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1

29APPENDIX

Catecholamines

and Serotonin

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 fluids. In thissection, 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


Metanephrine and Normetanephrineby LC-MS/MS


Vanillylmandelic Acid by HPLC/EC


Vanillylmandelic Acid by LC-MS/MS


Homovanillic Acid (3-Methoxy-4-

Hydroxyphenylacetic Acid) by HPLC/EC


Homovanillic Acid by LC-MS/MS

Method 29-12. Determination of Urinary 5-

Hydroxyindoleacetic Acid by Quantitative HPLC


5-Hydroxyindole-3-Acetic Acid by LC-MS/MS

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.

Copyright 2006, 1999, 1994, 1986 Elsevier Inc. All rights reserved.

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2 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

PrincipleFree catecholamines are first rapidly adsorbed from plasma onto

aluminum oxide at pH 8.6. After washing with water, the cate-

cholamines 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 fixed potential to form

an o-quinone:

clonidine is preferred because this drug does not produce a false-

positive result.

Catecholamines in general are unstable compounds and can be

readily oxidized. To prevent this oxidation, many procedures recom-

mend that blood samples be transported to the laboratory on ice,

centrifuged at 4 C within 30 minutes of collection, and the plasmaremoved 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 metabisulfite. Advocates of a more liberal and

less constrained method of collecting and processing blood cate-

cholamines recommend the use of heparin without added preserva-

tives. 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, 0.1mol/L. Mix 8.6mL 72% HClO4 with distilled

water to a final volume of 1 L.

3. Potassium hydroxide (KOH), 4 mol/L. Dissolve 112.2g of KOH

in 400mL 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 135mL 85% H3PO4 with distilled

water to a final 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 3000rpm.

d. Remove an aliquot of the supernatant and analyze for cate-

cholamines. 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 follow-

ing catecholamines into separate 200-mL volumetric flasks:

10.0mg epinephrine free base (MW 183.2); 10.0mg nor-

epinephrine free base (MW 169.2); 12.4mg dopamine HCl

(3-hydroxytyramine HCl, MW 189.6). Dissolve and dilute to

the mark with HClO4, 0.1mol/L, to give free-base concentrations

of 50mg/L. Store the stock calibrators at 4 C. Stable for at least

6 months.9. Catecholamine combination calibrator. Dilute 100mL of the

catecholamine stock calibrators to 100mL with HClO4, 0.1mol/

L, to give free-base concentrations of 50mg/L. Store at 4 C.

10. Stock internal standard, 3,4-dihydroxybenzylamine, 50mg/L.

Dissolve 16 mg 3,4-dihydroxybenzylamine hydrobromide

(DHBA HBr, MW 220.1) in HClO4, 0.1mol/L. Dilute to

200mL with HClO4, 0.1mol/L, in a volumetric flask. 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 tempera-

ture and flow 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. Standard-

ization of posture is essential because plasma catecholamine levelsincrease twofold to threefold when a supine subject assumes an

upright position. Most procedures recommend that morning speci-

mens 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


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Appendix 29 3

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

For example:

(16mg DHBA HBr)/(0.2L perchloric acid) (MW free base

= 139)/(MW salt= 220.1)= 50mg/L (free-base concentration)

11. Working internal standard, 3,4-dihydroxybenzylamine, 50mg/L.

Dilute 100mL of the DHBA HBr stock internal standard

(50mg/L) to 100mL with HClO4, 0.1mol/L. Store at 4 C.12. Catecholamine prerun calibrator. Dilute 500 mL of the cate-

cholamine combination calibrator and 1000 mL of the working

internal standard to 10mL with HClO4, 0.1mol/L. Prepare fresh

daily.

13. Tris buffer. Dissolve 45g of Trizma base and 5 g Na2EDTA in

approximately 200mL distilled water. Adjust to pH 8.6 by adding

drops of concentrated HCl, 12mol/L. Dilute to 250mL and filter.

Store at 4 C. Stable for 2 months.

14. Water, pH 7.0. Using a pH meter, adjust approximately 500mL

of distilled water to pH 7.0 with sodium hydroxide (0.1mol/L)

or phosphoric acid (0.1mol/L), as required. Check pH of H2O

just before use. Adjust as necessary.

15. Alumina (acid-washed aluminum oxide; Bioanalytical Systems,

West LaFayette, Ind., Catalog No. CF-8010). Store tightly cappedin a desiccator.

16. Mobile phase, acetonitrile-citrate mixture. Dissolve 20.59g

trisodium citrate dihydrate in 880mL distilled water. Using a

pH meter, adjust the solution to pH 6.50 0.02 with H3PO4as required. Add 120 mL acetonitrile and mix well. Filter the

buffer mixture through a Millipore filtration device using a

0.45Tm (Type HA) membrane filter. Release vacuum imme-

diately after filtration is complete. Store tightly capped at room

temperature.

17. HPLC column wash reagent. Using a pH meter, adjust 1L

distilled water to pH 3 with H3PO4 (2mol/L) as required.

Instrumentation

A commercially available HPLC system equipped with a 15 cm4.6-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: 1cm/min

2. Inject 100mL 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

confirm that the system is at equilibrium.)

b. Epinephrine retention time is approximately 5 to 6 minutes

c. Epinephrine is clearly separated from the solvent front.

d. Epinephrine peak height is 30% to 50% of full scale.

e. All peak heights are within 5% on repeat injection.

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 4.0mL of each patient sample and control into labeled

96 16.8-mm extraction tubes.

7. Pipette 20, 40, and 80mL of the catecholamine combina-

tion calibrator into labeled tubes containing 4.0 mL of the

catecholamine-free plasma.

8. To all tubes add 80mL of the working internal standard solution

(50 mg/L).

9. Using a measuring spoon, transfer two level spoonfuls oalumina (about 60 to 80mg) to each tube.

10. Add 2mL 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 supernatan

from each tube using a vacuum aspirator connected to a vacuum

trap.

14. Using a squirt bottle, add about 1mL 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 withoudisturbing 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, 0.1mol/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 3000rpm. (Note: Do not

allow the supernatant extract to remain in contact with the

alumina for more than 30 minutes. Otherwise, degradation o

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 fo

up to 24 hours before injection into the HPLC system. (NoteExtracts should not be injected until the baseline has been estab-

lished. Check regularly for baseline shifts, and rezero the outpu

of the detector as necessary.)

21. Introduce 100mL of each standard, patient specimen,and contro

into the HPLC column. (Note: The remaining extract can be

used for dilutions as required.)

Chromatogram

Figure A29-1 shows a representative chromatogram of cate

cholamines extracted from plasma control.Continued


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4 Appendix 29

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

Calculations

1. Check sample identification 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 thatconnect these baseline points. (Note: Disregard small valleys

between partially fused peaks.)

4. For each sample, measure the peak heights to the nearest 0.1cm.

Record results on the appropriate worksheet as the example shows

below:

Calibrating Compound Retention Time, min Peak Heights, cm

Epinephrine 5.4 9.0

Norepinephrine 6.7 6.1

Dopamine 7.4 1.8

Internal standard 8.2 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).

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 500pg/mL calibrator

determined from a calibration curve

50= free-base concentration of catecholamine com-

bination 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: 152000pg/mL

epinephrine: 152000 pg/mLdopamine: 152000 pg/mL

When calculated values exceed these limits, the patient extract is

diluted with HClO4, 0.1mol/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

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Appendix 29 5

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.

PrincipleThis method allows measurement of picomolar plasma con-

centrations 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 con-

centrations of free O-methylated amine metabolites into a purified

low volume form appropriate for injection onto the HPLC appara-

tus. 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 first activated by washing with methano-

lic potassium hydroxide to bring up the negative charges on the

column matrix. The positively charged O-methylated amine metabo-

lites are thereby selectively adsorbed onto the activated cationexchange 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 flow of mobile

phase through the system. Separation on the HPLC column is facil-

itated by hydrophobic and ionic interactions between components

of the C18 analytical column stationary phase, mobile phase, andO-methylated amines.

The mobile phase provides an adjustable vehicle for passage of

the analytes of interest through the analytical column to the coulo-

metric detector. Adjustments to the composition of the mobile phase,

specifically the pH and concentrations of octane sulfonate and ace-

tonitrile, allow fine-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 sul-

fonate concentration, the retention of positively charged analytes of

interest may be altered in relation to other uncharged or changedspecies 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-methy-

lated amines and potentially interfering substances by modifying the

overall extent of hydrophobic interactions. Further changes to reten-

tion 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 tripleelectrode coulometric system, consisting of a conditioning cell and

an analytical cell connected to the Coulochem detector. The func-

tions of the electrochemical detection system are the detection and

quantification of the O-methylated amines after their separation and

elution on the analytical column. The first electrode in the condi-

tioning 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 nor-

mally 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 first electrode in

the conditioning cell. Outputs from the third electrochemical cell at

a reducing potential are generated as chromatographic recordingssuitable for subsequent analysis. Sample concentrations are calcu

lated from peak heights of the amine metabolites relative to those o

calibrators and corrected according to the procedural recovery of an

internal standard.


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 bloodat 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 a-70 C or colder until analysis. Samples requiring shipping should

be kept frozen using suitable amounts of dry ice packaged in styro

foam 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 establishedcauses of direct analytical interference from common over the

counter or prescribed medications (see below). However, occasiona

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 insufficiency. Therefore to minimize the appear-

ance of chromatographic interferences from dietary substances

samples of blood should be obtained after an overnight fast (wate

and decaffeinated soft drinks are permissible).Continued


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6 Appendix 29


Electrochemical Detectioncontd

Reagents

1. Calibrators: -normetanephrine HCl, -metanephrine HCl,

-methoxytyramine HCl, 4-hydroxy-3-methoxybenzylamine

HCl (all analytical grade); 4-hydroxy-3-ethoxyphenylethanola-

mine, special NIH synthesis. Store refrigerated. Expiration3years.

2. Prepare all calibrators to contain 1mg/mL stock calibrator (free

base) solutions in 0.2 mol/L acetic acid then dilute each of them

to separate 100ng/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

1.5mL Eppendorf tubes at -70 C to -80 C. Expiration2 years.

3. Mobile phase:

a. Acetonitrile, HPLC grade (no fixed source). Store at room tem-

perature. Expiration3 years.

b. 1-Octane sulfonic acid, ultrapure grade (Sigma O 0133). Store

at room temperature. Expiration3 years.

c. Phosphoric acid, HPLC grade (no fixed source). Store at room

temperature. Expiration3 years.d. Sodium phosphate monobasic (NaH2PO4 H2O), analytical

grade (no fixed source). Store at room temperature. Expira-

tion3 years.

e. Ethylenediamine-tetraacetic acid disodium salt: Dihydrate

(EDTA), analytical grade (Sigma ED2SS) Store at room tem-

perature. Expiration3 years.

f. Mobile phase stock solution: Add to a clean 1 L bottle 138g of

sodium phosphate monobasic, 1.28g of octane sulfonate, and

100mg EDTA. Dilute to 1L with deionized water and stir

thoroughly to dissolve. Store at room temperature. Expira-

tion6 months.

g. Mobile phase working solution: Add to a clean 1L bottle,

100mL of mobile phase stock solution and 50 to 90mL of

HPLC grade acetonitrile (75mL is often most appropriate fora new columnsee note below). Dilute to 950mL with milli-

Q 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 final volume of 1 L

with milli-Q water and Millipore filter through a 0.22 mm type

of GV (Millipore) filter 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 200mg

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 5.0g KOH (MW 56)

in 500mL of methanol or mix 89mL 1 mol/L methanolic

potassium hydroxide solution with 411mL methanol. Store at

room temperature. Expiration6 months.

c. Ten mmol/L acetic acid in methanol: Add 570 mL glacial acetic

acid to 1000mL of deionized H2O (10mmol/L acetic acid).

Mix together 900mL 10mmol/L acetic acid with 100mL

methanol. Store at room temperature. Expiration6 months.

d. Ammonium phosphate 10mmol/L, pH 8.5: Dissolve 1.15 g

monobasic ammonium phosphate (MW 115.03) in 800mL

deionized H2O. Adjust the pH to 8.5 with 5% ammonium

hydroxide (5 mL ammonium hydroxide in 95mL H2O).Adjust

volume to 1L with deionized H2O and add 0.05g EDTA. Store

at room temperature. Expiration6 months.

e. Ten percent ammonium hydroxide/methanol solution (1 : 3):Add together 90mL deionized H2O, 10mL concentrated

ammonium hydroxide solution, and 300mL HPLC grade

methanol. Made fresh on day of use. Expiration1 day.

f. Acetic acid, 0.2mol/L: Dilute 12 mL of glacial acid with milli-

Q water to 1L. Store at room temperature. Expiration1 year.

HPLC Instrumentation

1. Isocratic solvent delivery system (HPLC pump): Should be rela-

tively 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 100mL sample onto the analytical column (Waters

717 plus autosampler is recommended, Waters Chromatography,

Milford, Mass.).3. Analytical column: 5-mm particle size 4.6 250mm C18 reversed-

phase 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 diver-

sion 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 Refrig-

erated Vapor Trap, both from Thermo Savant, Holbrook, N.Y.).

Quality Control

Each extraction and HPLC run should include a water blank, a QCsample (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 3000g for 10


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Appendix 29 7



minutes to pellet fibrin and other particulate matter (Note: It is

essential that samples are free of particulate matter for their

efficient passage through extraction columns).

2. Number extraction columns, 13 100mm culture tubes, and

sample sheet according to the sequence of samples to beextracted and run on the HPLC.

3. Wash extraction columns with 5 mL NH4OH:methanol under

light vacuum (1 to 5mmHg). Do not allow columns to dry out.

4. Activate columns with 2mL KOH: methanol under reduced

vacuum (1 to 5mmHg). Again do not allow columns to dry out.

5. Wash columns with 2mL deionized H2O under reduced vacuum

(1 to 5mmHg). Again do not allow columns to dry out.

6. Add to columns 1mL deionized H2O, 70mL of 0.2mol/L acetic

acid, then the water (for blank) or plasma sample (2mL) fol-

lowed 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).

7. Wash columns with 2mL acetic acid: methanol under reduced

vacuum and dry the column out by increasing the vacuum to-20mmHg for 2 minutes.

8. Wash columns with 2mL ammonium phosphate solution under

reduced vacuum.

9. Wash columns with 2mL deionized H2O under reduced vacuum.

Elute all H2O.

10. Rotate the vacuum manifold to collect samples into the num-

bered 13 100mm glass culture tubes and check for correct

positioning of all needles.

11. Elute samples into culture tubes with 2 mL of NH4OH:

methanol. Use gravity and finally vacuum to elute all the

NH4OH:methanol.

12. Dry the sample down using a vacuum concentrator.

13. Dissolve the residue in 150mL of 0.2 mol/L acetic acid and inject

140mL onto the HPLC system.

High-Performance Liquid Chromatography

1. Establish optimal operating conditions of the chromatographic

and detection systems. Suggested conditions are as follows:

Flow rate: 1.0mL/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 de-

tecting 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 uponinjection of the series of calibrators. Figure A29-2 shows repre-

sentative 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

distinct from the amperometric mode of electrochemical detection

used in many other procedures. Most commercially available elec-

trochemical detectors feature glassy carbon or wall jet cells that

usually enable charge transfer of only 1% to 5% of the analyte tha

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 gener-

ally requires oxidation. The detector required for the method

described here features flow-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 defined as coulometric as distinct from amperometric.

The advantage of coulometric detection for measurements o

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 first in

a series of electrodes thereby provides a mechanism to reversibly

oxidize the analytes of interest while screening out potentially inter

fering impurities by irreversible oxidation. The reversibly oxidizedanalytes of interest may then be detected at a reducing potentia

without interference from any irreversibly oxidized impurities in the

sample.

The above features of coulometric detection enable generation o

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 concen-

trations 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% confidence limits (i.e., 2 standard deviations above and below

the geometric mean).

Geometric Upper Reference Lower Reference

Analyte Mean Limit Limit

Normetanephrine

(pg/mL) 45 112 18

(nmol/L) 0.25 0.61 0.10

Metanephrine

(pg/mL) 27 61 12(nmol/L) 0.14 0.31 0.06

Adult Reference Intervals Adjusted for Age and Gender

The reference intervals shown below are adjusted for a significant

influence of gender on plasma concentrations of metanephrine and

a significant influence of age on plasma normetanephrine and tota

normetanephrine. Data were log-transformed before analysis, and

upper and lower reference intervals were determined from the 95%

confidence limits.Continued


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8 Appendix 29



Upper

Geometric Reference Lower Reference

Analyte Mean Limit Limit

Normetanephrine

Age

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Appendix 29 9



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. Quantification of unconjugated meta-

nephrines in human plasma without interference by aceta-

minophen. 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 concen-

trated eluate is analyzed using liquid chromatography-tandem

mass spectrometry (LC-MS/MS) and quantified using stable iso-

tope-labeled internal standards, d3-metanephrine (d3-MN) and d3-

normetanephrine (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

specific 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 epi-nephrine and norepinephrine,respectively.They are produced by the

actions of the enzyme catechol-O-methyltransferase (COMT). The

membrane-bound form of the enzyme is specifically 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 identification of patients with

pheochromocytoma.


Use a 100 16mm lavender stopper vacutainer containing 15mg

EDTA. Ten milliliters of blood is collected and centrifuged at

2000rpm for 10 minutes. The plasma is removed and placed in

15mL conical centrifuge tubes and is immediately frozen at -20 C

until time of analysis. The specimen is stored at -20 C for short-term storage. Store at -70 C if long-term storage is required.

Reagents

1. -Metanephrine HCl, Sigma Chemical Co. M-8625 (or equiv-

alent >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 Laborato-

ries, 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 expirationdate.

5. Oasis TM HLB 1cc (30mg) 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 a

room temperature. Stable until expiration date.

8. Water, HPLC Grade, Fisher Scientific W5-4. Store at room tem

perature. Stable until expiration date.

9. Formic acid, Sigma F-0507. Store at room temperature. Stable

until expiration date.

10. Trifluoroacetic acid (TFA), Pierce 28901. Store at room temper

ature. 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 (1.0mg/mL). Weigh ou

11.8mg of metanephrine HCl and 12.0mg of normetanephrine

HCl. Transfer to a 10mL volumetric flask 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 9.9mL RO H2O. Store at 2 C to 8 C. Stable 3

months.

15. Analytical working dilution II (100ng/mL). Add 100mL analyti-

cal working I to 9.9mL RO H2O. Store at 2 C to 8 C. Stable3 months.

16. Analytical working dilution III (10ng/mL). Add 1.0 mL analyti

cal working II to 9.0mL RO H2O. Store at 2 C to 8 C. Stable 3

months.

17. Analytical working dilution IV (1ng/mL). Add 1.0mL analytica

working III to 9.0mL RO H2O. Store at 2 C to 8 C. Stable 3

months.

18. Calibrators 1 to 6. Pipette stripped serum and analytical calibra

tor (see chart) into 12 75 tubes; store at -20 C.Continued


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10 Appendix 29


LC-MS/MScontd

1. Internal stock standard solution (1.0 mg/mL). Weigh out 11.8 mg

of d3-metanephrine HCl and 12.0mg of d3-normetanephrine

HCl. Transfer to a 10mL volumetric flask and add RO H2O to

volume. Store at -20 C. Stable 12 months.

2. Internal working dilution I (10 mg/mL). Add 100mL stock stan-

dard to 9.9mL RO H2O. Store at 2 C to 8 C. Stable 3 months.

3. Internal working dilution II (100ng/mL). Add 100mL internal

working I to 9.9mL RO H2O. Store at 2 C to 8 C. Stable 3

months.4. Internal working dilution III (10ng/mL). Add 1.0mL internal

working II to 9.0mL 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% triflu-

oroacetic acid. Weigh out 120mg ammonium acetate and trans-

fer to a 1 L flask. Add 600mL RO H2O and 400mL acetonitrile.

Mix with magnetic stir bar until in solution. Add 60mL formic

acid and 40 mL trifluoroacetic acid. Store at room temperature.

Stable 2 weeks.

6. Injector rinse solution: 75/25 (v/v) methanol/RO H2O. Combine

750mL methanol and 250mL of RO H2O.

Consumables1. Plastic tubes, 13 75mm, Sarstedt.

2. Disposable culture tubes, 10 75mm, Fisher Scientific 14-961-25.

3. Pipette tips, 1000 mL and 200 mL, Continental Lab Products 2167,

2007.

4. Disposable plastic transfer pipettes, Fisher Scientific 13-711-7.

5. Autosampler vials, National Scientific Co., Target DP Catalog

No. C4000-1, or Fisher Scientific minivials 0.2mL Catalog No.

0334064 with snap-it-seal cap Catalog No. 0337749.

6. Caps, National Scientific Co., DP blue cap & TS septa, Catalog No.

C4000-54B.

7. Inserts, National Scientific Co., 0.3mL, Catalog No. C4010-630.

Instrumentation

1. Gilson Pipetman pipettes, 1000mL adjustable, 200mL 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, 15cm 4.6mm, 5 mm, Phenomenex

9. Guard column: Security Guard CN, 2cm 2.1 mm, 5 mm,

Phenomenex

10. Offline HPLC pump for flushing column after analysis

Calibration

The calibration uses a six point extracted calibrator curve over a con-

centration range of 0.25 to 10 nmol/L. Calibration curves are gener-

ated with each assay. The Analyst software package calculates the area

counts and plots a calibration curve for the analyte/IS area countratio 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 per-

formed with each assay. The spiked plasma control pools are made

by adding calibrators to plasma pools so that the final concentrations

of MN and NMN are approximately 0.3, 0.6; 1.0, 2.0; and 2.0,

4.0nmol/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 20times over multiple days in conjunction with the current pools.

Values are recorded using Levy-Jennings control charts and moni-

tored 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 final

interpretation made by the laboratory supervisor or director. General

QC rules in effect are:

All controls within 2 SD limits: Release results

One control exceeds 2 SD limits: First time, release results

Subsequent occurrences,

take corrective action

One control exceeds 3 SD limits: Take corrective action

Two or more controls exceed 2 SD limits: Take corrective action

Corrective action may include: (1) evaluating results, including

repeats and linearity; (2) evaluating control pool and/or reagents; (3)

repeating the assay; or (4) notifying supervisor or lead personnel.

Procedure

Sample Preparation

1. Invert plasmas to mix, and place 1.0mL of patient or control

plasma in a labeled 12 75mm Sarstedt tube.

2. Add 50mL of internal standard working dilution III to each tube

using an Eppendorf repeater pipette.

Concentration Concentration Volume Analytical Volume Analytical Volume Analytical

Calibrator Metanephrine Normetanephrine Stripped Serum Std IV (1 ng/mL) Std III (10 ng/mL)

1 0.25nmol/L 0.27 nmol/L 1mL 50mL


3 1.0nmol/L 1.08 nmol/L 1mL 200mL 4 2.5nmol/L 2.7 nmol/L 1mL 50mL


6 10.0nmol/L 10.8nmol/L 1mL 200mL


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Appendix 29 11


LC-MS/MScontd

Solid Phase Extraction

1. Load Oasis HLB SPE cartridges on to the Supelco vacuum man-

ifold and pull vacuum at 5 in Hg for all steps. Precondition the

cartridges with 1 mL methanol followed by 1mL RO H2O.

2. Add the plasma/IS mixture with a plastic disposable transferpipette.

3. Wash with 2mL of RO H2O.

4. Place an empty 12 75 mm disposable culture tube in the mani-

fold rack for each SPE cartridge. Place manifold rack into mani-

fold and verify that all tubes are in line with a SPE connection.

Elute MN and NMN with 1mL methanol. Discard Oasis car-

tridges 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 50mL 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: PMET

Acquisition method: PMET

Flow 1.2 mL/min

Inj volume 30 mL

Run time 8.0 min

Pre/Postinjection flushes 0/5

3. Mobile phase: 40/60 (v/v) acetonitrile/HPLC grade water1.5mmol/L ammonium acetate. 0.06% formic acid, 0.04% triflu-

oroacetic acid. HPLC column: LUNA CN, 15cm 4.6mm, 5 mm,

Phenomenex Guard column: Security Guard CN, 2 cm 2.1mm,

5 mm, Phenomenex.

MS/MS Parameters:

Parameter MN d3-MN NMN d3-NMN

MRM pairs 180.0/ 183.3/ 166.2/ 169.2/

148.0 151.0 134.0 137.0

Dwell times 500 500 500 500

Nebulizer gas (NEB) 15

Curtain gas (CUR) 7

Collision gas (CAD) 4

Nebulizer current (NC) 3Temperature (TEM) 500

Declustering potential 50 V

(DP)

Focusing potential (FP) 175 V

Entrance potential (EP) 10 V

Collision energy (CE) 25 V

Collision cell exit 2 V

potential (CXP)

After analysis is complete, flush columns with 50/50 acetonitrile/

water at 1mL/min for 30 minutes. This will help to extend the

column life. This can be performed with an off-line HPLC pump.

Calculations

Results are automatically calculated by the LC-MS/MS software and

printed as nmol/L plasma. If a volume other than 1mL plasma

is used, the value should be edited appropriately. For example: i

500 mL is used, the final result should be multiplied by 2.

Reference Intervals

Metanephrine:

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12 Appendix 29


LC-MS/MScontd

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.

CorrelationN Slope Y-intercept Coefficient

Metanephrine 92 0.87 0.05 0.95

(nmol/L)

Normetanephrine 132 0.90 -0.06 0.93

(nmol/L)

7. Reference intervals: An independent study was conducted using

healthy volunteers to obtain a reference interval for the LC-

MS/MS method. Results matched the existing reference intervals

for the HPLC-EC method.

8. Carryover: There are no problems due to carryover on the LC-

MS/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 chromatography-

tandem 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 pheochro-mocytoma. 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 first 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 affinity 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:

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 detec-

tion system. As shown earlier, each catecholamine passing through

the detector cell undergoes a rapid two-electron oxidation at a fixed

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 flow rate, this oxidation current is directly propor-

tional 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

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:


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Appendix 29 13

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

peak heights and retention times. To calculate sample concentra-

tions, peak height ratios relative to the internal standard (dihydroxy-

benzylamine) for unknowns are compared with those of the

calibrations.

Specimen Collection and StorageAll 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 con-

taining 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 indefinitely.

Reagents1. 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 final 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 final 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 tem-

perature and prepare every 2 weeks.

9. Catecholamine stock calibrator. Weigh and transfer the follow-

ing catecholamines to a 500-mL volumetric flask: 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 HClO40.1 mol/L, in a volumetric flask 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 octy

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 filtration apparatus using a

2-mm membrane filter. Degas for 1 hour with a vacuum and then

add methanol (100 mL/L buffer). Stir the mixture for 10 minutesat 40 C. Further degas the mixture for 10 minutes by slowly bub

bling 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 o

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 octy

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 Laborato

ries). 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 abou

2.5 cm.e. Store packed columns at 4 C after carefully sealing both tip

and cap with parafilm. Stable for at least 1 year.

17. Nitrogen gas, prepurified, 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


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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 isadjusted to pH 3 with hydrochloric acid, 6 mol/L, and spiked with

the catecholamine stock calibrator, 10 mL per L of urine. Six-

milliliter 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 900g.

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 ontoseparate 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.


11. Establish optimal operating conditions of the chromatographicand 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 rep-

resentative chromatogram of catecholamines extracted from a

urine calibrator.

Calculations

1. Identify catecholamine and internal standard peaks by relative

retention times (in minutes). Measure peak heights (nA) directlyfrom the chromatographic tracings.

Example:

Retention Time Peak Heights,ht

Calibrating Compound (min) (nA)

Norepinephrine 4.5 56

Epinephrine 5.5 14

Internal standard 6.4 18

Dopamine 9.0 108

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

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 identified on the chromatogram.


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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 finely divided particles of graphite and a binder thatis 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 elec-

trode performance with minimal noise. Paraffin 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, dis-

solves paraffin oil; carbon paste using paraffin 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 transferproperty, are comparable for both electrodes. However, the carbon-

paste 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) Norepinephrine

Children

0-1 0-10 (0-59)

1-2 1-17 (6-100)

2-4 4-29 (24-171)

4-7 8-45 (47-266)

7-10 13-65 (77-384)

10-15 15-80 (89-473)

Adults

>15 15-80 (89-473)

Age (yr) Epinephrine

Children

0-1 0-2.5 (0-14)1-2 0-3.5 (0-19)

2-4 0-6.0 (0-33)

4-7 0.2-10 (1-55)

7-10 0.2-10 (1-55)

10-15 0.5-20 (3-109)

Adults

>15 0.5-20 (3-109)

Age (yr) Dopamine

Children

0-1 0-85 (0-555)

1-2 10-140 (65-914)

2-4 40-260 (261-1697)

4-7 65-400 (424-2612)7-10 65-400 (424-2612)

10-15 65-400 (424-2612)

Adults

>15 65-400 (424-2612)

Daily Excretion Relative to Creatinine

mg/g Creatinine

Age (yr) Norepinephrine

0-1 up to 0.31

1-4 up to 0.29

4-10 up to 0.11

10-18 up to 0.11

>18 up to 0.11

Age (yr) Epinephrine

0-1 up to 0.38

1-4 up to 0.08

4-10 up to 0.09

10-18 up to 0.06

>18 up to 0.04

Age (yr) Dopamine

0-1 up to 1.29

1-4 up to 1.22

4-10 up to 0.72

10-18 up to 0.45

>18 up to 0.35

ReferenceMoyer TP, Jiang NS, Tyce GM, Sheps SG. Analysis for urinary cate-

cholamines by liquid chromatography with amperometric detec-

tion: methodology and clinical interpretation of results. Clin

Chem 1979;25:256-63.


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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, Uni-

versity of Utah School of Medicine, Salt Lake City, Utah)

PrincipleAn 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 significantly greater

instrument throughput and improved specificity.

The specificity of this sample preparation method is based on

the difference in affinity to PBA of CATS and potentially interferingcompounds 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 accord-

ingly for each multiple reaction monitoring (MRM) transition

specified in the acquisition scan. Alternating the collision cell volt-ages produces consistent product ion response ratios for qualitative

confirmation of the catecholamines. The conditions employed

produce satisfactory sensitivity and specificity for the selective

detection of CATS in urine.


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). Alka-

line specimens (pH> 7) and specimens stored at ambient tempera-

ture 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 stabil-

ity 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

metabisulfite (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 flask. Stir and add 100 mg of sodium metabisulfite.

Mix until the solid is dissolved completely. Stable for 1 week at20 C.

5. Catecholamine stock calibrators. Epinephrine, norepinephrine,

or dopamine (1.0 g/L) in acetic acid/sodium metabisulfite

diluent (AASM).Stock solutions for each catecholamine are pre-

pared 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 com-

pletely. Stable for 1 year at -70 C.

6. Catecholamine working calibrator. Epinephrine, norepineph-

rine, and dopamine (0.5 mg/L). In a 50-mL volumetricflask 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-d 3, 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 isdissolved 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 flask, 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 thelimit 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).


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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 dis-

solved. 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 totalvolume 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 flask.

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., Belle-

fonte, 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 neg-

ative 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 acidto this tube. This sample is not extracted.

3. Quantitatively add into empty tubes 30 mL of the working inter-

nal 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 gfor 3 minutes.

9. Label a clean tube for each calibrator, control, and urine speci-men. Add 50 mL of 0.08 mol/L acetic acid and 500 mL of 1-

octanol 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 gfor 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 0.4 mL/min

LC column effluent split flow 60-80%

Column temperature Ambient

Injection volume 3-5 mL

Number of syringe washes Preinjection: 5

Postinjection: 5

The mass spectrometer is tuned for unit resolution of Q1 and Q3Voltages are optimized for maximum sensitivity.

MRM transitions monitored (m/z):

Epinephrine 184 to 107, 184 to 77

Epinephrine-d3 187 to 110

Norepinephrine 170 to 107, 170 to 77

Norepinephrine-d3 173 to 110

Dopamine 154 to 91, 154 to 65Dopamine-d4 158 to 95

Transitions m/z 184 to 107, 170 to 107, and 154 to 91 are quantita-

tive, 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, cali

brators, and controls for acceptable peak shapes and area counts

2. The coefficient 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 mus

be within the range established by the calibration. This is typically50% of the average of the ion-mass ratios observed in the cali-

brators 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 o

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, normetanephrineContinued


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18 Appendix 29

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

A

C

HEB

GD

IF

0 1 2 3Time min

0 1 2 3Time min

0 1 2 3Time min

Figure A29-4 Chromatograms of an extracted urine sample containing 12 mg/L of epinephrine,

29mg/L of norepinephrine, and 66mg/L of dopamine.The analytes elute at the same retention

time. Ion transitions: epinephrine (A) m/z 184 to 107 and (B) m/z 184 to 77; epinephrine-d3(C) m/z 187 to 110; norepinephrine (D) m/z 170 to 107 and (E) m/z 170 to 77;

norepinephrine-d3 (F) m/z 173 to 110; dopamine (G) m/z 154 to 91 and (H) m/z 154 to 65;

and dopamine-d4 (I) m/z 158 to 95.


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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

0.1mol/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, 3.2mm I.D. 10cm

(Bioanalytical Systems, Catalog No. MF 6213), and an amperomet-

ric 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,

0.05mol/L, and processed for immediate use (Bio-Rad Laboratories,

Catalog Nos. C-390-25 and C-395-25).

Procedure

Hydrolysis1. 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 2.0mL of calibrator, controls, and patient samples into

labeled 16 150-mm test tubes. Add 2.0mL distilled water and

200mL internal standard solution to each tube. Mix each tube

briefly in a vortex mixer.

3. Add HCl (2mol/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

Parafilm.

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 anion-

exchange column for each calibrator, control, and sample.

Prepare the columns, about five 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 nec-

essary. Adjust slope to 4.00 using the pH 4 buffer.

9. To each tube, add 5.0mL ammonium pentaborate buffer and

300mL NaOH (0.5mol/L). Mix in a vortex mixer. Check that thepH is between 6.0 and 7.0. If necessary, adjust pH with 100-mL

aliquots of NaOH (0.5 mol/L) or acetic acid (1.0mol/L).

10. Pour each sample into the cation-exchange column assigned to

it and allow to drain completely.

11. Add 10mL 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 anion-

exchange column and insert it through the polycolumn support

so that it is held in place.

13. Pipette 8.0mL of NaOH (2mol/L) into each upper cation-

exchange column and allow it to drain completely through both

columns. Remove and discard the cation-exchange columns.

14. Rinse each anion-exchange column with 5mL of distilled water.

15. Replace the waste reservoir with a tube rack containing 16150-mm test tubes. Make sure that the columns are located

above marked tubes so that no sample will be spilled.

16. Dispense 5.0mL of elution buffer into each column. Allow the

columns to drain completely and then discard them.

17. Add 400mL acetic acid (1 mol/L) to each tube and then mix them

in a vortex mixer. If necessary, the eluates may be covered with

Parafilm and stored overnight at room temperature.Eluates may

also be stored for 1 week at 4 C.


18. Establish the optimal operating condition of the chromato-

graphic and detection systems. Suggested conditions are:

Flow rate: 1.0 mL/min

Potential: 800 mVSensitivity: 20 or 50 n

chapter 029

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