Chapter 029

APPENDIX
29
Catecholamines and Serotonin

Thomas G. Rosano, Ph.D., Graeme Eisenhofer, Ph.D., Ronald J. Whitley, Ph.D., Ravinder Jit Singh, Ph.D., Mark M. Kushnir, M.S., Elizabeth L. Frank, Ph.D., D.A.B.C.C., F.A.C.B., Mark J. Magera, B.S., M.A., Dietrich Matern, M.D., and Piero Rinaldo, M.D., Ph.D.
SELECTED METHODS FOR ANALYSIS OF CATECHOLAMINES, SEROTONIN, AND THEIR METABOLITES
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
METHOD 29-1 Determination of Plasma Free Catecholamines by HPLC/EC

Submitted by Ronald J. Whitley, Ph.D. Department of Pathology and Laboratory Medicine, College of Medicine, University of Kentucky Medical Center, Lexington, Kentucky Principle Free catecholamines are rst rapidly adsorbed from plasma onto aluminum oxide at pH 8.6. After washing with water, the catecholamines are then desorbed with a small volume of perchloric acid. Subsequent separation of the individual catecholamines is achieved by HPLC under optimum isocratic conditions. A silica-based cation exchange column is used as the stationary phase, and an organic/ aqueous buffer mixture, pH 6.5, is used as the mobile phase. A thin-layer glassy carbon working electrode in conjunction with an Ag/AgCl reference electrode is used as the amperometric detection system. Each catecholamine passing through the detector cell undergoes a rapid two-electron oxidation at a xed potential to form an o-quinone: clonidine is preferred because this drug does not produce a falsepositive result. Catecholamines in general are unstable compounds and can be readily oxidized. To prevent this oxidation, many procedures recommend that blood samples be transported to the laboratory on ice, centrifuged at 4 C within 30 minutes of collection, and the plasma removed and frozen at -70 C until analysis. Two anticoagulants are commonly used, heparin and ethylenediaminetetraacetic acid (EDTA), with or without the addition of antioxidants such as glutathione or metabisulte. Advocates of a more liberal and less constrained method of collecting and processing blood catecholamines recommend the use of heparin without added preservatives. Catecholamine values from heparinized blood appear to be quite stable after separation (24 hours at 20 C, 48 hours at 4 C, 1 month at -20 C). At -70 C, catecholamines in heparin or EDTA plasma are stable for at least 8 months without the addition of preservatives. Reagents 1. Perchloric acid (HClO4), 72%, analytical grade. 2. Perchloric acid, 0.1 mol/L. Mix 8.6 mL 72% HClO4 with distilled water to a nal volume of 1 L. 3. Potassium hydroxide (KOH), 4 mol/L. Dissolve 112.2 g of KOH in 400 mL distilled water and dilute to 0.5 L. Store tightly capped. Stable for 1 year at room temperature. 4. Phosphoric acid (H3PO4), 85%, analytical grade. 5. Phosphoric acid, 2 mol/L. Mix 135 mL 85% H3PO4 with distilled water to a nal volume of 1 L. 6. Acetonitrile, HPLC grade. 7. Catecholamine-free plasma. Obtain one unit of frozen plasma from a blood bank (approximately 300 mL). Remove residual catecholamines as follows: a. Thaw and transfer the plasma to 50-mL centrifuge tubes. b. Incubate all tubes in a water bath at 56 C for 60 minutes. c. Centrifuge all tubes for 10 minutes at 3000 rpm. d. Remove an aliquot of the supernatant and analyze for catecholamines. If catecholamines are present, discard the entire unit of plasma. Otherwise, pipette 4.0-mL aliquots of the catecholamine-free plasma into storage vials. e. Freeze all aliquots. Store at -70 C. Stable for 1 month. 8. Catecholamine stock calibrator. Weigh and transfer the following catecholamines into separate 200-mL volumetric asks: 10.0 mg epinephrine free base (MW 183.2); 10.0 mg norepinephrine free base (MW 169.2); 12.4 mg dopamine HCl (3-hydroxytyramine HCl, MW 189.6). Dissolve and dilute to the mark with HClO4, 0.1 mol/L, to give free-base concentrations of 50 mg/L. Store the stock calibrators at 4 C. Stable for at least 6 months. 9. Catecholamine combination calibrator. Dilute 100 mL of the catecholamine stock calibrators to 100 mL with HClO4, 0.1 mol/ L, to give free-base concentrations of 50 mg/L. Store at 4 C. 10. Stock internal standard, 3,4-dihydroxybenzylamine, 50 mg/L. Dissolve 16 mg 3,4-dihydroxybenzylamine hydrobromide (DHBA HBr, MW 220.1) in HClO4, 0.1 mol/L. Dilute to 200 mL with HClO4, 0.1 mol/L, in a volumetric ask. Store at 4 C. Stable for at least 6 months. Note: The free-base concentration is calculated by multiplying the mass of the salt by the molecular weight ratio of free base to salt.
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
Appendix 29
METHOD 29-1 Determination of Plasma Free Catecholamines by HPLC/ECcontd

For example: (16 mg DHBA HBr)/(0.2 L perchloric acid) (MW free base = 139)/(MW salt = 220.1) = 50 mg/L (free-base concentration) Working internal standard, 3,4-dihydroxybenzylamine, 50 mg/L. Dilute 100 mL of the DHBA HBr stock internal standard (50 mg/L) to 100 mL with HClO4, 0.1 mol/L. Store at 4 C. Catecholamine prerun calibrator. Dilute 500 mL of the catecholamine combination calibrator and 1000 mL of the working internal standard to 10 mL with HClO4, 0.1 mol/L. Prepare fresh daily. Tris buffer. Dissolve 45 g of Trizma base and 5 g Na2EDTA in approximately 200 mL distilled water. Adjust to pH 8.6 by adding drops of concentrated HCl, 12 mol/L. Dilute to 250 mL and lter. Store at 4 C. Stable for 2 months. Water, pH 7.0. Using a pH meter, adjust approximately 500 mL of distilled water to pH 7.0 with sodium hydroxide (0.1 mol/L) or phosphoric acid (0.1 mol/L), as required. Check pH of H2O just before use. Adjust as necessary. Alumina (acid-washed aluminum oxide; Bioanalytical Systems, West LaFayette, Ind., Catalog No. CF-8010). Store tightly capped in a desiccator. Mobile phase, acetonitrile-citrate mixture. Dissolve 20.59 g trisodium citrate dihydrate in 880 mL distilled water. Using a pH meter, adjust the solution to pH 6.50 0.02 with H3PO4 as required. Add 120 mL acetonitrile and mix well. Filter the buffer mixture through a Millipore ltration device using a 0.45 Tm (Type HA) membrane lter. Release vacuum immediately after ltration is complete. Store tightly capped at room temperature. HPLC column wash reagent. Using a pH meter, adjust 1 L distilled water to pH 3 with H3PO4 (2 mol/L) as required. b. c. d. e. Epinephrine retention time is approximately 5 to 6 minutes. Epinephrine is clearly separated from the solvent front. Epinephrine peak height is 30% to 50% of full scale. All peak heights are within 5% on repeat injection.
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
Appendix 29
METHOD 29-1 Determination of Plasma Free Catecholamines by HPLC/ECcontd

7. Calculate concentrations of unknown catecholamines as follows: a. Estimate a linear regression line for the data set and calculate unknown concentrations. b. Manual calculation: CAT = U/S 50 0.04/4.0 1000 where: CAT = unknown catecholamine concentration (pg/mL) U = unknown catecholamine peak height ratio S = peak height ratio for 500 pg/mL calibrator determined from a calibration curve 50 = free-base concentration of catecholamine combination calibrator (50 mg/L) 0.04 = volume of catecholamine calibrator applied to alumina (mL) 4.0 = volume of patient sample or control applied to alumina (mL) 1000 = conversion factor (mg/L to pg/mL) 8. Linearity: norepinephrine: 152000 pg/mL epinephrine: 152000 pg/mL dopamine: 152000 pg/mL When calculated values exceed these limits, the patient extract is diluted with HClO4, 0.1 mol/L, and the analysis repeated started at procedure step 21. Comments 1. When calculated values are less than the linearity limits listed previously, results are reported as <15 pg/mL. 2. Labetalol (Normadyne and Trandate) interferes chemically with this assay. Reference Intervals
Norepinephrine Supine (30 min) Sitting (15 min) Standing (30 min) Epinephrine Supine (30 min) Sitting (15 min) Standing (30 min) Dopamine Supine (30 min) Sitting (15 min) Standing (30 min) pg/mL (pmol/L) <87 (<475) <87 (<475) <87 (<475) pg/mL (pmol/L) <50 (<273) <60 (<328) <90 (<491) pg/mL (pmol/L) 110410 (6502423) 120680 (7094019) 125700 (7394137)
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.
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
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
Appendix 29
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
Appendix 29
(A) Standards HMBA NMN EHPEA MN NMN = 500 pg MN = 500 pg MTY = 500 pg
(B) Normal 36 yr old female HMBA EHPEA
(C) 31 yr old female with MEN-2 a HMBA NMN EHPEA
MTY
NMN = 43 MN = 31 pg
NMN = 345 pg MN = 205 pg/m
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
Upper Reference Limit
Lower Reference Limit
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
95 0.52 125.00 0.68
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
102 0.52 73 0.37
16 0.08 11 0.06
Appendix 29
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.
METHOD 29-3 Determination of Plasma Free Metanephrine and Normetanephrine by LC-MS/MS

Submitted by Ravinder J. Singh, Ph.D. Department of Laboratory Medicine and Pathology, Mayo Clinic and Foundation, Rochester, Minn. Principle Free metanephrine (MN) and normetanephrine (NMN) are extracted from plasma using solid phase extraction. The concentrated eluate is analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and quantied using stable isotope-labeled internal standards, d3-metanephrine (d3-MN) and d3normetanephrine (d3-NMN). Analytes and internal standards are ionized using atmospheric pressure chemical ionization and are detected in the multiple reaction monitoring (MRM) mode using the specic transitions for m/z 180 to m/z 148, m/z 166 to m/z 134, m/z 183 to m/z 151, and m/z 169 to m/z 137, respectively. Metanephrine and normetanephrine are the metabolites of epinephrine and norepinephrine, respectively. They are produced by the actions of the enzyme catechol-O-methyltransferase (COMT). The membrane-bound form of the enzyme is specically highly expressed in pheochromocytes of the tumor. Measurements of plasma metanephrines have been found to be more sensitive than those of plasma catecholamines for the identication of patients with pheochromocytoma. Specimen Collection and Storage Use a 100 16 mm lavender stopper vacutainer containing 15 mg EDTA. Ten milliliters of blood is collected and centrifuged at 2000 rpm for 10 minutes. The plasma is removed and placed in 15 mL conical centrifuge tubes and is immediately frozen at -20 C until time of analysis. The specimen is stored at -20 C for shortterm storage. Store at -70 C if long-term storage is required. Reagents 1. -Metanephrine HCl, Sigma Chemical Co. M-8625 (or equivalent >98% purity). Store at 2 C to 8 C. Stable until expiration date. 2. -Normetanephrine HCl, Sigma Chemical Co. N-7127 (or equivalent >98% purity). Store at 2 C to 8 C. Stable until expiration date. 3. d3-Metanephrine HCl-a,a,b-d3, Cambridge Isotope Laboratories, Inc. Store at 2 C to 8 C. Stable until expiration date. 4. d3-Normetanephrine HCl-a,a,b-d3, Medical Isotopes, Inc., Pelham, NH. Store at 2 C to 8 C. Stable until expiration date. 5. Oasis TM HLB 1 cc (30 mg) extraction cartridges, Waters WAT094225. Store at room temperature. Stable until expiration date. 6. Charcoal Stripped Serum, SeraCare, Inc. HS-230. Store at 2 C to 8 C. Stable until expiration date. 7. Methanol (HPLC grade), EM Science MX0488-1 (4L). Store at room temperature. Stable until expiration date. 8. Water, HPLC Grade, Fisher Scientic W5-4. Store at room temperature. Stable until expiration date. 9. Formic acid, Sigma F-0507. Store at room temperature. Stable until expiration date. 10. Triuoroacetic acid (TFA), Pierce 28901. Store at room temperature. Stable until expiration date. 11. Ammonium acetate, Sigma A-7330. Store at 2 C to 8 C. Stable until expiration date. 12. Acetonitrile (HPLC grade), Aldrich 27,071-7 (2 L). Store at room temperature. Stable until expiration date. 13. Analytical stock calibrator solution (1.0 mg/mL). Weigh out 11.8 mg of metanephrine HCl and 12.0 mg of normetanephrine HCl. Transfer to a 10 mL volumetric ask and add RO H2O (water processed by reverse-osmosis) to volume. Store at -20 C. Stable 12 months. 14. Analytical working dilution I (10 mg/mL). Add 100 mL stock calibrator to 9.9 mL RO H2O. Store at 2 C to 8 C. Stable 3 months. 15. Analytical working dilution II (100 ng/mL). Add 100 mL analytical working I to 9.9 mL RO H2O. Store at 2 C to 8 C. Stable 3 months. 16. Analytical working dilution III (10 ng/mL). Add 1.0 mL analytical working II to 9.0 mL RO H2O. Store at 2 C to 8 C. Stable 3 months. 17. Analytical working dilution IV (1 ng/mL). Add 1.0 mL analytical working III to 9.0 mL RO H2O. Store at 2 C to 8 C. Stable 3 months. 18. Calibrators 1 to 6. Pipette stripped serum and analytical calibrator (see chart) into 12 75 tubes; store at -20 C.
Continued
10
Appendix 29
METHOD 29-3 Determination of Plasma Free Metanephrine and Normetanephrine by LC-MS/MScontd

Calibrator 1 2 3 4 5 6 Concentration Metanephrine 0.25 nmol/L 0.5 nmol/L 1.0 nmol/L 2.5 nmol/L 5.0 nmol/L 10.0 nmol/L Concentration Normetanephrine 0.27 nmol/L 0.54 nmol/L 1.08 nmol/L 2.7 nmol/L 5.4 nmol/L 10.8 nmol/L Volume Analytical Stripped Serum 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL Volume Analytical Std IV (1 ng/mL) 50 mL 100 mL 200 mL Volume Analytical Std III (10 ng/mL) 50 mL 100 mL 200 mL
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
One control exceeds 3 SD limits: Two or more controls exceed 2 SD limits:
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.
Appendix 29
11

Solid Phase Extraction 1. Load Oasis HLB SPE cartridges on to the Supelco vacuum manifold and pull vacuum at 5 in Hg for all steps. Precondition the cartridges with 1 mL methanol followed by 1 mL RO H2O. 2. Add the plasma/IS mixture with a plastic disposable transfer pipette. 3. Wash with 2 mL of RO H2O. 4. Place an empty 12 75 mm disposable culture tube in the manifold rack for each SPE cartridge. Place manifold rack into manifold and verify that all tubes are in line with a SPE connection. Elute MN and NMN with 1 mL methanol. Discard Oasis cartridges after each use. 5. Evaporate the eluate in a Zymark TurboVap LV evaporator at 45 C and 15 psi for 20 minutes. 6. Reconstitute with 50 mL methanol and transfer to autosampler vials. Refrigerate until analysis. Extracted specimens are stable for up to 3 days. LC-MS/MS 1. Build the sample batch using the Analyst software and inject the preview standard to insure that the instrument is operating properly and that the chromatography is resolving the peaks of interest. (See API 3000 Tandem Mass Spectrometer Operating Procedure.) 2. Continue building the load and start the run.
Project name: Acquisition method: Flow Inj volume Run time Pre/Postinjection ushes PMET PMET 1.2 mL/min 30 mL 8.0 min 0/5
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
12
Appendix 29

6. Correlation: A comparison between the Mayo LC-MS/MS method (Y) and the Mayo HPLC-EC method (X) was performed. Linear regression results are indicated below.
N Metanephrine (nmol/L) Normetanephrine (nmol/L) 92 132 Slope 0.87 0.90 Y-intercept 0.05 -0.06 Correlation Coefcient 0.95 0.93
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.
METHOD 29-4 Determination of Urinary Free Catecholamines by HPLC/EC

Submitted by Ronald J. Whitley, Ph.D. Department of Pathology and Laboratory Medicine, College of Medicine, University of Kentucky Medical Center, Lexington, Ky. Principle After protein precipitation with perchloric acid, an aliquot of a 24-hour urine collection (preserved in acid) is rst applied to a weak-acid cation-exchange resin. Unconjugated catecholamines are selectively adsorbed at pH 6.5 and then eluted with dilute boric acid (pH 4.0). An intermediate water wash removes interfering urine impurities. Subsequent resolution of the individual catecholamines is achieved by reversed-phase, paired-ion HPLC under optimized isocratic conditions. Alkyl-bonded silica is used as the nonpolar stationary phase, and an organic/aqueous buffer mixture (pH 2.8) is used as the polar mobile phase. To enhance the afnity of the polar catecholamines for the hydrophobic stationary phase, an ion of opposite charge (octyl sodium sulfonate) is also included in the mobile phase. This counter-ion is capable of forming uncharged ion pair conjugates with catecholamine cations before partitioning into the lipophilic stationary phase: This separation mechanism may also be described by postulating that the counter-ion itself partitions into the stationary phase with its ionic groups oriented at the surfaces:
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
Appendix 29
13
METHOD 29-4 Determination of Urinary Free Catecholamines by HPLC/ECcontd

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 All antihypertensive medications should be withheld from the patient for at least 2 days before and during specimen collection. If the patient cannot be completely removed from a drug regimen, the test may still be performed as long as the results are evaluated with an understanding of the expected physiological response to the drug or drugs. A complete 24-hour urine sample is collected in a gallon jug containing 10 mL hydrochloric acid (HCl), 6 mol/L, as a preservative. The specimen should be refrigerated during collection. The total urine volume is measured and recorded; a 100-mL aliquot is stored in the refrigerator until the test is performed, or may be frozen and stored indenitely. Reagents 1. Perchloric acid (HClO4), 72%, analytical grade. 2. Phosphoric acid (H3PO4), 85%, analytical grade. 3. Sodium phosphate (Na2HPO4), 10 mmol/L. Dissolve 1.42 g anhydrous Na2HPO4 in distilled water to nal volume of 1 L. Stable at 4 C for 3 months. 4. Potassium phosphate (KH2PO4), 10 mmol/L. Dissolve 1.36 g KH2PO4 in distilled water to nal volume of 1 L. Stable at 4 C for 3 months. 5. Phosphate buffer, 10 mmol/L, pH 6.5. Mix 32 mL Na2HPO4 (10 mmol/L) and 68 mL KH2PO4 (10 mmolL) to give 100 mL of a phosphate buffer, pH 6.5. Stable at 4 C for 3 months. 6. Disodium EDTA (Na2EDTA), 2.7 mmol/L. Dissolve 1 g Na2EDTA in 1 L distilled water. Stable at room temperature for 3 months. 7. Sodium hydroxide (NaOH), 0.5 mol/L. Dissolve 20.0 g NaOH in distilled water and dilute to 1 L. Store tightly capped. Prepare monthly. 8. Boric acid (H3BO3), 0.65 mol/L. Dissolve 40 g H3BO3 in 800 mL distilled water. Warm in a 50 C water bath to aid dissolution. Allow to cool and dilute to 1 L with water. Store at room temperature and prepare every 2 weeks. 9. Catecholamine stock calibrator. Weigh and transfer the following catecholamines to a 500-mL volumetric ask: 30.7 mg norepinephrine bitartrate monohydrate (MW 346.3), 9.1 mg epinephrine bitartrate (MW 333.3), and 55.8 mg dopamine HCl (MW 189.6). Dissolve and dilute to the mark with HClO4, 0.1 mol/L, to give the following free-base concentrations: norepinephrine, 30 mg/L; epinephrine, 10 mg/L; and dopamine, 90 mg/L. Store the stock calibrator at 4 C. Stable for at least 6 months. Note: Free-base concentrations are calculated by multiplying the mass of the catecholamine salt by the molecular weight ratio of free-base to salt. For norepinephrine: (30.7 mg norepinephrine bitartrate)/(0.5 L perchloric acid) (MW free-base = 169.2)(MW salt = 346.3) = 30 mg/L (free-base concentration) 10. Internal standard, 3,4-dihydroxybenzylamine, 10 mgL. Dissolve 8 mg of 3,4-dihydroxybenzylamine hydrobromide (MW 220.1) in perchloric acid, 0.1 mol/L. Dilute to 500 mL with HClO4, 0.1 mol/L, in a volumetric ask to give a free-base concentration of 10 mg/L. Store at 4 C. Stable for at least 6 months. 11. Catecholamine working calibrator. Add 50 mL of the catecholamine stock calibrator and 50 mL of the internal standard solution to 7.0 mL H3BO3, 0.65 mol/L. Prepare fresh daily. 12. Methanol, spectrograde. 13. Sodium octyl sulfate. 14. Phosphoric acid, 0.1 mol/L. Dilute 6.9 mL H3PO4 (85%) to 1 L with distilled water. Store at room temperature. 15. Mobile phase, methanol/phosphate buffer (10:90). Dissolve 21.5 g KH2PO4, 74 mg Na2EDTA, and 100 mg sodium octyl sulfate in 1 L distilled water. Add 3.3 mL H3PO4 (85%) and dilute to 2 L with distilled water. Buffer is phosphate, 0.1 mol/L, pH 2.8; octyl sodium sulfate, 50 mg/L; and Na2EDTA, 0.1 mmol/L. Filter the buffer through a Millipore ltration apparatus using a 2-mm membrane lter. Degas for 1 hour with a vacuum and then add methanol (100 mL/L buffer). Stir the mixture for 10 minutes at 40 C. Further degas the mixture for 10 minutes by slowly bubbling nitrogen gas through the mixture. Store tightly capped at room temperature. (Note: Changes in the composition of the mobile phase will be necessary as the HPLC column ages and as catecholamine retention times decrease. To maintain peak integrity and a consistent retention time, the concentration of methanol is decreased and that of the ion-pair reagent increased during the lifetime of the column [5 to 6 months]. With a new column, begin with the mobile phase containing 50 mg/L octyl sulfate and 10% methanol. Adjust the methanol concentration to sharpen peaks and to optimize retention times. Allow 1 to 2 hours between adjustments for the column to equilibrate with the mobile phase. Full equilibration requires 3 to 4 hours. Overnight equilibration may be more convenient.) 16. Bio-Rex 70 exchange resin (50 to 100 mesh, Bio-Rad Laboratories). Prepacked columns may be obtained from Bio-Rad. Alternatively, the resin may be washed and packed into plastic columns as follows: a. With gentle manual agitation, wash the resin with successive volumes of hydrochloric acid, 3 mol/L; NaOH, 3 mol/L; acetic acid, 3 mol/L; ammonium acetate, 1.0 mol/L (pH 6.5); and ammonium acetate, 0.1 mol/L (pH 6.5). b. Prepare a slurry of about 3 g resin and 15 mL ammonium acetate buffer, 0.1 mol/L (pH 6.5). c. Pipette the resin slurry into a plastic chromatography column, 1 cm I.D. 8 cm in length. d. Allow the resin to pack in each column to a height of about 2.5 cm. e. Store packed columns at 4 C after carefully sealing both tip and cap with paralm. Stable for at least 1 year. 17. Nitrogen gas, prepuried, 99.995% minimum purity. Instrumentation A commercially available HPLC system equipped with a 25 0.4 cm (I.D.) reversed-phase C18 (average particle diameter, 5 or 10 mm) column and an electrochemical detector is suitable.
Continued
14
Appendix 29

Controls Three urine controls are included in every run. For example, Bio-Rad Lyphochek Quantitative Urine Control, Levels 1 and 2 (Bio-Rad Clinical Diagnostics Group, Hercules, Calif.) are reconstituted each day and processed for immediate use. A 24-hour urine collection from a healthy individual is also used as a control pool. This urine is adjusted to pH 3 with hydrochloric acid, 6 mol/L, and spiked with the catecholamine stock calibrator, 10 mL per L of urine. Sixmilliliter aliquots of the spiked urine are frozen in polypropylene vials and stored for future test runs. Procedure Sample Preparation 1. Into separate 50-mL centrifuge tubes, pipette 25.0 mL of each urine specimen and control. 2. Add 1.0 mL concentrated HClO4. Cap and vortex mix for 15 seconds. Allow to stand for 10 minutes and then centrifuge for 5 minutes at 900 g. 3. Pipette 5.0 mL of each supernatant into 50-mL plastic beakers. Store the remaining volume of the supernatants in the freezer. 4. Transfer 5.0 mL of phosphate buffer, 10 mmol/L (pH 6.5), to a 50-mL beaker and add 50 mL of the catecholamine stock calibrator. 5. To all beakers, add 50 mL of the internal standard solution (3,4-dihydroxybenzylamine, 10 mg/L) and 15 mL of Na2EDTA, 2.7 mmol/L. Adjust the pH to 6.5 0.1 with NaOH, 0.5 mol/L, or H3PO4, 0.1 mol/L. Ion-Exchange Chromatography 6. Prepare one Bio-Rex 70 ion-exchange column for each urine specimen, urine control, and calibrator to be analyzed. Insert the column into a suitable support rack and allow to drain into a drainage tray. 7. Quantitatively transfer the entire contents of each beaker onto separate columns, then rinse each beaker with 5 mL of water. Apply the rinse solutions to the respective columns and allow to drain completely. 8. Wash the columns with two 10-mL portions of water to remove urine contaminants. 9. When completely drained, add 7.0 mL H3BO3, 0.65 mol/L, to each column and collect the eluate in clean polypropylene vials, 17 100 mm. 10. Cap and mix the vials by inversion. Note: At this time, samples may be stored at 4 C for up to 48 hours before injection into the HPLC system. High-Performance Liquid Chromatography 11. Establish optimal operating conditions of the chromatographic and detection systems. Suggested conditions are as follows: Flow rate: 0.8 mL/min Potential: 720 mV Sensitivity: 100 nA/V Chart speed: 1 cm/min 12. Before the introduction of specimens onto the reversed-phase HPLC column, adjust the pH of each sample to 2.8 0.1 with H3PO4. Inject 100 mL of each mixture. Figure A29-3 shows a representative chromatogram of catecholamines extracted from a urine calibrator.
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
Appendix 29
15

where 0.05 = volume (mL) of catecholamine stock calibration applied to ion-exchange column 5.0 = volume (mL) of deproteinized urine applied to exchange column 25.0 = 24-hour urine aliquot (mL) 26.0 = total volume (mL) of deproteinized mixture (urine and perchloric acid) 4. Calculate 24-hour catecholamine excretion: CAT/d = CAT TV where CAT, mg/d = catecholamine excretion per day CAT, mg/mL = unknown catecholamine concentration TV, mL = 24-hour urine volume Comments Amperometric detectors for catecholamine analysis by HPLC consist of carbon-paste or glassy-carbon working electrodes. Carbon paste consists of very nely divided particles of graphite and a binder that is mixed thoroughly to the consistency of a semidry paste. The binder holds the particles of graphite together as an integral unit and should be inert, both chemically and physically, to maintain sensitive electrode performance with minimal noise. Parafn oil and silicone grease have proved most satisfactory and are popular in this regard. However, the choice of the binder depends on its solubility in the mobile phase; acetonitrile, a constituent of many mobile phases, dissolves parafn oil; carbon paste using parafn oil as binder would be obviously unsuitable under this condition. Glassy carbon has also come into use as a general-purpose electrode for amperometric detectors. Glassy carbon is a hard, brittle, solvent-impervious electrode material. Because it is 100% carbon, the chemical and physical resistance of glassy carbon to all mobile phases is unequaled. Detector characteristics, such as linearity and electron transfer property, are comparable for both electrodes. However, the carbonpaste electrode under optimal operating conditions provides more sensitive detection than that obtained using the glassy-carbon electrode. Reference Intervals
Urinary Daily Excretion mg/d (nmol/d) Age (yr) Children 0-1 1-2 2-4 4-7 7-10 10-15 Norepinephrine 0-10 (0-59) 1-17 (6-100) 4-29 (24-171) 8-45 (47-266) 13-65 (77-384) 15-80 (89-473) Adults >15 Age (yr) Children 0-1 1-2 2-4 4-7 7-10 10-15 Adults >15 Age (yr) Children 0-1 1-2 2-4 4-7 7-10 10-15 Adults >15 65-400 (424-2612) 0.5-20 (3-109) Dopamine 0-85 (0-555) 10-140 (65-914) 40-260 (261-1697) 65-400 (424-2612) 65-400 (424-2612) 65-400 (424-2612) 15-80 (89-473) Epinephrine 0-2.5 (0-14) 0-3.5 (0-19) 0-6.0 (0-33) 0.2-10 (1-55) 0.2-10 (1-55) 0.5-20 (3-109)
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.
16
Appendix 29
METHOD 29-5 Determination of Urinary Free Catecholamines by LC-MS/MS

Submitted by Mark M. Kushnir, M.S. (ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah) and Elizabeth L. Frank, Ph.D. (Department of Pathology, ARUP Laboratories, University of Utah School of Medicine, Salt Lake City, Utah) Principle An internal standard, phenylboronic acid (PBA) in a buffer solution, and extraction solvent are added to calibrators, controls, and patient samples. Samples are vortexed, centrifuged, and the organic phase is transferred to a new set of tubes. Acetic acid solution and 1-octanol are added to the tubes and catecholamines are extracted into aqueous solution. The organic phase is discarded, the aqueous solution is transferred into autosampler vials, and aliquots are injected onto the liquid chromatograph-tandem mass spectrometer (LC-MS/MS). Advantages of the LC-MS/MS method for catecholamine (CATS) analysis compared with commonly used high performance liquid chromatography (HPLC) methods include signicantly greater instrument throughput and improved specicity. The specicity of this sample preparation method is based on the difference in afnity to PBA of CATS and potentially interfering compounds present in the sample matrix. CATS react with PBA in an alkaline solution that promotes the existence of the boronate ion, Ph2B(OH)-, a reactive form of PBA. Compounds containing vicinal cis-diol groups, such as those present in CATS, covalently bind to the boronate ion with the concomitant release of a molecule of water. The product PBA-catecholamine complex is polar and must be coupled with an ion-pairing reagent, such as tetraoctylammonium bromide, to be extracted into a nonpolar solvent. The complexation reaction requires a pH above 8.5, while release of CATS from the complex takes place in acidic solution. Collision energies were selected to provide an optimal signal for each product ion. Voltages on the collision cell are alternated accordingly for each multiple reaction monitoring (MRM) transition specied in the acquisition scan. Alternating the collision cell voltages produces consistent product ion response ratios for qualitative conrmation of the catecholamines. The conditions employed produce satisfactory sensitivity and specicity for the selective detection of CATS in urine. Specimen Collection and Storage An aliquot (3.0 mL) from a random or 24-hour urine collection is required. The sample should be stored at refrigerator temperature during a timed collection and until testing is performed. The total 24-hour urine volume should be recorded (accurate to 5 mL). Alkaline specimens (pH > 7) and specimens stored at ambient temperature are not acceptable. Adjusting the pH to 2-3 by adding acid, such as 6 mol/L HCl, to the sample during collection can enhance stability of the catecholamines. Specimens are stable frozen for 6 months. The minimum sample volume is 1.0 mL. Reagents 1. Acetic acid (C2H4O2, 0.08 mol/L). Add 0.5 mL of glacial acetic acid to 50 mL of deionized water and mix. Bring the total volume to 100 mL with deionized water. Stable for 2 months at 20 C. 2. Formic acid (CH2O2, 0.007 mol/L). To prepare 500 mL of this reagent, measure 500 mL of deionized water into a 1 L beaker. Stir and add 150 mL of formic acid. Mix for 15 minutes. Stable for 5 days at 20 C. 3. 1-Octanol (C8H18O). 4. Stock calibrator diluent. Acetic acid (0.08 mol/L)/sodium metabisulte (Na2S2O5, 0.005 mol/L) solution. To prepare 100 mL of this reagent, volumetrically add 100 mL of 5 mL/L acetic acid to a 150-mL ask. Stir and add 100 mg of sodium metabisulte. Mix until the solid is dissolved completely. Stable for 1 week at 20 C. 5. Catecholamine stock calibrators. Epinephrine, norepinephrine, or dopamine (1.0 g/L) in acetic acid/sodium metabisulte diluent (AASM). Stock solutions for each catecholamine are prepared in separate tubes. Using a 5 mL volumetric pipette, add exactly 5.0 mL of AASM diluent to a screw top test tube labeled with the name of the calibrator. Weigh 5.00 mg of epinephrine (C9H13NO3), 9.43 mg of norepinephrine bitartrate salt (C8H11NO3C4H6O6), or 6.19 mg of dopamine hydrochloride (C8H11NO2HCl). Transfer the solid into a labeled tube. Cap and mix using a rocker or vortex until the solid is dissolved completely. Stable for 1 year at -70 C. 6. Catecholamine working calibrator. Epinephrine, norepinephrine, and dopamine (0.5 mg/L). In a 50-mL volumetric ask place 40 mL of methanol and 25 mL each of epinephrine, norepinephrine, and dopamine stock calibrator solutions. Add methanol to a total volume of 50 mL and mix. Stable for 6 months at -70 C. 7. Catecholamine stock internal standard solutions. Epinephrine-d3, norepinephrine-d3, and dopamine-d4 (1.0 g/L) in AASM. Stock solutions for each internal standard are prepared in separate tubes. Weigh 5.00 mg of epinephrine-d3 (C9H10D3NO3), 9.36 mg of norepinephrine-d3 bitartrate salt (C8H8D3NO3C4H6O6), or 6.16 mg dopamine-d4 hydrochloride (C8H7D4NO2HCl). Transfer the solid into the corresponding tube. Using a 5-mL volumetric pipette, add exactly 5.0 mL of AASM diluent to a screw top test tube labeled with the name of the deuterated catecholamine standard. Cap and mix using a rocker or vortex until the solid is dissolved completely. Stable for 2 years at -70 C. 8. Catecholamine working internal standard. Epinephrine-d3 and norepinephrine-d3 (1.5 mg/L) and dopamine-d4 (3.0 mg/L). In a 50-mL volumetric ask, place 40 mL of methanol and 75 mL of each epinephrine-d3 and norepinephrine-d3 stock internal standard solutions and 150 mL of dopamine-d4 stock internal standard solution. Add methanol to a total volume of 50 mL and mix. Stable for 1 year at -70 C. 9. Catecholamine-free urine. Catecholamine-free urine is prepared by incubation of a nonpathological urine sample at alkaline pH. Adjust 300 mL of urine to pH > 13 using sodium hydroxide (NaOH, 2 mol/L). Incubate the urine at 40 C to 45 C for 48 hours. Analyze an aliquot of the urine using this procedure. If the concentrations of catecholamines in the urine are above the limit of detection of the method, incubate the sample for an additional 24 hours. If the catecholamine concentrations are less than or equal to the limit of detection, dilute urine with water to 600 mL. Adjust the pH to between 3 and 4 with hydrochloric acid (HCl, 6 mol/L). Stable for 6 months at -70 C. 10. Diphenylboric acid 2-aminoethyl ester (C14H16BNO, PBA) solution. In a 1 L beaker dissolve 106 g of ammonium chloride (NH4Cl) and 5 g of ethylenediaminetetraacetic acid (C10H16N2O8, EDTA) in 900 mL of water. Adjust the pH of the solution to 8.95 0.05 with ammonium hydroxide (NH4OH).
Appendix 29
17
METHOD 29-5 Determination of Urinary Free Catecholamines by LC/MS/MScontd

Dissolve 0.5 g of PBA in this solution and add deionized water to a total volume of 1000 mL. Mix until PBA is completely dissolved. Stable for 3 months at 4 C. 11. Tetraoctylammonium bromide (C32H68NBr, TOAB) solution. TOAB (11 mmol/L) in heptane (C7H16). Into a 1 L beaker add 10 mL of 1-octanol and 6 g of TOAB. Add heptane to a total volume of 1000 mL. Mix until completely dissolved. Stable for 3 months at 4 C. 12. Mobile phase. Tetrahydrofuran (THF, C4H8O)/formic acid (0.007 mol/L), (60 : 40). To prepare 500 mL of mobile phase, pour 300 mL of THF into a 1 L beaker. Add 200 mL of 0.07 mol/L aqueous formic acid. Mix. Stable for 3 days at 20 C. 13. Needle wash solution. Methanol (CH4O)/2-propanol (C3H8O)/water, (60 : 20 : 20). To prepare 1000 mL of needle wash solution, pour 600 mL of methanol into a 1000 mL ask. Add 200 mL of 2-propanol and then add 200 mL of deionized water. Mix. Stable for 5 days at 20 C. Instrumentation A commercially available HPLC instrument equipped with a column (Allure Basix, 50 mm 2 mm, 5 mm particles; Restek Corp., Bellefonte, Pa.); a tandem mass spectrometer with ion spray interface. Quality Control Two commercial urine controls (Bio-Rad Laboratories, Irvine, Calif.) are included in every run. Catecholamine-free urine serves as a negative control. Procedure Sample Preparation 1. Label a 2-mL microcentrifuge tube for each calibrator, control, and urine specimen. 2. Prepare an unextracted calibrator by aliquoting 6 mL of working calibrator in an empty tube. Add 50 mL of 10 mL/L acetic acid to this tube. This sample is not extracted. 3. Quantitatively add into empty tubes 30 mL of the working internal standard solution. 4. Prepare a calibration curve at concentrations of 10, 25, 100, and 200 mg/L. Add 300 mL of catecholamine-free urine in each tube. Aliquot to the labeled microcentrifuge tubes 6, 15, 60, and 120 mL of working calibrator solution. 5. Aliquot 300 mL of each urine sample in labeled microcentrifuge tubes. 6. Add 900 mL of TOAB extraction solvent to each tube. 7. Add 750 mL of PBA/buffer solution to each tube. 8. Vortex tubes on high speed for 3 minutes; centrifuge at 15,000 g for 3 minutes. 9. Label a clean tube for each calibrator, control, and urine specimen. Add 50 mL of 0.08 mol/L acetic acid and 500 mL of 1octanol to each tube. 10. Transfer 700 mL of the organic layer from each sample to the appropriately labeled tube. 11. Vortex samples on high speed for 3 minutes; centrifuge at 15,000 g for 3 minutes. 12. Using vacuum aspiration, remove the upper, organic layer from each sample tube. 13. Transfer the aqueous solution from each sample to labeled autosampler vials. High Performance Liquid Chromatography-Tandem Mass Spectrometry Inject the unextracted calibrator, extracted calibrators, controls, and samples. Suggested operating conditions:
Flow rate LC column efuent split ow Column temperature Injection volume Number of syringe washes 0.4 mL/min 60-80% Ambient 3-5 mL Preinjection: 5 Postinjection: 5
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
18
Appendix 29
METHOD 29-5 Determination of Urinary Free Catecholamines by LC-MS/MScontd
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.
Appendix 29
19
METHOD 29-5 Determination of Urinary Free Catecholamines by LC-MS/MScontd

salsolinol, serotonin, and theophylline. None of the evaluated compounds interfered with the method performance. Reference Intervals
Concentration mg/g creatinine mg/day (nmol/day) (mmol/mol of creatinine) 0-25 (0-136) 0-100 (0-591) 60-440 (392-2872) 0-20 (0-12.3) 0-45 (0-30.1) 0-250 (0-184.4)
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.
Analyte Epinephrine Norepinephrine Dopamine
METHOD 29-6 Determination of Urinary Metanephrine and Normetanephrine by HPLC/EC

Submitted by Thomas G. Rosano, Ph.D. Department of Pathology and Laboratory Medicine, Albany Medical College and Hospital, Albany, N.Y. Principle An aliquot of urine is combined with an internal standard (4-Omethyldopamine), and the mixture is hydrolyzed with acid to convert all the metanephrines to their free unconjugated forms. Isolation of metanephrines from the sample matrix is accomplished using two disposable minicolumns. The hydrolyzed urines, diluted with an ammonium pentaborate buffer, are rst applied to a weak acid cation-exchange resin. The adsorbed metanephrines are then eluted with ammonium hydroxide directly onto a strong quaternary amine anion-exchange column, which separates the metanephrines from nonphenolic amines and more hydrophobic phenolic compounds. The nal elution uses a weak ammonium acetate buffer; the eluate is then injected onto a reversed-phase HPLC system. Electrochemical detection is used to quantify metanephrine and normetanephrine separately. Specimen Collection and Storage A complete 24-hour urine specimen is collected in a clean container containing acid as a stabilizing preservative (e.g., 10 mL of HCl, 6 mol/L). The specimen should be refrigerated during collection. Overnight or untimed (random) urine collections should be acidied to a pH between 1 and 3 immediately after collection. The total urine volume is measured and recorded after collection, and a 50-mL aliquot is reserved for analysis. Samples are refrigerated until the test is performed or are stored frozen indenitely. Reagents 1. Combination normetanephrine and metanephrine calibrator (Bio-Rad Laboratories, Catalog No. 195-6021). Reconstitute the lyophilized urine calibrator with 25 mL of HCl, 0.05 mol/L. Let stand 10 minutes, then agitate gently to dissolve. Reconstituted calibrator is stable 30 days at 2 C to 8 C. 2. Internal standard, 4-O-methyldopamine (Bio-Rad Laboratories, Catalog No. 195-6022). Reconstitute lyophilized calibrator with 25 mL of HCl, 0.05 mol/L. Agitate gently to dissolve. Stable for 3 months at 2 C to 8 C. 3. HPLC column test solution for metanephrines (Bio-Rad Laboratories, Catalog No. 195-6024). Store refrigerated. Dilute 20 mL in 8 mL of mobile phase before each use. 4. Cation resin columns lled with weakly acidic cation-exchange resin (Bio-Rad Laboratories, Catalog No. 195-6017). Store at 2 C to 8 C. 5. Anion resin columns lled with strongly basic anion-exchange resin (Bio-Rad Laboratories, Catalog No. 195-6018). Stable at room temperature. 6. NaOH, 0.5 mol/L. Dissolve 2.0 g NaOH in 80 mL of distilled water. Dilute to 100 mL with water and mix. Stable at room temperature. 7. Hydrochloric acid, 2 mol/L. Under a fume hood, slowly add 50 mL of concentrated hydrochloric acid to 250 mL of distilled water with continuous mixing. Continue stirring for 5 minutes or until the solution cools to room temperature. Stable at room temperature. 8. Hydrochloric acid, 0.05 mol/L. Dilute 1.0 mL concentrated hydrochloric acid to 250 mL with distilled water and mix. Stable at room temperature. 9. Ammonium pentaborate buffer. Dissolve 40 g ammonium pentaborate and 1.0 g Na2EDTA in 900 mL distilled water. Dilute to 1 L. Mix well and transfer to a 1000-mL dispenser set to deliver 4.0 mL. Stable at room temperature up to 3 months. 10. NH4OH, 2 mol/L. Under a fume hood, add 135 mL of concentrated NH4OH to 865 mL distilled water. Mix well and transfer to a 1000-mL dispenser bottle set to deliver 8.0 mL. Stable at room temperature up to 3 months. 11. Acetic acid, 1 mol/L. Mix 5.7 mL of glacial acetic acid with 94.3 mL of distilled water. Stable at room temperature. 12. Elution buffer. Dissolve 15.4 g of ammonium acetate in 950 mL distilled water. Adjust the pH to 6.00 0.05 using 1 mol/L of acetic acid. Dilute to 1 L with water and transfer to a 1000-mL dispenser set to deliver 5.0 mL. Stable at 2 C to 8 C for 3 months. 13. Monochloroacetic acid (ClCH2COOH) mobile-phase solution. Dissolve 9.4 g of ClCH2COOH, 3.3 g of NaOH, 1.0 g of Na2EDTA, and 0.3 g of sodium octyl sulfate in 1 L of water. Adjust the pH to 3.0 to 3.1 with ClCH2COOH. Add 75 mL of acetonitrile (CH3CN). Store at room temperature for 3 months. 14. Citric acid mobile-phase solution. Dissolve 250 mg of NaOH, 18.61 mg of Na2EDTA, 21.0 g of citric acid monohydrate, and 0.9 mL of diethylamine in 500 mL of water. Add 90 mL of acetonitrile. Dilute to 1 L with deionized water and adjust the pH to 2.55 with solid NaOH pellets.
Continued
20
Appendix 29
METHOD 29-6 Determination of Urinary Metanephrine and Normetanephrine by HPLC/ECcontd

15. Working mobile-phase solution. Mix equal volumes of the 0.1 mol/L monochloroacetic acid and the 0.1 mol/L citric acid solutions. Filter and degas before use. The combined mobile phase is stable for 3 months at room temperature. Instrumentation A commercially available HPLC system equipped with a 3-mm phase II reversed-phase ODS (C18) cartridge column, 3.2 mm I.D. 10 cm (Bioanalytical Systems, Catalog No. MF 6213), and an amperometric detector are suitable. Quality Control Two urine controls are included in every run. Bio-Rad normal and abnormal urine controls are reconstituted each day with HCl, 0.05 mol/L, and processed for immediate use (Bio-Rad Laboratories, Catalog Nos. C-390-25 and C-395-25). Procedure Hydrolysis 1. Set the pH meter calibration at 7.00 using pH 7 buffer. Adjust the pH meter slope calibration to 1.00 using pH 1 buffer. 2. Pipette 2.0 mL of calibrator, controls, and patient samples into labeled 16 150-mm test tubes. Add 2.0 mL distilled water and 200 mL internal standard solution to each tube. Mix each tube briey in a vortex mixer. 3. Add HCl (2 mol/L), drop by drop, to each tube until obtaining a pH between 0.8 and 1.0. Rinse the pH electrode with distilled water after measuring each sample. Recheck the pH 1 buffer every two to three samples. 4. To minimize evaporative losses, cover the tubes loosely with Paralm. 5. Place the tubes in a boiling water bath under a hood for 30 minutes to allow complete hydrolysis of metanephrines. 6. Remove tubes from the water bath and allow them to cool to room temperature. Ion-Exchange Chromatography 7. Label and prepare one cation-exchange column and one anionexchange column for each calibrator, control, and sample. Prepare the columns, about ve or six at a time, by shaking them up and down until completely suspending the resin. Allow the resin to settle. Remove the caps and then snap off the tips. Allow the columns to drain into the waste reservoir. 8. Recheck the pH 7 buffer and adjust pH meter calibration if necessary. Adjust slope to 4.00 using the pH 4 buffer. 9. To each tube, add 5.0 mL ammonium pentaborate buffer and 300 mL NaOH (0.5 mol/L). Mix in a vortex mixer. Check that the pH is between 6.0 and 7.0. If necessary, adjust pH with 100-mL aliquots of NaOH (0.5 mol/L) or acetic acid (1.0 mol/L). 10. Pour each sample into the cation-exchange column assigned to it and allow to drain completely. 11. Add 10 mL of distilled water to each column and allow to drain completely. 12. Place a polycolumn support over each anion-exchange column. Move the appropriate cation-exchange column over the anionexchange column and insert it through the polycolumn support so that it is held in place. 13. Pipette 8.0 mL of NaOH (2 mol/L) into each upper cationexchange column and allow it to drain completely through both columns. Remove and discard the cation-exchange columns. 14. Rinse each anion-exchange column with 5 mL of distilled water. 15. Replace the waste reservoir with a tube rack containing 16 150-mm test tubes. Make sure that the columns are located above marked tubes so that no sample will be spilled. 16. Dispense 5.0 mL of elution buffer into each column. Allow the columns to drain completely and then discard them. 17. Add 400 mL acetic acid (1 mol/L) to each tube and then mix them in a vortex mixer. If necessary, the eluates may be covered with Paralm and stored overnight at room temperature. Eluates may also be stored for 1 week at 4 C. High-Performance Liquid Chromatography 18. Establish the optimal operating condition of the chromatographic and detection systems. Suggested conditions are: Flow rate: 1.0 mL/min Potential: 800 mV Sensitivity: 20 or 50 nA/V Chart speed: 1 cm/min 19. Inject 10 mL of the test mix into the HPLC system to check chromatographic retention times and detector sensitivity. 20. Inject 20 mL of each extract into the HPLC system. Figure A29-5 shows a representative chromatogram of metanephrines extracted from human urine. Calculations 1. Identify metanephrine, normetanephrine, and internal standard peaks by comparing retention times of unknown peaks with retention times of known peaks in the test mix. Measure peak heights to nearest 0.5 mm. Verify that the internal standard peak
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.
Appendix 29
21
METHOD 29-6 Determination of Urinary Metanephrine and Normetanephrine by HPLC/ECcontd

height for each unknown is at least 75% of the internal standard peak height for the calibrators. 2. Calculate the ratio of peak heights (peak height of unknown/peak height of internal standard) for each urine sample, urine control, and calibrator. 3. Calculate metanephrine and normetanephrine concentrations for controls and unknowns: MN or NMN = Ru/Rc C c where MN = unknown concentration of metanephrine (mg/L) NMN = unknown concentration of normetanephrine (mg/L) Ru = peak height ratio of unknown Rc = peak height ratio of corresponding calibrator Cc = concentration of corresponding calibrator (mg/L) 4. Calculate 24-hour excretion: MN or NMN (mg/d) = MN or NMN (mg/L) 24-hour urine volume (L) 5. Calculate 24-hour excretion rate: MN or NMN (mg/g creatinine) = MN or NMN (mg/L)/Urine creatinine concentration (g/L) Reference Intervals
Age 0-3 mo 4-6 mo 7-9 mo 10-12 mo 1-2 y 2-6 y 6-10 y 10-16 y Adult Normetanephrine, Free plus Conjugated mg/d mg/g Creatinine 47-156 31-111 42-109 23-103 32-118 50-111 47-176 53-290 105-354 1535-3355 737-2194 592-1046 271-1117 350-1275 104-609 103-452 96-411 Age 0-3 mo 4-6 mo 7-9 mo 10-12 mo 1-2 y 2-6 y 6-10 y 10-16 y Adult Metanephrine, Free plus Conjugated mg/d mg/g Creatinine 5.9-37 6.1-42 12.0-41 8.5-101 6.7-52 11-99 54-138 39-242 74-297 202-708 156-572 150-526 148-651 40-526 74-504 121-319 46-307
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.
METHOD 29-7 Determination of Urinary Metanephrine and Normetanephrine by LC-MS/MS

Submitted by Ravinder J. Singh, Ph.D. Department of Laboratory Medicine and Pathology, Mayo Clinic and Foundation, Rochester Minn. Principle Urine samples are acidied and hydrolyzed for 20 minutes in a boiling water bath. Metanephrine and normetanephrine are extracted from the specimens using Waters HLB Oasis cartridges. The metanephrine and normetanephrine are eluted from the cartridges using 20% methanol and analyzed by LC-MS/MS using multiple reaction monitoring. Deuterated metanephrine (d3-metanephrine) and deuterated normetanephrine (d3normetanephrine) are added before hydrolysis as internal standards. The following ion pairs are used for analysis:
Metanephrine: Normetanephrine: d3-metanephrine: d3-normetanephrine: 180/148 166/134 183/151 169/137
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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Appendix 29
METHOD 29-7 Determination of Urinary Metanephrine and Normetanephrine by LC-MS/MScontd

Urine Preservative Collection Options Important Note: The addition of preservative or application of temperature controls must occur within 4 hours of completion of the collection or an acceptable preservative must be added at the start of the collection. Urine specimens that require that the preservative be added at the start of the collection are noted by an *(asterisk).
Ambient: Refrigerated: Frozen: 6 mol/L HCl Acetic Acid 50%: Na2CO3: Toluene: HNO3: Boric Acid: Thymol: No Yes* Yes* Yes* Preferred* Yes* Yes* Yes* Preferred* Yes*
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.
Appendix 29
23

Final Calibrator Conc. ng/mL 10 50 200 1000 5000 Preview Std. Blank mL Working Internal Std. 25 25 25 25 25 25 mL 20 mg/mL Std. mL 2.0 mg/mL Std. 25 100 50 250 50 mL 0.2 mg/mL Std. 50
mL -20% Methanol 925 950 875 925 725 925 725
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
24
Appendix 29
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
Dilution Straight 5 10 20 40 Straight 2 5 10 20
% Expected 102% 88% 106% 101% 111% 105% 105% 109%
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.
Appendix 29
25

Measured Normetanephrine ng/mL 4640 957 458 248 132 Expected Normetanephrine ng/mL 928 464 232 116 Measured Normetanephrine ng/mL 214 112 41 22 10.5 Expected Normetanephrine ng/mL 107 42.8 21.5 10.75
Dilution Straight 5 10 20 40
% Expected 103% 99% 107% 114%
Dilution Straight 2 5 10 20
% Expected 105% 96% 101% 98%
Calibration Curves for Analysis of Metanephrine and Normetanephrine.
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
Specimen Type Unpreserved Acetic acid Boric acid
Metanephrine % Difference Range -10.3% to 14.7% -9.5% to 10.0% -6.5% to 31.7%
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%
Normetaphrine Average % Difference 0.2% 9.3% 3.1%
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.
26
Appendix 29
METHOD 29-8 Determination of Urinary Vanillylmandelic Acid by HPLC/EC

Submitted by Thomas G. Rosano, Ph.D. Department of Pathology and Laboratory Medicine, Albany Medical College and Hospital, Albany, N.Y. Principle An aliquot of urine is mixed with an internal standard (isovanillylmandelic acid, iso-VMA) and then applied to a strongly basic anionexchange resin that has been preconditioned with an acetate buffer. Uric acid and hydrophobic acids, such as HVA, are removed from the cleanup column during an acetic acidethanol wash step. VMA and iso-VMA are eluted with H3PO4 and subsequently resolved by reversed-phase HPLC under optimized isocratic conditions. The VMA concentration is determined electrochemically using an amperometric detection system. Specimen Collection and Storage A complete 24-hour urine collection is recommended because variations in VMA excretion occur throughout the day. Shorter collection times may be useful provided that VMA results are expressed per milligram of creatinine. The urine should be collected in a clean container that contains acid as a stabilizing preservative (e.g., 10 mL of HCl, 6 mol/L, for a 24-hour collection). The specimen should be refrigerated during collection. The total urine volume is measured and recorded; a 50-mL aliquot is stored at 4 C until the test is performed or may be frozen and stored indenitely. No dietary restrictions during urine collection are necessary. Reagents 1. Resin column lled with strongly basic anion-exchange resin (Bio-Rad Laboratories, Catalog No. 195-5005). 2. Sodium acetate, 2 mol/L. Dissolve 82 g of sodium acetate in distilled water to a nal volume of 0.5 L. This solution is stable for 1 month at 4 C. 3. Acetic acid, 2 mol/L. Dilute 57.5 mL glacial acetic acid in distilled water to a nal volume of 0.5 L. 4. KH2PO4, 1 mol/L. Dissolve 68.05 g KH2PO4 in distilled water to a nal volume of 0.5 L. This solution is stable for 1 month at 4 C. 5. NaOH, 0.5 mol/L. Dissolve 2.0 g NaOH in distilled water and dilute to 0.1 L. Store tightly capped. 6. HCl, 0.05 mol/L. Dilute 1.0 mL of concentrated HCl in distilled water to a nal volume of 0.25 L. 7. Acetate buffer, 0.08 mol/L, pH 6.1. Mix 40 mL of sodium acetate, 2 mol/L, with 1.2 mL of acetic acid, 2 mol/L. Dilute to 1 L. Adjust the pH to 6.10 0.05 with acetic acid, 2 mol/L, or NaOH, 0.5 mol/L. This solution is stable for 1 month at 4 C. 8. Wash reagent, acetic acidethanol. Mix 250 mL acetic acid, 2 mol/L, and 250 mL ethanol, 95%. This solution is stable for 1 month at 4 C. 9. Elution reagent, H3PO4. Dilute 250 mL of H3PO4 (2 mol/L) in distilled water to a nal volume of 1 L. 10. VMA calibration solutions. a. Stock calibrator of VMA, 1 mg/mL. Accurately weigh 10 mg of VMA (Sigma Chemical Co., Catalog No. H0131) and dissolve in 10 mL of HCl, 0.05 mol/L, in a volumetric ask. Store at 4 C. This solution is stable for 6 months under refrigeration. b. Working calibrators, 5, 10, and 20 mg/L. Dilute 50, 100, and 200 mL of the stock calibrator to 10 mL with HCl, 0.05 mol/L. Prepare fresh before use. 11. Internal standard, iso-VMA, 1 mg/mL. Dissolve 10 mg of DL-3hydroxy-4-methoxymandelic acid (Sigma Chemical Co., Catalog No. H3139) in 10 mL of HCl, 0.05 mol/L. Store at 4 C. This solution is stable for 6 months at 4 C. 12. Mobile phase, ethanol/phosphate buffer. Mix 40 mL KH2PO4 (1 mol/L) with 5 mL of phosphoric acid, 2 mol/L. Dilute to 1 L with distilled water. Adjust the pH to 3.02 0.02 with H3PO4, 2 mol/L, or NaOH, 0.5 mol/L. Add 16 mL absolute ethanol. Mix. Filter and degas before use. Store tightly capped. This solution is stable for 1 month at room temperature. Instrumentation A commercially available HPLC system, equipped with a 4.6 mm I.D. > 25 cm reversed-phase C18 column (average particle diameter, 5 mm), and either a glassy-carbon or porous-graphite cell, adaptable for use in electrochemical detection, is required for this method. Procedure Sample Preparation 1. Pipette 3.0 mL of calibrators, controls, and patient urine samples into labeled 16 150-mm tubes. 2. To each tube, add 200 mL of the internal standard solution (isoVMA), 5.0 mL of acetate buffer (0.08 mol/L, pH 6.1), and 0.1 mL NaOH (0.5 mol/L). 3. Mix well and check pH. pH must be in the range of 5.0 to 7.0. If necessary, adjust pH with 0.1-mL aliquots of NaOH (0.5 mol/L) or acetic acid (2 mol/L). 4. Label one anion-exchange resin column for each calibrator, control, and urine sample to be run. Prepare the columns, about ve or six at a time, by shaking them up and down until the resin is completely suspended. 5. Insert each column into a suitable support rack and allow the resin to settle. 6. Remove caps, snap off the tips, and allow the columns to drain into a waste drainage tray. 7. Rinse each column three times with 5 mL of acetate buffer (0.08 mol/L, pH 6.1). Discard the eluates. 8. Quantitatively transfer urine samples into the appropriate columns and allow to drain completely. 9. Rinse each sample tube with 5 mL of acetate buffer. Apply the rinse solution to the respective columns and allow to drain completely. 10. Wash each column with 9.0 mL of wash reagent and allow to drain completely. 11. Replace the drainage tray with a rack containing clean 16 150-mm tubes. Make sure that columns are located above marked tubes so that sample will not be spilled. Sample Analysis 12. Dispense two 9-mL portions of elution reagent into each column. Collect the eluates. Allow the columns to drain completely, discard columns, cover tubes with Paralm, and mix the tubes thoroughly. (Note: The eluates may be stored overnight at room temperature and at -20 C for longer periods of storage.)
Appendix 29
27
METHOD 29-8 Determination of Urinary Vanillylmandelic Acid by HPLC/ECcontd

13. Establish the optimal operating conditions of the chromatographic and detection systems. Suggested conditions for a dualcell porous graphite electrochemical detector are as follows:
Flow rate: Potential of detector cell No. 1: Potential of detector cell No. 2: Chart speed: 2.0 mL/min 180 mV 410 mV 1.0 cm/min
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.
METHOD 29-9 Determination of Urinary Vanillylmandelic Acid by LC-MS/MS

Submitted by Mark J. Magera, M.A., Dietrich Matern, M.D., and Piero Rinaldo, M.D., Ph.D. Department of Laboratory Medicine and Pathology, Mayo Clinic and Foundation, Rochester, Minn Principle Hydrophilic-lipophilic balance (HLB) solid phase extraction (SPE) is performed on a 1 mL aliquot of either a 24-hour or random urine collection. The SPE column is eluted with 1 mL of methanol. The eluate is evaporated at 30 C under nitrogen, and the residue reconstituted in 1 mL of a mobile phase consisting of 15% methanol in 0.05% aqueous formic acid. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is performed by injecting 10 mL of the reconstituted specimen onto an AmideC16 HPLC column. The mobile phase ow rate is 1.0 mL/min, with the LC column efuent ow diverted so that approximately 200 mL/min is delivered to the MS/MS electrospray probe tip. VMA elutes apart from the bulk of the specimen matrix at a retention time of approximately 1.5 minutes. VMA is quantitated using a stable isotope labeled internal standard (2H3-VMA) from calibration over a concentration range 1.03 to 20 mg/L. Specimen Collection and Storage The preferred specimen is a 24-hour urine collected into 25 mL of 50% glacial acetic acid. For low urine volumes, and infants and children under the age of 5 years, use 15 mL of 50% glacial acetic acid. Boric, nitric, and hydrochloric acids are also acceptable preservatives if the pH of the collected specimen is in the range 1 to 5. A random or 4-hour urine collection is acceptable on pediatric (up to 14 years) submissions if sent to the laboratory immediately after collection. Random urine samples collected on lter paper are also acceptable, but the
Continued
28
Appendix 29
METHOD 29-9 Determination of Urinary Vanillylmandelic Acid by LC-MS/MScontd

sensitivity of the method is greater on an aliquot from a 24-hour specimen than on random specimens or dried urine on lter paper. Dried lter paper specimens also typically exhibit more variability. Reagents 1. Methanol, HPLC grade. 2. Formic acid (HCOOH), 98% to 100%, analytical ACS reagent grade. 3. LC-MS/MS mobile phase, 15% methanol in 0.05% aqueous formic acid. Prepare as needed by mixing 150 mL of methanol with 850 mL of reverse-osmosis water and add 0.50 mL of formic acid, mixing well. 4. VMA internal standard (VMA-IS), 0.500 mg/mL. Prepare by dissolving 5.0 mg of vanillylmandelic acid-ring-2H3 in 10 mL of 0.1 mol/L HCl in a volumetric ask. Store refrigerated. Stable for 6 months. 5. VMA stock calibrator, 1.00 mg/mL. Prepare by dissolving 10.0 mg VMA in 10 mL of 0.1 mol/L HCl in a volumetric ask. Store refrigerated. Stable for 6 months. 6. VMA working calibrator, 20 mg/L. Prepare by diluting 2.0 mL VMA stock calibrator to 100 mL with reverse-osmosis water in a volumetric ask, mixing well. Store refrigerated. Stable for 6 months. 7. Hydrochloric acid, concentrated, analytical grade. 8. Hydrochloric acid, 0.1 mol/L. Prepare by diluting 4.2 mL concentrated HCl to 500 mL with reverse-osmosis water in a volumetric ask, mixing well. Store at room temperature. Stable indenitely. 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. SPE columns are Oasis HLB (Waters, No. WAT094225) 1 mL containing 30 mg sorbent packing. 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 VMA is generally 1 to 4 mg/L. The elevated urine control is prepared by spiking the urine with approximately 18 mL of the VMA 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 VMA and standard deviation are calculated from a minimum of 10 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 VMA Quality Report if indicated. Any control values that are >3 SD require a completed VMA 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. Procedure HPLC Operating Parameters 1. LC-MS/MS HPLC pump Mobile phase ow rate: 1.0 mL/min Pressure limit: set at 3000 psi Refer to manual for maintenance and trouble shooting of pump problems 2. LC-MS/MS Autosampler Injection volume = 10 mL MS/MS Operating Parameters 1. Electrospray source voltage = -5000 V 2. Source assist gas (zero grade air) = setting 40 3. Source drying gas (zero grade air) = setting 40 4. Source temperature = 280 C 5. Orice = -51 V 6. Collision energy = 27 V 7. Collision gas (nitrogen) = setting 3 8. Q1/Q3 resolution = unit mass (0.7 FWHM) 9. Selected reaction monitoring (SRM) mode transitions (Q1/Q3): VMA = m/z 197 to m/z 137 2 H3-VMA = m/z 200 to m/z 140 Sample Analysis 1. Check the samples for acceptable pH. Properly collected specimens will have a pH in the range 1 to 5. Collect an aliquot of urine on pediatric samples (<15 years) for creatinine determination. Urine-soaked lter paper specimens can be analyzed after reconstitution of the urine in reverse-osmosis water, results being expressed as mg/mg creatinine. The method for reconstitution is as follows: Cut a strip from the lter paper about 4 cm wide. Fanfold the strip and place it into a 10-mL glass screw-capped test tube. For dried urine specimens that appear visually dilute, a 6cm wide strip may be used. Add about 10 mL of RO water (water produced by reverse-osmosis) to the vial and cap. Let the urine elute from the lter paper in the vial at room temperature for at least 15 minutes, inverting occasionally. Do not shake; vigorous shaking or vortex is not advised as it tends to cause the lter paper to disintegrate, making subsequent steps difcult. After elution, aliquot the reconstituted specimen for creatinine determination, and use as directed in the subsequent steps. 2. To a 10- 75-mm tube, add 10 mL of VMA-IS; 600 mL of urine sample, or reconstituted urine from lter paper; and 600 mL of RO water. Vortex to mix. 3. The sample tubes are now ready for automated SPE. Using an automatic processor (Gilson ASPEC, or similar device), the SPE columns are conditioned by adding 1 mL methanol, which is allowed to drain through, followed by 1 mL of reverse-osmosis water. A 1-mL aliquot of the diluted urine specimen is added to the SPE column and allowed to drain through. Following a wash with 0.5 mL of reverse-osmosis water, VMA and 2H3-VMA are eluted in 1 mL of methanol.
Appendix 29
29
METHOD 29-9 Determination of Urinary Vanillylmandelic Acid by LC-MS/MScontd

4. The eluate tubes are removed from the processor and placed in an evaporator with a water bath at 30 C under a gentle stream of nitrogen. Evaporate the eluate tubes just to dry. 5. Remove the eluate tubes from the evaporator and let them cool to room temperature briey. Add 1.0 mL of VMA LC-MS/MS mobile phase. Vortex to dissolve the residue and mix well. Transfer a 200-mL aliquot of the solution to an autosampler vial and cap. Samples are now ready for analysis. Note: For specimens that are <0.01 mg/L, the amount of mobile phase used to reconstitute can be decreased to 100 mL. Calculations A 7-point calibration curve is run each time that a new internal standard solution (VMA-IS) is prepared. The calibrator concentrations are 1.03, 2.08, 4.17, 8.33, 12.50, 16.7, and 20.0 mg/L of VMA. The amount of VMA-IS added to each sample is 2.5 mg/L. Normal and elevated VMA controls are calculated against each new calibration and the current QC mean to validate the new calibration. Both controls must also be acceptable to validate the new calibration. VMA concentrations greater than 20 mg/L will be repeated at a dilution with water. The table below indicates how to prepare the calibrators, which are then prepared by SPE as for urine specimens.
STD 1 2 3 4 5 6 7 VMA-IS, mL 10 10 10 10 10 10 10 VMA Working Calibrator, mL 62 125 250 500 750 1000 1200 RO Water, mL 1138 1075 950 700 450 200 0 mg VMA 1.03 2.08 4.17 8.33 12.50 16.67 20.0 Theoretical Ratio 0.2500 0.5000 1.0000 2.0000 3.0000 4.0000 4.800
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.
METHOD 29-10 Determination of Urinary Homovanillic Acid (3-Methoxy-4-Hydroxyphenylacetic Acid) by HPLC/EC

Submitted by Thomas G. Rosano, Ph.D. Department of Pathology and Laboratory Medicine, Albany Medical College and Hospital, Albany, N.Y. Principle An aliquot of urine is mixed with an internal standard (iso-VMA) and then applied to a strongly basic anion-exchange resin that has been preconditioned with an acetate buffer. HVA is eluted with acetic acid in ethanol and subsequently resolved by reversed-phase HPLC under optimized isocratic conditions. The HVA concentration is determined electrochemically using an amperometric detection system. Specimen Collection and Storage A complete 24-hour urine collection is recommended because variations in HVA excretion occur throughout the day. Shorter collection times may be useful provided that HVA results are expressed per milligram of creatinine. The urine should be collected in a clean container that contains acid as a stabilizing preservative (e.g., 10 mL of HCl, 6 mol/L, for a 24-hour collection). The specimen should be refrigerated during collection. The total urine volume is measured and recorded; a 50-mL aliquot is stored at 4 C until the test is performed or may be frozen and stored indenitely. No dietary restrictions during urine collection are necessary. Urine may also be collected on lter paper. Reagents 1. Resin column lled with strongly basic anion-exchange resin (Bio-Rad Laboratories, Catalog No. 195-5005). 2. Sodium acetate, 2 mol/L. Dissolve 82 g of sodium acetate in distilled water to a nal volume of 0.5 L. This solution is stable for 1 month at 4 C. 3. Acetic acid, 2 mol/L. Dilute 57.5 mL of glacial acetic acid in distilled water to a nal volume of 0.5 L. 4. KH2PO4, 1 mol/L. Dissolve 68.05 g KH2PO4 in distilled water to a nal volume of 0.5 L. This solution is stable for 1 month at 4 C. 5. NaOH, 0.5 mol/L. Dissolve 2.0 g NaOH in distilled water and dilute to 0.1 L. Store tightly capped. 6. HCl, 0.05 mol/L. Dilute 1.0 mL of concentrated HCl in distilled water to a nal volume of 0.25 L.
Continued
30
Appendix 29
METHOD 29-10 Determination of Urinary Homovanillic Acid (3-Methoxy-4-Hydroxyphenylacetic Acid) by HPLC/ECcontd

7. Acetate buffer, 0.08 mol/L, pH 6.1. Mix 40 mL of sodium acetate, 2 mol/L, with 1.2 mL of acetic acid, 2 mol/L. Dilute to 1 L. Adjust the pH to 6.10 0.05 with acetic acid, 2 mol/L, or NaOH, 0.5 mol/L. This solution is stable for 1 month at 4 C. 8. Elution reagent, acetic acid/ethanol. Mix 250 mL acetic acid (2 mol/L), 250 mL H2O, and 500 mL ethanol (95%). This solution is stable for 1 month at 4 C. 9. HVA calibration solutions (Bio-Rad Laboratories, Catalog No. 195-5006). Reconstitute with 25 mL 0.05 mmol/L HCl. Let stand 10 minutes, then gently agitate to dissolve. Stable 30 days at 2 C to 8 C. 10. Internal standard, iso-VMA (Bio-Rad Laboratories, Catalog No. 195-5213). Reconstitute with 25 mL of methanol. Agitate gently to dissolve. Stable 3 months at 2 C to 8 C. 11. Mobile phase, ethanol/phosphate buffer. Combine 965 mL of deionized H2O, 20 mL 1 mmol/L KH2PO4, and 15 mL 2 mol/L phosphoric acid. Mix well and allow 5 minutes for equilibration. Check pH on a pH meter. If necessary, adjust pH to 2.3 0.05 with 2 mol/L phosphoric acid or 0.5 mol/L sodium hydroxide. Add 90 mL of ethanol (100%), 5 mL methanol, and 25 mL isopropanol and mix another 2 to 3 minutes. Filter by suction through a 0.45-mm lter and store capped at 2 C to 8 C. Instrumentation A commercially available HPLC system, equipped with a 4.6-mm I.D. 25-cm, reversed-phase C18 column (average particle diameter, 5 mm), and either a glassy-carbon or porous-graphite cells, adaptable for use in electrochemical detection, is required. Procedure Sample Preparation 1. Pipette 3.0 mL of calibrators, controls, and patient urine samples into labeled 16 150-mm tubes. 2. To each tube, add 200 mL of the internal standard solution (isoVMA), 5.0 mL of acetate buffer (0.08 mol/L, pH 6.1), and 0.1 mL NaOH (0.5 mol/L). 3. Mix well and check pH. pH must be in the range of 5.0 to 7.0. If necessary, adjust pH with 0.1-mL aliquots of NaOH (0.5 mol/L) or acetic acid (2 mol/L). Isolation of Analyte 4. Label one anion-exchange resin column for each calibrator, control, and urine sample to be run. Prepare the columns, about ve or six at a time, by shaking them up and down until the resin is completely suspended. 5. Insert each column into a suitable support rack and allow the resin to settle. 6. Remove caps, snap off the tips, and allow the columns to drain into a waste drainage tray. 7. Rinse each column three times with 5 mL of acetate buffer (0.08 mol/L, pH 6.1). Discard the eluates. 8. Quantitatively transfer urine samples into the appropriate columns and allow to drain completely. 9. Rinse each sample tube with 5 mL of acetate buffer. Apply the rinse solution to the respective columns and allow to drain completely. 10. Replace the drainage tray with a rack containing clean 16 150mm tubes. Make sure that columns are located above marked tubes so that sample will not be spilled. 11. Dispense 2 6 mL of elution reagent into each column and allow column to drain completely. Note: The eluates may be stored overnight at room temperature and at -20 C for longer periods of storage. High-Performance Liquid Chromatography Analysis 12. Establish the optimal operating conditions of the chromatographic and detection systems. Suggested conditions for a dualcell porous graphite electrochemical detector are as follows:
Flow rate: Potential of detector cell No.1: Potential of detector cell No.2: Chart speed: 1.0 mL/min 180 mV 460 mV 1.0 cm/min
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.
Appendix 29
31
METHOD 29-10 Determination of Urinary Homovanillic Acid (3-Methoxy-4-Hydroxyphenylacetic Acid) by HPLC/ECcontd

4. From the HVA calibrator curve, determine the concentrations of unknowns (mg/L). 5. Calculate 24-hour HVA excretion by multiplying HVA concentration (mg/L) by 24-hour urine volume (L). 6. Calculate HVA excretion normalized for creatinine excretion by dividing HVA concentration (mg/L) by urine creatinine (g/L). Reference Intervals
Age (yr) 3-6 6-10 10-16 16-83 mg/d 1.4-4.3 2.1-4.7 2.4-8.7 1.4-8.8 mmol/d 8-24 12-26 13-48 8-48 mg/g Creatinine 5.4-15.5 4.4-11.5 3.3-10.3 mmol/mol Creatinine 3.4-9.6 2.7-7.1 2.0-6.4
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.
METHOD 29-11 Determination of Urinary Homovanillic Acid by LC-MS/MS

Submitted by Mark J. Magera, M.A., Dietrich Matern, M.D., and Piero Rinaldo, M.D., Ph.D. Department of Laboratory Medicine and Pathology, Mayo Clinic and Foundation, Rochester, Minn. Principle Octadecyl (C18) solid phase extraction (SPE) is performed on a 1 mL aliquot of either a 24-hour or random urine collection. The SPE column is eluted with 1 mL of methanol. The eluate is evaporated at 30 C to 40 C under nitrogen and the residue reconstituted in 1 mL of 30% acetonitrile in 0.05% aqueous acetic acid. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is performed by autosampler injection of 10 mL of the reconstituted specimen onto an AmideC16 HPLC column. The mobile phase ow rate is 1.0 mL/ min, with the LC column efuent ow diverted so that approximately 200 mL/min is delivered to the MS/MS electrospray probe tip. Homovanillic acid (HVA) elutes apart from the bulk of the specimen matrix at a retention time of approximately 1.5 minutes. HVA is quantitated using a custom synthesized stable isotope labeled internal standard (13C618O-HVA) from calibration over a concentration range 0.52 to 16.7 mg/L. Specimen Collection and Storage The preferred specimen is a 24-hour urine collected into 25 mL of 50% glacial acetic acid. For low urine volumes, and infants and children under the age of 5 years, use 15 mL of 50% glacial acetic acid. Boric, nitric, and hydrochloric acids are also acceptable preservatives if the pH of the collected specimen is in the range 1 to 5. A random or 4-hour urine collection is acceptable on pediatric (up to 14 years) submissions if sent to the laboratory immediately after collection. Random urine samples collected on lter paper are also acceptable, but the sensitivity of the method is greater on an aliquot from a 24-hour specimen than on random specimens or dried urine on lter paper. Dried lter paper specimens also typically exhibit more variability. Reagents 1. Acetonitrile, HPLC grade. 2. Acetic acid, glacial (CH3COOH), analytical grade. 3. LC-MS/MS mobile phase, 30% acetonitrile in 0.05% aqueous acetic acid. Prepare as necessary by mixing 600 mL of acetonitrile with 1400 mL of reverse-osmosis water and add 1 mL glacial acetic acid, mixing well. 4. HVA internal standard (HVA-IS), 0.500 mg/mL. Prepare by dissolving 5.0 mg of 13C618O-HVA in 10 mL of 0.1 mol/L HCl in a volumetric ask. Store refrigerated. Stable for 3 months. 5. HVA stock calibrator, 1.00 mg/mL. Prepare by dissolving 10.0 mg HVA in 10 mL of 0.1 mol/L HCl in a volumetric ask. Store refrigerated. Stable for 3 months. 6. HVA working calibrator, 20 mg/L. Prepare by diluting 2.0 mL HVA stock calibrator to 100 mL with reverse-osmosis water in a volumetric ask, mixing well. Store refrigerated. Stable for 3 months. 7. Hydrochloric acid, concentrated, analytical grade. 8. Hydrochloric acid, 0.1 mol/L. Prepare by diluting 4.2 mL concentrated HCl to 500 mL with reverse-osmosis water in a volumetric ask, mixing well. Store at room temperature. Stable indenitely. 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: (1) Bond Elut C18 (Varian No. 1210-2001), 1mL (100mg); (2) Oasis HLB, (Waters) 1 mL (30 mg); and (3) Strata X, 33 mm polymeric sorbent (Phenomenex No. 8B-S100-TAK), 1mL (30 mg). Additional equipment used includes an automated SPE sample processor (Gilson ASPEC, or equivalent), and an evaporator (Zymark Turbo-Vap).
Continued
32
Appendix 29
METHOD 29-11 Determination of Urinary Homovanillic Acid by LC-MS/MScontd

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 HVA is generally 1 to 4 mg/L. The elevated urine control is prepared by spiking the urine with approximately 18 mL of the HVA 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 HVA and standard deviation are calculated from a minimum of 10 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 HVA Quality Report if indicated. Any control values that are >3 SD require a completed HVA 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. Procedure HPLC Operating Parameters 1. LC-MS/MS HPLC pump Mobile phase ow rate: 1.0 mL/min Pressure limit: set at 3000 psi Refer to manual for maintenance and trouble shooting of pump problems 2. LC-MS/MS autosampler Injection volume = 10 mL MS/MS Operating Parameters 1. Electrospray source voltage = -5000 V 2. Source assist gas (zero grade air) = setting 40 3. Source drying gas (zero grade air) = setting 40 4. Source temperature = 250 C 5. Orice = -27 V 6. Collision energy = 15 V 7. Collision gas (nitrogen) = setting 3 8. Q1/Q3 resolution = unit mass (0.7 FWHM) 9. Selected reaction monitoring (SRM) mode transitions (Q1/Q3): HVA = m/z 181 to m/z 137 13 C618O-HVA = m/z 189 to m/z 145 Sample Analysis 1. Check the samples for acceptable pH. Properly collected specimens will have a pH in the range 1 to 5. Collect an aliquot of urine on pediatric samples (<15 yr) for creatinine determination. Urinesoaked lter paper specimens can be analyzed after reconstitution of the urine in reverse-osmosis water, results being expressed as mg/mg creatinine. The method for reconstitution is as follows: Cut a strip from the lter paper, about 4 cm wide. Fan-fold the strip and place it into a 10 mL glass screw-capped test tube. For dried urine specimens that appear visually dilute, a 6-cm wide strip may be used. Add about 10 mL of RO water (water prepared by reverse-osmosis) to the vial and cap. Let the urine elute from the lter paper in the vial at room temperature for at least 15 minutes, inverting occasionally. Do not shake as vigorous shaking or vortex tends to cause the lter paper to disintegrate, making subsequent steps difcult. After elution, aliquot the reconstituted specimen for creatinine determination, and use as directed in the subsequent steps. To a 10- 75-mm tube, add 10 mL of HVA-IS, 1200 mL of urine sample, or reconstituted urine from lter paper. Vortex to mix. The sample tubes are now ready for automated SPE. Using an automatic processor (Gilson ASPEC, or similar device), the SPE columns are conditioned by the adding 1 mL methanol, which is allowed to drain through, followed by 1 mL of reverse-osmosis water. A 1 mL aliquot of the diluted urine specimen is added to the SPE column and allowed to drain through. Following a wash with 0.5 mL of reverse-osmosis water, HVA and 13C618O-HVA are eluted in 1 mL of methanol. The eluate tubes are removed from the processor and placed in an evaporator with a water bath at 30 C under a gentle stream of nitrogen. Evaporate the eluate tubes just to dry. Remove the eluate tubes from the evaporator and let them cool to room temperature briey. Add 1.0 mL of HVA LC-MS/MS mobile phase. Vortex to dissolve the residue and mix well. Transfer a 200 mL aliquot of the solution to an autosampler vial and cap. Samples are now ready for analysis. Note: For specimens that are <0.01 mg/L, the amount of mobile phase used to reconstitute can be decreased to 100 mL.
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.)
Appendix 29
33
METHOD 29-11 Determination of Urinary Homovanillic Acid by LC-MS/MScontd

Calculations A 7-point calibration curve is run each time that a new internal standard solution (HVA-IS) is prepared. The calibrator concentrations are 0.52, 1.04, 2.08, 4.16, 8.33, 16.7, and 20.0 mg/L of HVA. The amount of HVA-IS added to each sample is 4.17 mg/L. Normal and elevated HVA controls are calculated against each new calibration and the current QC mean to validate the new calibration. Both controls must also be acceptable to validate the new calibration. HVA concentrations greater than 20 mg/L will be repeated at a dilution with water. The table below indicates how to prepare the calibrators, which are then prepared by SPE as for urine specimens.
STD 1 2 3 4 5 6 7 HVA-IS, mL 10 10 10 10 10 10 10 HVA Working Calibrator, mL 31 62 125 250 500 1000 1200 OR Water, mL 1169 1138 1075 950 700 200 0 mg/L HVA 0.52 1.04 2.08 4.16 8.33 16.7 20.0 Theoretical Ratio 0.1250 0.2500 0.5000 1.0000 2.0000 4.0000 4.8000
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.
METHOD 29-12 Determination of Urinary 5-Hydroxyindoleacetic Acid by Quantitative HPLC

Submitted by Ronald J. Whitley, Ph.D. Department of Pathology and Laboratory Medicine, College of Medicine, University of Kentucky Medical Center, Lexington, Ky. Principle Diluted urine samples are analyzed without sample preparation using reversed-phase HPLC with electrochemical detection. Specimen Collection and Storage A 24-hour urine specimen is recommended, since 5-HIAA concentrations in random urine specimens are extremely variable. Shorter (or longer) collection times may be acceptable provided results are expressed per milligram of creatinine. Because 5-HIAA is light sensitive, urine should be collected in 2-L brown polypropylene containers. The specimen may be collected with or without preservatives. In view of conicting reports, however, the addition of a stabilizing preservative to the urine container is encouraged (e.g., 0.5 g boric acid; 10 mL HCl, 6 mol/L; 25 mL glacial acetic acid). Most importantly, the specimen should be refrigerated during collection. On receipt in the laboratory, the urine specimen is thoroughly mixed and the total volume measured and recorded. If the specimen is not collected with an acid preservative, then the pH of the urine may now be adjusted to between 2 and 3 by addition of HCl, 6 mol/L. Acidied urine can be stored at 4 C for 2 to 4 weeks and for longer periods of time at -20 C. False-negative results may occur in patients taking phenothiazine drugs. The ingestion of foods high in hydroxyindole content (e.g., bananas, avocados, red plums, eggplants, pineapples, kiwi fruit, walnuts, hickory nuts, and tomatoes) or cough medications containing glycerol guaiacolate may lead to false-positive results. Therefore these drugs and foods should be restricted 3 to 4 days before and during the collection. Reagents 1. Acetic acid, 6 mol/L. Dilute 34.5 mL of glacial acetic acid to 100 mL with distilled water. Stable for 1 year at room temperature. 2. Acetate buffer, 0.1 mol/L, pH 5. Transfer 1.5 mL of acetic acid, 6 mol/L, and 1.3 g of sodium acetate to a 250-mL volumetric ask. Dissolve and dilute to the mark with distilled water. Stable for 1 year at room temperature. 3. 5-HIAA calibrators. a. Stock calibrator, 500 mg/L. Weigh and transfer 25 mg of 5HIAA to a 50-mL volumetric ask. Dissolve in 40 mL of water. Adjust the pH to <3 with concentrated HCl. Dilute to the mark with water. Store in a dark brown bottle at 4 C. Discard after 1 month. b. Working calibrator, 50 mg/L. Dilute 1 mL of the stock solution to 10 mL with water. Prepare fresh before use. 4. Mobile phase. Add the following reagents to 1500 mL of distilled water: 54 mL of concentrated NH4OH, 69 mL of glacial acetic acid, and 0.2 g of Na2EDTA. Adjust pH to 5.1 with either acetic acid
Continued
34
Appendix 29
METHOD 29-12 Determination of Urinary 5-Hydroxyindoleacetic Acid by Quantitative HPLCcontd

(6 mol/L) or NH4OH (6 mol/L). Then add 20 mL of methanol and 20 mL of acetonitrile. Dilute to 2 L with distilled water, mix, and lter. Degas for 15 minutes. Prepare fresh daily. Instrumentation A commercially available HPLC system equipped with a 25-cm 4.6mm, reversed-phase C18 column (average particle diameter 5 mm) and a glassy-carbon electrochemical detector is suitable. Quality Control Two urine controls are included in every run. For example, Bio-Rad Lyphochek Quantitative Urine Control, Levels 1 and 2 (Bio-Rad Clinical Diagnostics Group, Hercules, Calif.) are reconstituted each day with distilled water. Procedure 1. Pipette 4 mL of sodium acetate buffer into labeled 13 100-mm tubes. 2. Add 100 mL of working HIAA calibrator, specimens, and urine controls to their respective tubes. Mix in a vortex mixer. 3. Establish operating conditions of the chromatographic and detection systems. Suggested conditions are:
Potential: Sensitivity: Flow rate: Chart speed: 500 mV 100 mA/V 1.0 mL/min 0.5 cm/min
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.
Appendix 29
35
METHOD 29-13 Determination of Urinary 5-Hydroxyindole-3-Acetic Acid by LC-MS/MS

Submitted by Mark J. Magera, M.A., Dietrich Matern, M.D., and Piero Rinaldo, M.D., Ph.D. Department of Laboratory Medicine and Pathology, Mayo Clinic and Foundation, Rochester, Minn. Principle Octadecyl (C18) solid phase extraction (SPE) is performed on a 0.5mL aliquot of a 24-hour urine collection. The SPE column is eluted with 1 mL of 20% acetonitrile in 0.05% aqueous formic acid. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is performed by autosampler injection of 10 mL of the reconstituted specimen onto an AmideC16 HPLC column. The mobile phase ow rate is 1.0 mL/min, with the LC column efuent ow diverted so that approximately 200 mL/min is delivered to the MS/MS electrospray probe tip. 5-hydroxyindole-3-acetic acid (5-HIAA) elutes apart from the bulk of the specimen matrix at a retention time of approximately 1.5 minutes. 5-HIAA is quantitated using a custom synthesized stable isotope labeled internal standard (2H6-HIAA) from calibration over a concentration range up to 150 mg/L. Specimen Collection and Storage The preferred specimen is a 24-hour urine collected into 25 mL of 50% glacial acetic acid. For low urine volumes, and infants and children under the age of 5 years, use 15 mL of 50% glacial acetic acid. Other urine preservatives are also acceptable, including boric, nitric, and hydrochloric acids along with sodium carbonate, toluene, and thymol. 5-HIAA is stable in properly preserved refrigerated specimens for up to 7 days and stable for long periods if specimens are kept frozen at -20 C. Spot (random) or nonpreserved urine collections are unacceptable. Foods containing high levels of serotoninincluding walnuts, hickory nuts, plantain, pineapple, bananas, kiwi, plums, tomatoes, avocados, dates, grapefruit, cantaloupe, or honeydew melonshould not be consumed for at least 48 hours before urine collection. Additionally, some drugs affecting serotonin levels will affect 5-HIAA levels. Reagents 1. Methanol, HPLC grade. 2. Acetonitrile, HPLC grade. 3. Formic acid (HCOOH), 98% to 100%, analytical ACS reagent grade. 4. LC-MS/MS mobile phase, 20% acetonitrile in 0.05% aqueous formic acid. Prepare as necessary by mixing 200 mL of acetonitrile with 800 mL of reverse-osmosis water and add 0.500 mL formic acid, mixing well. 5. Acetic acid, glacial (CH2COOH), analytical grade. 6. Acetic acid, 1%. Prepare by diluting 10 mL of glacial acetic acid to 1000 mL with reverse-osmosis water in a volumetric ask. Store at room temperature. Stable indenitely. 7. 5-HIAA internal stock standard (HIAA-IS), 1 mg/mL. Prepare by dissolving 25.0 mg of 2H6-HIAA in 25 mL of methanol in a volumetric ask. Store refrigerated. Stable for 6 months. 8. 5-HIAA stock calibrator, 1.00 mg/mL. Prepare by dissolving 100 mg 5-HIAA in 100 mL of 1% acetic acid in a volumetric ask. Store refrigerated. Stable for 6 months. 9. HIAA-IS working calibrator, 10 mg/L. Prepare by diluting 2.0 mL HIAA-IS stock calibrator to 200 mL with 1% acetic acid in a volumetric ask, mixing well. Store refrigerated. Stable for 1 month. 10. 5-HIAA working calibrators. Prepare using the following table. Stable for 3 months when stored refrigerated in amber bottles.
5-HIAA Working Calibrator Conc. (mg/mL) Blank (0) 5 12.5 25 150 Amount (mL) of 1 mg/mL Stock 5-HIAA Calibrator 1 1.25 5 15 Total Volume (mL) 1% Acetic Acid 200 100 200 100
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
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Appendix 29
METHOD 29-13 Determination of Urinary 5-Hydroxyindole-3-Acetic Acid by LC-MS/MScontd

Procedure HPLC Operating Parameters 1. LC-MS/MS HPLC pump Mobile phase ow rate: 1.0 mL/min Pressure limit: set at 3000 psi Refer to manual for maintenance and trouble shooting of pump problems 2. LC-MS/MS autosampler Injection volume = 10 mL MS/MS Operating Parameters 1. Electrospray source voltage = -5000 V 2. Source assist gas (zero grade air) = setting 30 3. Source drying gas (zero grade air) = setting 55 4. Source temperature = 250 C 5. Orice = -36 V 6. Collision energy = 13 V 7. Collision gas (nitrogen) = setting 3 8. Q1/Q3 resolution = unit mass (0.7 FWHM) 9. Selected reaction monitoring (SRM) mode transitions (Q1/Q3): 5-HIAA = m/z 190 to m/z 146 2 H6-HIAA = m/z 196 to m/z 152 Sample Analysis 1. To a 10 75-mm tube, add 500 mL of working HIAA-IS and 500 mL of urine sample or 5-HIAA calibrator (see Reagents 10.) Vortex to mix. 2. The sample tubes are now ready for automated SPE. Using an automatic processor (Gilson ASPEC, or similar device), the SPE columns are conditioned by adding 1 mL of methanol, which is allowed to drain through, followed by 1 mL of reverse-osmosis water. 1 mL of the diluted urine specimen is added to the SPE column and allowed to drain through. Following a wash with 0.5 mL of reverse-osmosis water, 5-HIAA and 2H6-HIAA are eluted in one mL of LC-MS/MS mobile phase (see Reagents). 3. Vortex the eluate tubes to dissolve the residue. Transfer a 200-mL aliquot of the solution to an autosampler vial and cap. Samples are now ready for analysis. Calculations A 5-point calibration curve is run each time that a new internal standard solution (HIAA-IS) is prepared. The calibrator concentrations are 0.00, 5.00, 12.50, 25.00, and 150.00 mg/mL of 5-HIAA. The amount of HIAA-IS added to each sample is 5 mg. Normal and elevated 5-HIAA controls are calculated against each new calibration and the current QC mean to validate the new calibration. Both controls must also be acceptable to validate the new calibration. 5-HIAA concentrations greater than 150 mg/L will be repeated at a dilution with water. The observed 5-HIAA concentration in mg/L is converted to mg 5-HIAA/24 hours by multiplying by the 24-hour urine volume in liters. Comments 1. Normally 1% to 3% of dietary tryptophan is metabolized to serotonin. However, as much as 60% of this tryptophan is converted to serotonin in the intestinal carcinoid syndrome, argentafnoma. 5-HIAA is the major metabolite of serotonin and is excreted in the urine. 2. Any 5-HIAA value >150 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. 5-HIAA values less than 0.1 mg/L are reported as <0.1 mg/L. 3. 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 6 mg 5-HIAA/24 hours Reference Kroll CA, Magera MJ, Helgeson JK, Rinaldo P. Liquid chromatographic-tandem mass spectrometric method for the determination of 5-hydroxyindole-3-acetic acid in urine. Clin Chem 2002; 48:204951.

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APPENDIX

Catecholamines and Serotonin

SELECTED METHODS FOR ANALYSIS OF CATECHOLAMINES, SEROTONIN, AND THEIR METABOLITES

METHOD 29-1 Determination of Plasma Free Catecholamines by HPLC/EC

METHOD 29-1 Determination of Plasma Free Catecholamines by HPLC/ECcontd

METHOD 29-1 Determination of Plasma Free Catecholamines by HPLC/ECcontd

(C) 31 yr old female with MEN-2 a HMBA NMN EHPEA

NMN = 345 pg MN = 205 pg/m

Upper Reference Limit

Lower Reference Limit

95 0.52 125.00 0.68

102 0.52 73 0.37

METHOD 29-3 Determination of Plasma Free Metanephrine and Normetanephrine by LC-MS/MS

METHOD 29-3 Determination of Plasma Free Metanephrine and Normetanephrine by LC-MS/MScontd

One control exceeds 3 SD limits: Two or more controls exceed 2 SD limits:

METHOD 29-3 Determination of Plasma Free Metanephrine and Normetanephrine by LC-MS/MScontd

METHOD 29-3 Determination of Plasma Free Metanephrine and Normetanephrine by LC-MS/MScontd

METHOD 29-4 Determination of Urinary Free Catecholamines by HPLC/EC

METHOD 29-4 Determination of Urinary Free Catecholamines by HPLC/ECcontd

METHOD 29-4 Determination of Urinary Free Catecholamines by HPLC/ECcontd

METHOD 29-4 Determination of Urinary Free Catecholamines by HPLC/ECcontd

METHOD 29-5 Determination of Urinary Free Catecholamines by LC-MS/MS

METHOD 29-5 Determination of Urinary Free Catecholamines by LC/MS/MScontd

METHOD 29-5 Determination of Urinary Free Catecholamines by LC-MS/MScontd

METHOD 29-5 Determination of Urinary Free Catecholamines by LC-MS/MScontd

Analyte Epinephrine Norepinephrine Dopamine

METHOD 29-6 Determination of Urinary Metanephrine and Normetanephrine by HPLC/EC

METHOD 29-6 Determination of Urinary Metanephrine and Normetanephrine by HPLC/ECcontd

METHOD 29-6 Determination of Urinary Metanephrine and Normetanephrine by HPLC/ECcontd

METHOD 29-7 Determination of Urinary Metanephrine and Normetanephrine by LC-MS/MS

METHOD 29-7 Determination of Urinary Metanephrine and Normetanephrine by LC-MS/MScontd

METHOD 29-7 Determination of Urinary Metanephrine and Normetanephrine by LC-MS/MScontd

mL -20% Methanol 925 950 875 925 725 925 725

METHOD 29-7 Determination of Urinary Metanephrine and Normetanephrine by LC-MS/MScontd

Dilution Straight 5 10 20 40 Straight 2 5 10 20

% Expected 102% 88% 106% 101% 111% 105% 105% 109%

METHOD 29-7 Determination of Urinary Metanephrine and Normetanephrine by LC-MS/MScontd

% Expected 103% 99% 107% 114%

% Expected 105% 96% 101% 98%

Calibration Curves for Analysis of Metanephrine and Normetanephrine.

Specimen Type Unpreserved Acetic acid Boric acid

Metanephrine % Difference Range -10.3% to 14.7% -9.5% to 10.0% -6.5% to 31.7%

Normetaphrine Average % Difference 0.2% 9.3% 3.1%

METHOD 29-8 Determination of Urinary Vanillylmandelic Acid by HPLC/EC

METHOD 29-8 Determination of Urinary Vanillylmandelic Acid by HPLC/ECcontd

METHOD 29-9 Determination of Urinary Vanillylmandelic Acid by LC-MS/MS

METHOD 29-9 Determination of Urinary Vanillylmandelic Acid by LC-MS/MScontd

METHOD 29-9 Determination of Urinary Vanillylmandelic Acid by LC-MS/MScontd

METHOD 29-10 Determination of Urinary Homovanillic Acid (3-Methoxy-4-Hydroxyphenylacetic Acid) by HPLC/EC

METHOD 29-10 Determination of Urinary Homovanillic Acid (3-Methoxy-4-Hydroxyphenylacetic Acid) by HPLC/ECcontd

METHOD 29-10 Determination of Urinary Homovanillic Acid (3-Methoxy-4-Hydroxyphenylacetic Acid) by HPLC/ECcontd

METHOD 29-11 Determination of Urinary Homovanillic Acid by LC-MS/MS

METHOD 29-11 Determination of Urinary Homovanillic Acid by LC-MS/MScontd

METHOD 29-11 Determination of Urinary Homovanillic Acid by LC-MS/MScontd

METHOD 29-12 Determination of Urinary 5-Hydroxyindoleacetic Acid by Quantitative HPLC

METHOD 29-12 Determination of Urinary 5-Hydroxyindoleacetic Acid by Quantitative HPLCcontd

METHOD 29-13 Determination of Urinary 5-Hydroxyindole-3-Acetic Acid by LC-MS/MS

METHOD 29-13 Determination of Urinary 5-Hydroxyindole-3-Acetic Acid by LC-MS/MScontd

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