to collect samples into the numbered 13 100 mm glass culture tubes and check for correct positioning of all needles. Elute samples into culture tubes with 2 mL of NH4OH : methanol. Use gravity and nally vacuum to elute all the NH4OH : methanol. Dry the sample down using a vacuum concentrator. Dissolve the residue in 150 mL of 0.2 mol/L acetic acid and inject 140 mL onto the HPLC system. distinct from the amperometric mode of electrochemical detection used in many other procedures. Most commercially available electrochemical detectors feature glassy carbon or wall jet cells that usually enable charge transfer of only 1% to 5% of the analyte that passes by or across the electrode surface. Since most analytes, including catecholamines and their metabolites, are usually present in the reduced state, the charge transfer in amperometric detection generally requires oxidation. The detector required for the method described here features ow-through porous carbon cells, which allow oxidation or reduction of 100% of the analyte passing through the cell. Under these conditions of charge transfer, the electrode response is dened as coulometric as distinct from amperometric. The advantage of coulometric detection for measurements of catecholamines and metanephrines is that these analytes can be reversibly oxidized and reduced. In contrast, for other substances, oxidation is often irreversible. A preoxidation step at the rst in a series of electrodes thereby provides a mechanism to reversibly oxidize the analytes of interest while screening out potentially interfering impurities by irreversible oxidation. The reversibly oxidized analytes of interest may then be detected at a reducing potential without interference from any irreversibly oxidized impurities in the sample. The above features of coulometric detection enable generation of much cleaner chromatograms than would otherwise be possible for measurements of plasma free metanephrines. This is particularly useful for measurements of extremely low levels of analytes, such as catecholamines and metanephrines, where potentially interfering substances are often present in the sample at much greater concentrations than the analytes of interest. Reference Intervals Adult Reference Intervals The reference intervals below are determined using data from 178 normotensive and hypertensive individuals, including 100 men and 78 women, ages 18 to 72. Data were log-transformed before analysis, and upper and lower reference intervals were determined from the 95% condence limits (i.e., 2 standard deviations above and below the geometric mean).
Analyte Normetanephrine (pg/mL) (nmol/L) Metanephrine (pg/mL) (nmol/L) 27 0.14 61 0.31 12 0.06 45 0.25 112 0.61 18 0.10 Geometric Mean Upper Reference Limit Lower Reference Limit
2.
3. 4. 5. 6.
7.
8. 9. 10.
11.
12. 13.
High-Performance Liquid Chromatography 1. Establish optimal operating conditions of the chromatographic and detection systems. Suggested conditions are as follows: Flow rate: 1.0 mL/min Conditioning cell potential: +0.40 V (oxidization) Analytical cell 1 potential: +0.15 V Analytical cell 2 potential: -0.40 V (reduction) 2. Before the introduction of specimens onto the reversed-phase HPLC column, ensure that the background current at the detecting cell (i.e., the cell with the reducing potential) is below 0.02 mamp, that there is negligible baseline noise, and no injection artifacts or inappropriate additional chromatographic peaks upon injection of the series of calibrators. Figure A29-2 shows representative chromatograms of an injected mixture of standards (A), an extracted sample of plasma from a healthy volunteer (B), and an extracted sample of plasma from a patient with a small adrenal pheochromocytoma (C). Comments A distinct and important feature of the electrochemical detection used in this method is the use of a coulometric mode, which is
Adult Reference Intervals Adjusted for Age and Gender The reference intervals shown below are adjusted for a signicant inuence of gender on plasma concentrations of metanephrine and a signicant inuence of age on plasma normetanephrine and total normetanephrine. Data were log-transformed before analysis, and upper and lower reference intervals were determined from the 95% condence limits.
Continued
Copyright 2006, 1999, 1994, 1986 Elsevier Inc. All rights reserved.
Appendix 29
METHOD 29-2 Determination of Plasma Free Metanephrines by Liquid Chromatography With Electrochemical Detectioncontd
(A) Standards HMBA NMN EHPEA MN NMN = 500 pg MN = 500 pg MTY = 500 pg
(B) Normal 36 yr old female HMBA EHPEA
MTY
NMN = 43 MN = 31 pg
MN
NMN MN
10
20 30 Time (min)
10
30 20 Time (min)
10
20 30 Time (min)
Figure A29-2 A, Representative chromatogram of a solution of calibrators containing 500 pg of normetanephrine (NMN), 500 pg of metanephrine (MN), 2000 pg of 3-hydroxy-4methoxybenzylamine (HMBA) internal standard, and 2000 pg of 3-ethoxy-4hydroxyphenylethanolamine (EHPEA) internal standard, 3-methoxytyramine (MTY). B, Representative chromatogram after injection of an extracted 2 mL sample of plasma from a normal 36-year-old female. C, Representative chromatogram after injection of an extracted 2 mL sample of plasma from a normal 31-year-old female with a small adrenal pheochromocytoma.
Analyte Normetanephrine Age <38 yr (pg/mL) (nmol/L) Age 38 yr (pg/mL) (nmol/L) Metanephrine Male (pg/mL) (nmol/L) Female (pg/mL) (nmol/L)
Geometric Mean
7 to 18). Data were log-transformed before analysis, and upper and lower reference intervals were determined from the 95% condence limits).
Geometric Mean Upper Reference Limit Lower Reference Limit
40 0.23 51 0.28
17 0.09 21 0.11
Analyte Normetanephrine Boys (pg/mL) (nmol/L) Girls (pg/mL) (nmol/L) Metanephrine Boys (pg/mL) (nmol/L) Girls (pgl/mL) (nmol/L)
29 0.15 24 0.12
62 0.31 55 0.28
21 0.11 11 0.06
48 0.26 38 0.21
97 0.53 77 0.42
20 0.11 20 0.11
Pediatric Reference Intervals Adjusted For Sex The reference intervals below are determined using data from 43 boys (mean age 13, range 5 to 18) and 43 girls (mean age 12, range
39 0.20 30 0.15
16 0.08 11 0.06
Copyright 2006, 1999, 1994, 1986 Elsevier Inc. All rights reserved.
Appendix 29
METHOD 29-2 Determination of Plasma Free Metanephrines by Liquid Chromatography With Electrochemical Detectioncontd
References 1. Lenders JW, Eisenhofer G, Armando I, Keiser HR, Goldstein DS, Kopin IJ. Determination of metanephrines in plasma by liquid chromatography with electrochemical detection. Clin Chem 1993;39:97103. 2. Roden M, Raffesberg W, Raber W, Bernroider E, Niederle B, Waldhausl W, Gasic S. Quantication of unconjugated metanephrines in human plasma without interference by acetaminophen. Clin Chem 2001;47:10617. 3. The method outlined here is also detailed on the internet at http://www.catecholamine.org/labprocedures.html, accessed April 21, 2005.
Copyright 2006, 1999, 1994, 1986 Elsevier Inc. All rights reserved.
10
Appendix 29
1. Internal stock standard solution (1.0 mg/mL). Weigh out 11.8 mg of d3-metanephrine HCl and 12.0 mg of d3-normetanephrine HCl. Transfer to a 10 mL volumetric ask and add RO H2O to volume. Store at -20 C. Stable 12 months. 2. Internal working dilution I (10 mg/mL). Add 100 mL stock standard to 9.9 mL RO H2O. Store at 2 C to 8 C. Stable 3 months. 3. Internal working dilution II (100 ng/mL). Add 100 mL internal working I to 9.9 mL RO H2O. Store at 2 C to 8 C. Stable 3 months. 4. Internal working dilution III (10 ng/mL). Add 1.0 mL internal working II to 9.0 mL RO H2O. Store at 2 C to 8 C. Stable 3 months. 5. Mobile phase: 40/60 (v/v) acetonitrile/HPLC grade water, 1.5 mmol/L ammonium acetate, 0.06% formic acid, 0.04% triuoroacetic acid. Weigh out 120 mg ammonium acetate and transfer to a 1 L ask. Add 600 mL RO H2O and 400 mL acetonitrile. Mix with magnetic stir bar until in solution. Add 60 mL formic acid and 40 mL triuoroacetic acid. Store at room temperature. Stable 2 weeks. 6. Injector rinse solution: 75/25 (v/v) methanol/RO H2O. Combine 750 mL methanol and 250 mL of RO H2O. Consumables 1. Plastic tubes, 13 75 mm, Sarstedt. 2. Disposable culture tubes, 10 75 mm, Fisher Scientic 14-961-25. 3. Pipette tips, 1000 mL and 200 mL, Continental Lab Products 2167, 2007. 4. Disposable plastic transfer pipettes, Fisher Scientic 13-711-7. 5. Autosampler vials, National Scientic Co., Target DP Catalog No. C4000-1, or Fisher Scientic minivials 0.2 mL Catalog No. 0334064 with snap-it-seal cap Catalog No. 0337749. 6. Caps, National Scientic Co., DP blue cap & TS septa, Catalog No. C4000-54B. 7. Inserts, National Scientic Co., 0.3 mL, Catalog No. C4010-630. Instrumentation 1. Gilson Pipetman pipettes, 1000 mL adjustable, 200 mL adjustable 2. Eppendorf repeater pipette 3. Supelco vacuum manifold 4. PE Sciex API 3000 LC-MS/MS with Atmospheric Pressure Chemical Ionization Source 5. PE Series 200 isocratic LC pump 6. PE Series 200 LC autosampler with 100 position sample tray insert 7. Analyst software 1.1 P/N 027232A 8. HPLC column: LUNA CN, 15 cm 4.6 mm, 5 mm, Phenomenex
9. Guard column: Security Guard CN, 2 cm 2.1 mm, 5 mm, Phenomenex 10. Ofine HPLC pump for ushing column after analysis Calibration The calibration uses a six point extracted calibrator curve over a concentration range of 0.25 to 10 nmol/L. Calibration curves are generated with each assay. The Analyst software package calculates the area counts and plots a calibration curve for the analyte/IS area count ratio versus analyte/IS concentration ratio. Remove calibrator set from freezer and let thaw about 30 minutes. Follow sample extraction procedure. Controls Analysis of spiked plasma controls (low, medium, and high) is performed with each assay. The spiked plasma control pools are made by adding calibrators to plasma pools so that the nal concentrations of MN and NMN are approximately 0.3, 0.6; 1.0, 2.0; and 2.0, 4.0 nmol/L, respectively. The pool is assayed to verify the appropriate concentration and further diluted if necessary. When the desired level is obtained, it is mixed well and aliquoted into plastic tubes and kept at -20 C. Tolerance limits are established by assaying each pool level 20 times over multiple days in conjunction with the current pools. Values are recorded using Levy-Jennings control charts and monitored for trends and shifts. Controls that are not acceptable will result in repeating selected values or the entire assay. Controls and curve parameters are used as a measure of assay acceptability with nal interpretation made by the laboratory supervisor or director. General QC rules in effect are:
All controls within 2 SD limits: One control exceeds 2 SD limits: Release results First time, release results Subsequent occurrences, take corrective action Take corrective action Take corrective action
Corrective action may include: (1) evaluating results, including repeats and linearity; (2) evaluating control pool and/or reagents; (3) repeating the assay; or (4) notifying supervisor or lead personnel. Procedure Sample Preparation 1. Invert plasmas to mix, and place 1.0 mL of patient or control plasma in a labeled 12 75 mm Sarstedt tube. 2. Add 50 mL of internal standard working dilution III to each tube using an Eppendorf repeater pipette.
Copyright 2006, 1999, 1994, 1986 Elsevier Inc. All rights reserved.
Appendix 29
11
Calculations Results are automatically calculated by the LC-MS/MS software and printed as nmol/L plasma. If a volume other than 1 mL plasma is used, the value should be edited appropriately. For example: if 500 mL is used, the nal result should be multiplied by 2. Reference Intervals
Metanephrine: Normetanephrine: <0.50 nmol/L <0.90 nmol/L
Interpretation Elevated results may indicate pheochromocytoma. Results may also be mildly elevated because of hypertension. Conversion factors: 1 nmol/L MN = 0.20 ng/mL MN 1 ng/mL MN = 5 nmol/L MN 1 nmol/L NMN = 0.18 ng/mL NMN 1 ng/mL NMN = 5.6 nmol/L NMN Procedural Notes The organic solvents used in the extraction and mobile phases are ammable and give off toxic fumes; they should be handled with care and used in an exhaust hood. Use eye protection when working with acids or bases. Handle all patient specimens as potentially infectious. Formal procedures for biological and chemical safety can be found in the Laboratory Safety Manual. 1. Metanephrine precision:
Within Run N Mean SD CV 20 0.25 0.03 10.3% 20 1.2 0.1 5.1% 19 2.3 0.1 4.9% Between Run 45 0.59 0.07 12.5% 71 1.2 0.1 7.3% 54 2.1 0.2 7.7%
3. Mobile phase: 40/60 (v/v) acetonitrile/HPLC grade water 1.5 mmol/L ammonium acetate. 0.06% formic acid, 0.04% triuoroacetic acid. HPLC column: LUNA CN, 15 cm 4.6 mm, 5 mm, Phenomenex Guard column: Security Guard CN, 2 cm 2.1 mm, 5 mm, Phenomenex.
MS/MS Parameters: Parameter MRM pairs Dwell times Nebulizer gas (NEB) Curtain gas (CUR) Collision gas (CAD) Nebulizer current (NC) Temperature (TEM) Declustering potential (DP) Focusing potential (FP) Entrance potential (EP) Collision energy (CE) Collision cell exit potential (CXP) MN 180.0/ 148.0 500 15 7 4 3 500 50 V 175 V 10 V 25 V 2V d3-MN 183.3/ 151.0 500 NMN 166.2/ 134.0 500 d3-NMN 169.2/ 137.0 500
2. Normetanephrine precision:
Within Run N Mean SD CV 20 0.70 0.05 7.5% 20 2.2 0.1 5.3% 19 2.7 0.1 4.5% Between Run 45 0.59 0.06 10.0% 71 1.9 0.2 8.8% 54 2.8 0.4 13.4%
After analysis is complete, ush columns with 50/50 acetonitrile/ water at 1 mL/min for 30 minutes. This will help to extend the column life. This can be performed with an off-line HPLC pump.
MN: N = 20, mean = 0.15, SD = 0.01, CV = 9.5% NMN: N = 20, mean = 0.34, SD = 0.02, CV = 5.0% 4. Recovery: Three different levels of calibrator were spiked into four different plasma specimens. The mean recoveries relative to the internal standard were 99% and 105% with ranges of 89% to 110% and 96% to 119% for MN and NMN, respectively. Absolute recovery from the extraction averaged 72% for MN and 60% for NMN. 5. Linearity: Elevated plasma samples were serially diluted with water and extracted separately to determine linearity. Results showed good linearity between 10.6 and 0.3 nmol/L for MN, and 28 to 0.4 nmol/L for NMN. Results greater than the highest calibrator (10 nmol/L) will be reextracted using 1 : 10 dilution with RO H2O. 3. Functional sensitivity:
Continued
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7. Reference intervals: An independent study was conducted using healthy volunteers to obtain a reference interval for the LCMS/MS method. Results matched the existing reference intervals for the HPLC-EC method. 8. Carryover: There are no problems due to carryover on the LCMS/MS. Reuse of Oasis cartridges was not assessed. 9. Sample stability: MN and NMN in EDTA plasma are stable when refrigerated up to 1 week or when frozen and thawed one time.
References 1. Eisenhofer G. Free or total metanephrines for diagnosis of pheochromocytoma: what is the difference. Clin Chem 2001; 47:988-9. 2. Grouzmann E, Fathi M, Gillet M, de Torrente A, Cavadas C, Brunner H, Buclin T. Disappearance rate of catecholamines, total metanephrines, and neuropeptide Y from the plasma of patients after resection of pheochromocytoma. Clin Chem 2001;47: 1075-82. 3. Lagerstedt SA, OKane DJ, Singh RJ. Measurement of plasma free metanephrine and normetanephrine by liquid chromatographytandem mass spectrometry for diagnosis of pheochromocytoma. Clin Chem 2004;50:603-11. 4. Lenders JW, Pacak K, Walther MM, Linehan WM, Mannelli M, Friberg P, et al. Biochemical diagnosis of pheochromocytoma: which test is best, JAMA 2002;87:1427-34. 5. Taylor RL, Singh RJ. Validation of liquid chromatography/tandem mass spectrometry method for analysis of urinary conjugated metanephrine and normetanephrine for screening of pheochromocytoma. Clin Chem 2002;48 533-9.
The reversed-phase column then has the physical characteristics of a conventional ion-exchange resin. A thin-layer, glassy carbon or carbon-paste working electrode, in conjunction with a silver-silver chloride reference electrode and a stainless steel auxiliary electrode, is used as the amperometric detection system. As shown earlier, each catecholamine passing through the detector cell undergoes a rapid two-electron oxidation at a xed potential to form an o-quinone. The resulting current is converted to a voltage signal and monitored as a function of time. At a constant temperature and ow rate, this oxidation current is directly proportional to the concentration of the analyte. Catecholamine reference materials that have been previously checked for purity are used to calibrate the system on the basis of
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Figure A29-3 Representative chromatogram of catecholamines extracted from a urine calibrator. Norepinephrine (A), epinephrine (B), 3,4-dihydroxybenzylamine (C), and dopamine (D) retention are identied on the chromatogram.
Calculations 1. Identify catecholamine and internal standard peaks by relative retention times (in minutes). Measure peak heights (nA) directly from the chromatographic tracings. Example:
Calibrating Compound Norepinephrine Epinephrine Internal standard Dopamine Retention Time (min) 4.5 5.5 6.4 9.0 Peak Heights, ht (nA) 56 14 18 108
2. Calculate ratio of peak heights (peak height of catecholamine/ peak height of internal standard) for each urine specimen, urine control, and calibrator. 3. Calculate concentrations of unknown catecholamines: CAT = Ru/Rc Cc 0.0104 where CAT = unknown catecholamine concentration (mg/mL) Ru = peak height ratio for unknown catecholamine Rc = peak height ratio for corresponding catecholamine stock calibrator applied to ion-exchange resin Cc = free-base concentration of catecholamine stock calibrator solution (mg/L = mg/mL) Factor 0.0104 is derived as follows: 0.05/5.0 26.02/5.0 = 0.0104
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Daily Excretion Relative to Creatinine mg/g Creatinine Age (yr) 0-1 1-4 4-10 10-18 >18 Age (yr) 0-1 1-4 4-10 10-18 >18 Age (yr) 0-1 1-4 4-10 10-18 >18 Norepinephrine up to 0.31 up to 0.29 up to 0.11 up to 0.11 up to 0.11 Epinephrine up to 0.38 up to 0.08 up to 0.09 up to 0.06 up to 0.04 Dopamine up to 1.29 up to 1.22 up to 0.72 up to 0.45 up to 0.35
Reference Moyer TP, Jiang NS, Tyce GM, Sheps SG. Analysis for urinary catecholamines by liquid chromatography with amperometric detection: methodology and clinical interpretation of results. Clin Chem 1979;25:256-63.
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Copyright 2006, 1999, 1994, 1986 Elsevier Inc. All rights reserved.
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The mass spectrometer is tuned for unit resolution of Q1 and Q3. Voltages are optimized for maximum sensitivity. MRM transitions monitored (m/z):
Epinephrine Epinephrine-d3 Norepinephrine Norepinephrine-d3 Dopamine Dopamine-d4 184 to 107, 184 to 77 187 to 110 170 to 107, 170 to 77 173 to 110 154 to 91, 154 to 65 158 to 95
Transitions m/z 184 to 107, 170 to 107, and 154 to 91 are quantitative, and transitions m/z 184 to 77, 170 to 77, and 154 to 65 are qualitative. Results 1. Evaluate the chromatography of the unextracted calibrator, calibrators, and controls for acceptable peak shapes and area counts. 2. The coefcient of determination (R2) for the calibration curve should be greater than 0.990. 3. The catecholamine concentration of the negative control should be less than the limit of detection for each analyte. 4. Qualitative ion mass ratios of (m/z 184 to 77)/(m/z 184 to 107) for epinephrine, (m/z 170 to 77)/(m/z 170 to 107) for norepinephrine, and (m/z 154 to 65)/(m/z 154 to 91) for dopamine must be within the range established by the calibration. This is typically 50% of the average of the ion-mass ratios observed in the calibrators of the run. 5. Evaluate the results for acceptable chromatography, ion ratios, and area counts as compared with the calibrators. Calculations Calculations are performed using peak area ratios relative to the corresponding internal standard. Commercial data analysis software is used. Sample chromatograms for each analyte are shown in Figure A29-4. Notes Acidic specimens (pH < 2) may not give adequate results. The pH of such a sample can be adjusted and the sample can be reextracted and reanalyzed. Interferences Compounds evaluated for interference using this method: acetaminophen, caffeine, cimetidine, dexamethasone, diazepam, isoetharine, isoproterenol, labetalol, levodopa, metoclopramide, metanephrine, 3-methoxytyramine, methyldopa, normetanephrine,
Continued
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Time min Time min Time min Figure A29-4 Chromatograms of an extracted urine sample containing 12 mg/L of epinephrine, 29 mg/L of norepinephrine, and 66 mg/L of dopamine. The analytes elute at the same retention time. Ion transitions: epinephrine (A) m/z 184 to 107 and (B) m/z 184 to 77; epinephrine-d3 (C) m/z 187 to 110; norepinephrine (D) m/z 170 to 107 and (E) m/z 170 to 77; norepinephrine-d3 (F) m/z 173 to 110; dopamine (G) m/z 154 to 91 and (H) m/z 154 to 65; and dopamine-d4 (I) m/z 158 to 95.
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Reference Kushnir MM, Urry FM, Frank EL, Roberts WL, Shushan B. Analysis of catecholamines in urine by positive ion electrospray tandem mass spectrometry. Clin Chem 2002;48:323-31.
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Figure A29-5 Representative chromatogram of metanephrines extracted from human urine. Normetanephrine (A), metanephrine (B), 3-methoxytyramine (C), and 4-Omethyldopamine (D) retention are identied on chromatogram.
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Reference Adapted from Parker NC, Levtzow CB, Wright PW, Woodard LL, Chapman JF. Uniform chromatographic conditions for quantifying urinary catecholamines, metanephrines, vanillylmandelic acid, 5-hydroxyindoleacetic acid, by liquid chromatography, with electrochemical detection. Clin Chem 1986;32:1473-6.
A calibration curve, generated from 20% methanol spiked calibrators, is included with each batch of patient samples. Specimen Collection and Handling Requires 10 mL of urine (pediatric: 5 mL) from a 24-hour collection. Add 10 g (pediatric 3 g) of boric acid or 25 mL (pediatric 15 mL) of 50% acetic acid as a preservative at the start of the collection. Measure and record the volume of the 24-hour collection. Send specimen refrigerated in a plastic, 13-mL urine tube.
Continued
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10. Store the aliquot in use at 4 C. Expiration3 months 11. Working internal standard. Add 200 mL of the d3metanephrine/d3-normetanephrine stock internal standard to 19.8 mL of 20% methanol. Store at 4 C. Expiration2 weeks. 12. 4.5 mol/L HCl. Add 187.5 mL of concentrated HCl (EM Science, Catalog No. 7647-01-1) to RO H2O and dilute to 500 mL. Store at room temperature. Expiration6 months. 13. 5.0 mol/L NaOH. Dissolve 200 g of sodium hydroxide pellets (Fisher Scientic, Catalog No. S318-500) in RO H2O, cool and dilute to 1 L. Store at room temperature. Expiration6 months. 14. 2.0 mol/L Phosphate Buffer. Dissolve 284 g of Na2HPO4 (Fisher Scientic, Catalog No. S374-500) and 272 g KH2PO4 (Fisher Catalog No. P285-500) in RO H2O and dilute to 2000 mL. Consumables 1. Autosampler Vials: 0.2 mL, 8 mm vials (Kimble Glass Inc.) from Fisher: Catalog No. 0334064 200/pk. 2. Caps: 8 mm snap-it cap (National Scientic Co.) from Fisher: Catalog No. 0337749 100/pk or 1000/case. 3. Extraction Cartridges: Oasis HLB 1cc (30 mg) Extraction Cartridges from Waters: Catalog No. WATO94225. Instrumentation 1. A PE Sciex API 2000 Tandem mass spectrometer is used to monitor multiple ion pairs. 2. A Perkin Elmer HPLC system is used for sample separation and introduction into the mass spectrometer. 3. A computer with the Analyst Software from PE Sciex is used for data processing. 4. The analytical column is a Supelco Discovery RP Amide C16 HPLC column. 5.0 cm 4.6 mm, 5 mm. Catalog No. 505005. 5. The guard column is Supelco Discovery RP-AmideC16 Guard Column. 2 cm 2.1 mm, 5 mm. Catalog No. 505110. 6. The guard column holder is a Supelco Direct Connect Holder for 4.6 mm ID column. Catalog No. 55205. 7. Visiprep Solid Phase Extraction (SPE) Vacuum Manifold from Supelco: Catalog No. 57250-U. Calibration 1. 20 mg/mL metanephrine/normetanephrine stock calibrator. Add 20 mL of the metanephrine/normetanephrine stock calibrator to 980 mL of 20% methanol, mix and cap. 2. 2 mg/mL metanephrine/normetanephrine stock calibrator. Dilute 100 mL of the 20 mg/mL metanephrine/normetanephrine stock calibrator with 0.9 mL of 20% methanol. 3. 0.2 mg/mL metanephrine/normetanephrine stock calibrator. Dilute 100 mL of the 2 mg/mL metanephrine/normetanephrine stock calibrator with 0.9 mL of 20% methanol. 4. Prepare working calibrators according to the following table:
Reagents 1. Methanol, HPLC grade. 2. Chromatographic mobile phase. Dissolve 3.1 g ammonium acetate in 850 mL of RO H2O (water produced by reverseosmosis) and add 150 mL of methanol. Degas the solvent by placing on a magnetic stir plate, stirring under vacuum. Degas for 3 to 5 minutes or until bubble formation is slight. Store at room temperature. Expiration1 week. 3. Rinse solution. Combine 1500 mL of methanol and 500 mL of RO H2O. Degas as described for chromatographic mobile phase. Store at room temperature. Expiration1 week. 4. 20% Methanol-Cartridge elution solvent. Combine 800 mL of methanol and 3200 mL RO H2O; mix. Store at room temperature. Expiration1 month. 5. 70% Methanol-Cartridge wash solvent. Combine 2800 mL of methanol and 1200 mL RO H2O; mix. Store at room temperature. Expiration date1 month. 6. -Metanephrine hydrochloride. Sigma, catalog No. M-8625. Store as indicated on package label. 7. -Normetanephrine hydrochloride. Sigma, catalog No. N-7127. Store as indicated on package label. 8. Metanephrine/normetanephrine stock calibrator 1.0 mg/mL. Dissolve 11.845 mg -metanephrine hydrochloride and 11.992 mg -normetanephrine hydrochloride in 10 mL of 0.05 mol/L HCl to give a stock calibrator of 1 mg/mL metanephrine/normetanephrine. Store the stock calibrator in 1.0-mL aliquots at -70 C. ExpirationNo expiration. Store the aliquots in use at 4 C. Expiration3 months. 9. d3-Metanephrine/d3-Normetanephrine stock internal standard. Dissolve 10 mg of deuterated metanephrine and 20 mg deuterated normetanephrine in 10 mL of 0.05 mol/L HCl to give a 1 mg/mL d3-metanephrine-2 mg/mL d3-normetanephrine stock internal standard. Store the stock internal standard in 1.0-mL aliquots at -70 C. ExpirationNo expiration.
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Quality Control/Quality Assurance Donor urine collections are used for controls (abnormal control may need to be spiked to reach appropriate level). They are stored as 20-mL aliquots at -20 C for up to 2 years. Three urine controls are included in each assay. Procedure 1. Centrifuge a 2.0-mL portion of each 24-hour and random urine collection sample and sample control in appropriately labeled 12- 75-mm glass tubes to remove all sediment and particulate matter. 2. Transfer 1.0 mL of each urine sample and control to appropriately labeled 16- 125-mm glass tubes. 3. Add 50 mL of 4.5 mol/L HCl to each tube. 4. Add 25 mL of working internal standard to each tube and vortex. 5. Hydrolyze each tube for 20 minutes in a boiling water bath. 6. Remove the tubes from the water bath and allow samples to cool for 5 minutes at room temperature. 7. Add 50 mL of 5.0 mol/L NaOH to each tube and vortex. 8. Add 500 mL of 2 mol/L phosphate buffer. Check pH of each sample. The pH should be 7.0 (1.0). If pH is outside this range, add 4.5 mol/L HCl or 5.0 mol/L NaOH drops until pH is acceptable. 9. Prepare a Waters HBL Oasis cartridge for each control and sample as follows using the Visiprep SPE vacuum manifold described in the equipment section of this procedure. Add 1.0 mL of methanol and pull through the cartridge using a vacuum. Do not allow the cartridge to become dry (do not pull air through the cartridge for longer than 30 seconds). For slow owing cartridges, push the methanol through manually. Add 1.0 mL of RO H2O and pull through the cartridge using a vacuum. Do not allow the cartridge to become dry (do not pull air through the cartridge for longer than 30 seconds). For slow owing cartridges, push the RO H2O through manually.
10. Apply each control and sample to the appropriate cartridge. Apply slight vacuum, keeping the ow rate of the sample through the cartridge less than 2 mL/min. 11. Wash each cartridge with 1.0 mL of RO H2O. Apply slight vacuum, keeping the ow rate of the RO H2O through the cartridge less than 2 mL/min. 12. Place collection tubes in the extraction manifold. Add 1.0 mL of 20% methanol to each cartridge. Apply slight vacuum, keeping the ow rate of the 20% methanol through the cartridge less than 2.0 mL/min, collecting the eluate in the collection tubes. 13. Transfer the eluate of each control and sample to an appropriately labeled autosampler vial and apply cap. 14. To reuse the cartridges: wash each cartridge with 1 mL of 20% methanol, 2 mL of 70% methanol, and 2 mL of methanol. Cartridges may be used 5 times. 15. Prepare the calibration curve as described in Calibration. 16. Initiate analysis and quantitation of the assay on the PE Sciex 2000 Tandem Mass Spectrometer using Analyst software. Calculations For 24-hour samples: Metanephrine and normetanephrine concentration mg/24hr: Tandem MS value ng/mL TV/1000 = mg/24hr (TV = the total volume of urine in mL for the 24-hour period) For random samples: Report the answer as mg metanephrine/g creatinine: Tandem MS value ng/mL (creatinine value mg/dL 100) = mg/g Procedural Notes 1. Precision a. Intraassay, n = 20
Metanephrine Mean CV Normetanephrine Mean CV 61 ng/mL 7.8% 88 ng/mL 13.2% 717 ng/mL 3.9% 664 ng/mL 5.3% 3038 ng/mL 4.8% 3409 ng/mL 5.8%
b. Inter-assay
Metanephrine N Mean CV Normetanephrine N Mean CV 15 8.5 ng/mL 15.9% 15 11.4 ng/mL 13.9% 21 90 ng/mL 7.9% 21 107 ng/mL 8.4% 21 516 ng/mL 8.6% 21 606 ng/mL 6.6% 21 2933 ng/mL 7.5% 19 4854 ng/mL 5.7% Continued
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Metanephrine
d3-Metanephrine
Normetanephrine
d3-Normetanephrine
2. Recovery a. Metanephrine. Four samples were spiked with 144, 575, and 2300 ng/mL of metanephrine and assayed. Recovery range was 92% to 109% with a total average of 101%. Below are two samples from the recovery study.
Spike (ng/mL) 0 144 575 2300 0 144 575 2300 Measured Metanephrine ng/mL 53 205 677 2380 54 203 644 2310 % Recovery 106% 109% 101% 103% 103% 98%
3. Linearity. a. Metanephrine. Four samples were run straight, 2, 5, 20, 40 (diluted in RO H2O) and assayed. The expected range was 88% to 109% with a total average of 103%. Below are two samples from the linearity study.
Measured Metanephrine ng/mL 3080 629 272 163 78 130 72 27 14 7.1 Expected Metanephrine ng/mL 616 308 154 77 65 26 13 6.5
b. Normetanephrine. Four samples were spiked with 144, 575, and 2300 ng/mL of normetanephrine and assayed. Recovery range was 92% to 113% with a total average of 102%. Below are two samples from the recovery study.
Spike (ng/mL) 0 144 575 2300 0 144 575 2300 Measured Normetanephrine ng/mL 89 233 651 2530 114 266 733 2730 % Recovery 100% 98% 106% 106% 107% 113%
b. Normetanephrine. Four samples were run straight, 2, 5, 20, 40 (diluted in RO H2O) and assayed. The expected range was 95% to 113%, with a total average of 101%. Below are two samples from the linearity study.
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Dilution Straight 5 10 20 40
Dilution Straight 2 5 10 20
Metanephrine
Normetanephrine
1. Correlation with photometric total metanephrines. 100 samples were assayed on the photometric method, X (with normal spectral curves), and LC-MS/MS method, Y, and analyzed by linear regression. Y = 0.815 X - 0.0065; r = 0.82
Metaphrine Normetaphrine Average % Difference % Difference Range 0.4% 2.6% 9.6% -17.1% to 13.1% -3.6% to 17.1% -10.5% to 19.5%
2. Sample stability. a. Ambient. Stability was determined on unpreserved, acetic acid, and boric acid specimens. The following table outlines results at 7 days.
Specimen Type Unpreserved Acetic acid Boric acid Metanephrine % difference Range -1.7% to 20.5% -11.9% to 13.3% -4.4% to 4.9% Metaphrine Normetaphrine Average % Difference % Difference Range 4.7% 5.7% 0.5% -17.8% to 28.1% -3.4% to 16.4% 3.3% to 16.5% Normetaphrine Average % Difference 8.9% 11.0% 11.3%
a. Freeze-Thaw. Freeze-thaw was determined on acetic acid and boric acid specimens. The following table outlines results after three freeze-thaw cycles.
Specimen Type Acetic acid Boric acid Metanephrine % Difference Range -26.5% to 4.8% -20.3% to 5.1% Metaphrine Normetaphrine Average % Difference % Difference Range -9.4% -9.2% -13.7% to 3.5% -21.4% to -2.7% Normetaphrine Average % Difference -4.7% -12.6%
b. Refrigerated. Stability was determined on unpreserved, acetic acid, and boric acid specimens. The following table outlines results at 7 days.
Reference Taylor RL, Singh RJ. Validation of liquid chromatography-tandem mass spectrometry method for analysis of urinary conjugated metanephrine and normetanephrine for screening of pheochromocytoma. Clin Chem 2002;48:533-9.
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Copyright 2006, 1999, 1994, 1986 Elsevier Inc. All rights reserved.
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14. Inject 20 mL each of calibrator, control, and patient sample onto the HPLC column. Figure A29-6 shows a representative chromatogram of VMA extracted from human urine. Calculations 1. Identify VMA and internal standard peaks based on relative retention times (in minutes). Measure peak heights directly from the chromatographic tracings or obtain from the HPLC data processing system. 2. Calculate peak height ratios for each urine sample, urine control, and calibrator (peak height of VMA/peak height of internal standard). 3. Establish a linear regression curve from peak height ratios using the data obtained from the 5-, 10-, and 20-mg/L calibrators. 4. From the VMA calibrator curve, determine the concentrations of unknowns (mg/L). 5. Calculate 24-hour VMA excretion by multiplying VMA concentration (mg/L) by 24-hour urine volume (L). 6. Calculate VMA excretion normalized for creatinine excretion by dividing VMA concentration (mg/L) by urine creatinine (g/L). Reference Intervals
Age (yr) 3-6 6-10 10-16 16-83 mg/d 1.0-2.6 2.0-3.2 2.3-5.2 1.4-6.5 mmol/d 5-13 10-16 12-26 7-33 mg/g Creatinine 4.0-10.8 4.0-7.5 3.0-8.8 mmol/mol Creatinine 2.3-6.2 2.3-4.3 1.7-5.0
Figure A29-6 Representative chromatogram of vanillylmandelic acid (VMA) extracted from human urine.VMA (A) and iso-VMA (B) retention are identied on the chromatogram.
References 1. Binder SR, Sivorinovsky G. Measurement of urinary vanilmandelic acid and homovanillic acid by high-performance liquid chromatography with electrochemical detection following extraction by ion-exchange and ion-moderated partition. J Chromatogr 1984;336:173-88. 2. Premel-Cabic A, Turcant A, Allain P. Normal reference intervals for free catecholamines and their acid metabolites in 24-h urine from children, as determined by liquid chromatography with amperometric detection. Clin Chem 1986;32:1585-7.
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Copyright 2006, 1999, 1994, 1986 Elsevier Inc. All rights reserved.
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Comments 1. Any sample with a value <0.01 mg/L is repeated with a 100 mL mobile phase reconstitution volume. 2. Any value >20 mg/L must be diluted and brought into the linearity range. Subsequent results will be multiplied times the appropriate dilution factor and the results reported accordingly. 3. Quantitation of VMA has been valuable in the diagnosis and follow-up of patients with pheochromocytoma and related neurogenic tumors. 4. A blank (reverse-osmosis water substituted for urine sample) will be run after the elevated control in each analysis batch and reviewed for potential autosampler carryover. Reference Intervals
Age <1 year 1 year 2-4 years 5-9 years 10-14 years Adults VMA, urine <27 mg VMA/mg Creatinine <18 <13 <8.5 <7 <8 mg VMA/24 hours
Reference Magera MJ, Thompson AL, Matern D, Rinaldo P. A liquid chromatography-tandem mass spectrometry method for the determination of vanillylmandelic acid in urine. Clin Chem 2003;49: 825-6.
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13. Inject 20 mL each of calibrator, control, and patient sample onto the HPLC column. Figure A29-7 shows a representative chromatogram of HVA extracted from human urine. Calculations 1. Identify HVA and internal standard peaks based on relative retention times (in minutes). Measure peak heights directly from the chromatographic tracings or obtain from the HPLC data processing system. 2. Calculate peak height ratios for each urine sample, urine control, and calibrator (peak height of HMA/peak height of internal standard). 3. Establish a linear regression curve from peak height ratios using the data obtained from the 5-, 10-, and 20-mg/L calibrators.
Figure A29-7 Representative chromatogram of homovanillic acid (HVA) extracted from human urine. HVA (A) and iso-HVA (B) retention are identied on the chromatogram.
Copyright 2006, 1999, 1994, 1986 Elsevier Inc. All rights reserved.
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Reference Binder SR, Sivorinovsky G. Measurement of urinary vanilmandelic acid and homovanillic acid by high-performance liquid chromatography with electrochemical detection following extraction by ion-exchange and ion-moderated partition. J Chromatogr 1984; 336:173-88.
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2. 3.
4.
5.
Chromatograms Figure A29-8 shows representative selected reaction monitoring (SRM) chromatograms of HVA and HVA-IS extracted from urine.
Figure A29-8 LC-MS/MS analysis of HVA urine. Peak legend: 1, HVA; 2, 13C18 O-HVA (internal standard, 5 mg/L). A, HVA calibrator 1.04 mg/L; B, normal urine HVA 1.8 mg/L; C, elevated urine HVA 16.7 mg/L. (From Magera MJ, Stoor A, Helgeson JK, Matern D, Rinaldo P. Determination of homovanillic acid in urine by stable isotope dilution and electrospray tandem mass spectrometry. Clin Chim Acta 2001; 306:35-41.)
Copyright 2006, 1999, 1994, 1986 Elsevier Inc. All rights reserved.
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3. Urinary HVA, a metabolite of dopamine, is frequently measured to support a diagnosis of neuroblastoma and pheochromocytoma and is useful in monitoring treated patients. 4. A blank (reverse-osmosis water substituted for urine sample) will be run after the elevated control in each analysis batch and reviewed for potential autosampler carryover. 5. Administration of -dopa may result in elevated HVA values. Reference Intervals
Age <1 yr 1-2 2-5 5-l0 10-15 Adult HVA, urine <35 mg/mg creatinine <23 <13.5 <9.0 <12.0 <8 mg/24 hr
Comments 1. Any sample with a value <0.01 mg/L is repeated with a 100-mL mobile phase reconstitution volume. 2. Any value >20 mg/L must be diluted and brought into the linearity range. Subsequent results will be multiplied times the appropriate dilution factor and the results reported accordingly.
Reference Magera MJ, Stoor A, Helgeson JK, Matern D, Rinaldo P. Determination of homovanillic acid in urine by stable isotope dilution and electrospray tandem mass spectrometry. Clin Chim Acta 2001;306: 35-41.
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Appendix 29
Figure A29-9 Representative chromatogram of 5-hydroxyindoleacetic acid (5-HIAA) extracted from human urine. 5-HIAA (A) and internal standard (B) retention are identied on the chromatogram.
4. Inject 15 mL of each sample onto the HPLC column. Chromatogram Figure A29-9 shows a representative chromatogram of 5-HIAA extracted from urine. Calculations 1. Identify 5-HIAA based on relative retention times (in minutes). Measure peak heights directly from the chromatographic tracings or obtain from the HPLC data processing system. 2. Calculate concentrations of unknowns: HIAA = Hu/Hc Cc where HIAA = unknown 5-HIAA concentration (mg/L) Hu = peak height of unknown Hc = peak height of calibrator Cc = concentration of 5-HIAA working calibrator (50 mg/L) 3. Calculate 24-hour 5-HIAA excretion by multiplying 5-HIAA concentration (mg/L) by 24-hour urine volume (L). Comments 1. Specimens having a concentration greater than 50 mg/L must be diluted with water and repeated. 2. Inclusion of an internal standard may improve assay precision. 5Hydroxyindole-2-carboxylic acid is available commercially for
this purpose and can be added to all assay tubes before HPLC analysis (e.g., 100 mL of a 50-mg/L solution). The composition of the mobile phase, however, may need to be adjusted slightly to resolve the internal standard from interfering substances. Internal standard peaks are identied from the chromatographic tracing, and peak heights are measured. Peak height ratios are then calculated for each urine sample, urine control, and calibrator (peak height of 5-HIAA/peak height of internal standard). These peak height ratios are used to calculate concentrations of unknowns. Reference Intervals Representative reference intervals for adults using HPLC are as follows:
1-7 mg/d [6-37 mmol/d] 0-6.6 mg/g creatinine [0-3.9 mmol/mmol creatinine]
Reference Modied from Chou PP, Jaynes PK. Determination of urinary 5-hydroxyindole-3-acetic acid using solid-phase extraction and reversed-phase, high-performance liquid chromatography with electrochemical detection. J Chromatogr 1985;341:167-71.
Copyright 2006, 1999, 1994, 1986 Elsevier Inc. All rights reserved.
Appendix 29
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Instrumentation A commercially available bench-top tandem mass spectrometer (Applied Biosystems/SCIEX API 2000 MS/MS with Turbo IonSpray interface source, or equivalent) is suitable, accompanied by HPLC pump and autosampler (Perkin Elmer [PE] Series 200 isocratic LC pump, PE Series 200 LC autosampler, or equivalent). The HPLC column is a Discovery RP-AmideC16 (Supelco) 5 cm 4.6 mm, 5 mm particle size. Several SPE columns have been evaluated and validated and include: BondElut C18 Extraction Cartridges, (Varian No. 12102001), 1mL (100mg), Discovery DSC-18 (Supelco No.52602-U) 1 mL (100 mg) and C18 SPE Cartridge (Zorbax No.5184-3590), 1 mL (100 mg). The following alternate SPE columns may be used if the eluting solvent in the procedure is substituted with 30% acetonitrile in reverse-osmosis water: Strata C18-E (Phenomenex No. 8B-S001EAK-S), 1 mL (100 mg), or Oasis HLB (Waters, No. WAT094225), 1 mL (30 mg). Additional equipment used includes an automated SPE sample processor (Gilson ASPEC, or equivalent) and an evaporator (Zymark Turbo-Vap). Quality Control Two controls (normal, elevated) are included in every run. Both urine controls are prepared in-house. Urine containing acetic acid preservative is obtained (approximately 2 L per control). The normal urine control is prepared by simply aliquoting and freezing the urine. Normal urine 5-HIAA is generally 1 to 4 mg/L. The elevated urine control is prepared by spiking the urine with approximately 18 mL of the 5-HIAA stock calibrator per liter of urine. The target range is approximately 15 mg/L. The spiked urine is mixed well, aliquoted, and stored frozen. Controls are stable for 2 years when kept frozen. The mean 5-HIAA and standard deviation are calculated from a minimum of 20 interassay values for each control. Routine assay control values are entered into a spreadsheet (MS Excel) that automatically plots the value in relation to the mean. As a general rule, control values that fall within 2 SD of the mean are acceptable and require no further action. Any control values that are >2 SD and shifts/trends require review by a supervisor or designee, and a completed HIAA Quality Report if indicated. Any control values that are >3 SD require a completed HIAA Quality Report and the review of a supervisor or designee. The laboratory QC designee performs monthly compilation, review, and sign off of control values. The nal review of the control spreadsheets is performed by one of the laboratory supervisory staff on a monthly basis.
Continued
Copyright 2006, 1999, 1994, 1986 Elsevier Inc. All rights reserved.
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Appendix 29
Copyright 2006, 1999, 1994, 1986 Elsevier Inc. All rights reserved.