High performance liquid chromatographic analysis of urinary catecholamines employing amperometric detection.pdf

Bài báo khoa học

Clin Biochem 13 (6) 285-291 (1980)

High Performance Liquid Chromatographic Analysis of Urinary Catecholamines Employing Amperometric Detection:

References Values and Use in Laboratory Diagnosis of Neural Crest Tumors

S J SOLDIN, G LAM, A POLLARD, L C A L L E N and A G LOGAN

D e p a r t m e n t s , of Clinical Biochemistry, P h a r m a c o l o g y , and Medicine, University of T o r o n t o

and

D e p a r t m e n t of Biochemistry, Hospital for Sick Children, Mount Sinai Hospital, and T o r o n t o General Hospital

We have developed an high performance liquid

chromatographic procedure employing amperometric detection

for the measurement of urinary free norepinephrine, epine-

phrine and dopamine The between-day precision (C.V.) at

various concentrations of the above analytes varied from

4.8-11.6% There was negligible between-urine difference in the

percent recovery of norepinephrine and epinephrine but con-

siderable between-urine difference for dopamine The procedure

has been used to determine reference ranges in infants, children

and adults Its preliminary application to the laboratory detec-

tion of neural crest tumors is also described

Y O N F R A E N K E L (1) in 1886 s h o w e d that the concentra-

tion of catecholamines w a s increased in a patient with

"explosive hypertension" (pheochromocytoma) by

oxidising the tumor catechols to ortho-quinones which

have a characteristic red-brown color In 1957 the

pioneering w o r k of A r m s t r o n g and co-workers (2)

helped to identify the metabolic pathways involved, and

today the laboratory analysis of urinary catecholamines

and metabolites plays a central role in assisting the

clinician to arrive at a diagnosis of a neural crest tumor

T h e diagnosis of a pheochromocytoma in a patient

with hypertension is critical as the hypertension is

curable by resection of the tumor, but is almost always

fatal if the tumor escapes detection In order to

evaluate the laboratory tests of choice for the diagnosis

of neural crest tumors, w e felt it w a s necessary to first

develop improved analytical procedures, and then to

employ these procedures to assess their effectiveness in

diagnosis This approach w a s considered essential as

both our experience and that of other workers {3-5) have

s h o w n that m a n y chemical methods lack specificity and

Address correspondence to: Dr S J Soldin, Department of

Biochemistry, The Hospital for Sick Children, 555 University

give rise to both false positive and false negative results

A high performance liquid chromatographic pro- cedure for the simultaneous analysis of urinary

4 - h y d r o x y - 3 - m e t h o x y m a n d e l i c acid (VMA) and 4-hydroxy-3-methoxyphenylacetic acid (HVA) has been developed in this laboratory and has been recently described in the literature (6) The present paper describes an high performance liquid chromatographic analysis for urinary free norepinephrine, epinephrine and dopamine which has many features in common with the methods published by Riggin and Kissinger (7) and Moyer et al (8), and which has been used to determine reference ranges in infants, children, and adults Its preliminary application to the laboratory detection of patients with neural crest tumors is also described

MATERIALS AND M E T H O D S

W e used an high performance liquid chromatograph, series 2/2 {Perkin-Elmer Corp., Norwalk, C T 06856), and a 4 m m x 30

cm ~-Bondapak C18 column (Waters Associates, Inc., Milford,

M A 07157) The detector used was a Bioanalytical Systems Model LC-16 incorporating the TL-5 glassy carbon electrode (Bioanalytical Systems Inc., West Lafayette, IN 47906) con- nected to a Perkin-Elmer Model 023 recorder

DL-norepinephrine hydrochloride, DL-epinephrine, dopamine hydrochloride and Trizma base were obtained from the Sigma Chemical Company, St Louis, M O 63178 The 3,4-dihydroxybenzylamine was purchased from the Aldrich Chemical Company (Milwaukee, W I 53233) while heptane sulfonic acid was acquired from the Estman Kodak Company, Rochester, N Y 14650 Alumina Woelm Neutral: Akt 1 and BioRex 70 were purchased from ICN Nutritional Biochemicals, Cleveland, Ohio 44128 and from BioRad Laboratories, 32nd and Griffin, Richmond, California, respectively Thioglycolic acid was purchased from the Fisher Scientific Company, Fair Lawn,

N J 07410

A stock solution was prepared by dissolving 10 m g of each of norepinephrine, epinephrine, dihydroxybenzylamine and

2 8 6

nA

1 4 -

12

10

6 -

4 -

2 -

0

0

SOLDIN, LAM, POLLARD, ALLEN AND LOGAN

i

o', o12 o13 o14 o:5 0:6 o17 o18 o19

POTENTIAL (V)

Fig 1 Current obtained at different potentials on injection of 2.5, 3.~, 3.8 and 3.7 ng of norepinephrine (IJ, epinephrine (2), dihydroxybenzylamine {3), and dopa- mine {4] respectively employing the condi- tions described in the text

i

1.0

n A

24

2 2 -

2 0 -

1 8 -

1 6 -

14

12

1 0 -

8 -

6 -

4

2 -

n g I N J E C T E D

Fig 2 - Calibration curves f o r nore- pinephrine (1), epinephrine (2), dihydroxy- benzylamine (3), and dopamine (/~] employ- ing conditions described in the text

standard was prepared weekly by adding the following

volumes of stock standard solutions and diluting to 1 L with

0.100 N HCI: -

2.50 mL norepinephrine solution,

3.20 mL epinephrine solution,

3.84 mL dihydroxybenzylamine solution, and

3.69 mL dopamine solution

The stock solutions were found to be stable for at least 3

months at 4°C and the working standard solutions for at least 1

week at 4°C

All reagents for extraction of catecholamines were prepared

as described by Riggin and Kissinger (7) The extraction pro-

cedure used was identical to that described by these authors

except that we employed double the amount of internal stand-

ard, and performed 2 acetic acid extractions from the alumina

instead of 1 used by the original authors

Adjust the pH of 5 mL of urine to 3.0 with 1 N sodium hydroxide Add 15 mL OF 39 mM phosphate buffer pH 7.0, and

50 ~L of internal standard (3,4-dihydroxybenzylamine, 20 mg/L iBioRex 70, column dimensions 0.7 x 4.0 cm) Allow to drain completely and then wash the column with 10 mL of distilled water Add 1.5 mL of 0.7 M sulfuric acid and allow to drain Elute the catecholamines with 4 mL of 2 M ammonium sulfate solution Add 100 ~L of 4% thioglycolic acid and 500 ~L of 3 M Tris buffer pH 8.6 Add 100 mg of alumina and shake for 10 minutes Allow the alumina to settle and discard the superna- tant Wash the alumina with 3 mL of deionized water and discard the supernatant Elute the catecholamines from the alumina with 500 pL of i M acetic acid Repeat the last step and combine the acetic acid eluates Inject 20 ~L into the liquid

Buffer

T e m p e r a t u r e

Flow Rate

Sensitivity

Detector

Potential

HIGH P E R F O R M A N C E LIQUID CHROMATOGRAPHIC ANALYSIS OF URINARY CATECHOLAMINES

T A B L E 1

CHROMATOGRAPHIC CONDITIONS

TABLE 2 BETWEEN-DAY PRECISION

P r e s e n t Study Moyer et al (8) Compound Concentration (~g/L) %C.V

9515 ratio of same solutions

Room 3.0 mL/min Not mentioned Not mentioned

90/10 mixture of

70 mM phosphate buffer

pH 4.8 containing

0.1 mM EDTA & 2 mM

heptanesulfonic acid/

methanol

Room

2.0 mL/min

2 n A N

0.70 V

287

6 4 2

TIME (min)

n A

20

18

16

14

12

10

0

0

Fig 3 Chromatogram obtained f o r a hypertensive patient without a neural crest tumor 1, 2, 3 and 4 represent the peaks obtained for norepinephrine, epi- nephrine, d i h y d r o x y b e n z y l a m i n e , and dopamine respectively and correspond to

431 ng norepinephrine, 116 ng epinephrine and 990 ng dopamine in 5 m L of urine

)l,

Fig $ - Chromatogram obtained for a patient with pheochromocytoma 1, 2, 3 and 4 represent the peaks obtained f o r norepinephrine, epinephrine, dihydroxy- benzylamine and dopamine respectively and correspond to 4160 ng norepinephrine,

2100 ng epinephrine and 2110 ng dopamine

in 5 m L of urine

n A

20

18

16

14

12

10

6 4 2 0

TIME (min)

Ilttl u

/11tit -°

ill

i

~" 0

288 SOLDIN, LAM, POLLARD, ALLEN AND LOGAN

The chromatographic conditions employed (see Table 1) are

similar, though not identical, to those described by Moyer et

al (8)

RESULTS AND DISCUSSION

Detector Potential

F i g u r e 1 shows the c u r r e n t in n a n o a m p e r e s obtained

a t different p o t e n t i a l s on injecting 2.5, 3.2, 3.8 and 3.7 ng

of norepinephrine, epinephrine, d i h y r o x y b e n z y l a m i n e

and dopamine r e s p e c t i v e l y Monitoring the c u r r e n t at a

p o t e n t i a l of 0.70 V p r o v i d e s a sensitive a s s a y for all

t h e s e compounds H i g h e r p o t e n t i a l s increase baseline

noise and d e c r e a s e the specifity of the analysis

Column Temperature

T h e r e was no need to employ e l e v a t e d t e m p e r a t u r e s

as excellent c h r o m a t o g r a p h i c s e p a r a t i o n was achieved

at room t e m p e r a t u r e

Composition of the Mobile Phase

I n c r e a s i n g the methanol content of the mobile phase

from the 5O/o e m p l o y e d by M o y e r et al (8) to 10°/o con-

s i d e r a b l y r e d u c e d the c h r o m a t o g r a p h y time and provid-

ed s h a r p e r peaks

Precision

The p r o c e d u r e outlined was used to d e t e r m i n e the

b e t w e e n - d a y precision (Table 2) by the r e p e a t e d

analysis (n = 20) of urine s a m p l e s containing different

concentrations of norepinephrine, e p i n e p h r i n e and

dopamine The r e s u l t s show t h a t acceptable precision is

obtained at clinically i m p o r t a n t concentrations

Recovery Studies

The overall r e c o v e r y of n o r e p i n e p h r i n e , e p i n e p h r i n e ,

d i h y d r o x y b e n z y l a m i n e and dopamine from urine was

a s s e s s e d as follows:

Experiment A: The mean p e a k height (n = 10) of norepinephrine, epinephrine, and dopamine in each of four different urines was d e t e r m i n e d using the pro-

c e d u r e outlined

Experiment B: 1000 ng of each of n o r e p i n e p h r i n e ,

e p i n e p h r i n e , d i h y d r o x y b e n z y l a m i n e and d o p a m i n e

w e r e then a d d e d to 5 mL aliquots of the above u r i n e s and the analysis of each urine r e p e a t e d ten times The mean p e a k heights obtained w e r e again noted

S u b t r a c t i o n of the mean peak heights obtained in ex-

p e r i m e n t A from the means of those obtained in experi-

m e n t B gave the p e a k heights obtained for 1000 ng of each of n o r e p i n e p h r i n e , epinephrine, d i h y d r o x y b e n z y - lamine and dopamine

These mean p e a k heights w e r e then c o m p a r e d with those obtained by the d i r e c t injection of s t a n d a r d s (see

F i g u r e 2) and the overall r e c o v e r y calculated (Table 3) The use of a single acetic acid e x t r a c t i o n s t e p as recom-

m e n d e d by Riggin and K i s s i n g e r (7) led to only 80% of the r e c o v e r y values shown in Table 3 F o r this r e a s o n a second acetic acid e x t r a c t i o n was included in our pro- cedure

We e m p l o y e d the following approach to d e t e r m i n e the u r i n a r y free catecholamine c o n c e n t r a t i o n s in unknown samples W e d e t e r m i n e d the mean (n = 10)

p e a k h e i g h t r a t i o for each of n o r e p i n e p h r i n e ,

e p i n e p h r i n e and dopamine to d i h y d r o x y b e n z y l a m i n e for

s e v e r a l urines (see Table 3) The c o n c e n t r a t i o n of the above compounds a d d e d to the urines for this p u r p o s e was 1000 ng/5 mL of urine The following formula was then used to d e t e r m i n e the c o n c e n t r a t i o n of norepi-

TABLE 3

RECOVERY STUDIES

Mean %

recovery (n = 10)

Mean peak height ratio of analyte to internal standard (n = 10) Norepinephrine

Epinephrine

Dopamine

Dihydroxybenzylamine

43.0 43.6 46.0 41.0 39.0 34.5 37.7 34.0 38.0 37.0 61.6 53.0 51.5 47.0 52.0 46.5

0.9801 1.0103 0.9797 Mean 1.0038 1.0453

1.0680 0.9765 Mean 1.0083 1.0094

0.9796

0 4714 0.5918 0.8571 Mean 0.7517 0.8867

: 8 0 -

~ 7 0

Z 6 0

" ' 5 0

Z

+ 4 0

Z

~ 3o

Ill

Z

2 o

u

O

"1"

HIGH P E R F O R M A N C E LIQUID CHROMATOGRAPHIC ANALYSIS OF URINARY CATECHOLAMINES 289

INTERCEPT= 2.02

MEAN HPLC

NOREPINEPHRINE + EPINEPHRINE= 34.35

MEAN TOTAL

n = 51

I I I I i I I I

Fig 5 - Comparison of results ob- tained by the HPLC procedure for the analysis of norepinephrine and epineph- rine with those obtained for total catecholamines

T O T A L C A T E C H O L A M I N E S , u g / 2 4 hrs

TABLE 4

hypertensive

"Analyses performed on random urine samples obtained from healthy adults and children

"Analyses performed to rule out the diagnosis of pheochromocytoma in hypertensive patients Values in p a r e n t h e s e s are ~g/24 h

TABLE 5

Norepinephrine Epinephrine Dopamine VMA" HVA" Total Catecholamines

"95th percentile values for VMA and HVA in our laboratory are 6.0 and 7.6 mg/g creatinine respectively (19 years and over)

290 SOLDIN, LAM, POLLARD, ALLEN AND LOGAN

TABLE 6

URINARY EXCRETION OF N OREPINEPHRINE EPINEPHRINE, DOPAMINE 3-M ETHOXY-4-H YDROXYMANDELIC ACID ( V M A ) A N D 3-M ETHOXY-4-H Y-

DROXYPHENYLACETIC ACID { H V A ) IN PATIENTS WITH N E U R O B L A S T O M A

The age related 95th percentile values for VMA and HVA are in parentheses

nephrine, e p i n e p h r i n e and dopamine in unknown

samples:

P e a k height of unknown

( n o r e p i n e p h r i n e , e p i n e p h r i n e o r d o p a m i n e )

P e a k height of internal s t a n d a r d

1000

X

mean factor (see Table 3)

= ng of norepinephrine, e p i n e p h r i n e or dopamine in 5

mL of urine

As can be seen from Table 3 t h e r e was negligible

between-urine difference in both the mean p e r c e n t a g e

r e c o v e r y and the mean peak height ratio of a n a l y t e to

internal s t a n d a r d for n o r e p i n e p h r i n e and epinephrine

The overall peak height r a t i o mean of 1.0038 and 1.0083

for n o r e p i n e p h r i n e and epinephrine c o r r e s p o n d s to 1000

ng of these a n a l y t e s in 5 mL of urine, and is the mean

factor employed in the above calculations

T h e r e was considerable b e t w e e n - u r i n e difference in

both the mean p e r c e n t a g e r e c o v e r y of dopamine and

the mean peak height r a t i o of dopamine to internal stan-

dard Use of the mean factor of 0.7517 in the above may

under or o v e r e s t i m a t e the t r u e dopamine concentration

by as much as 27%

F i g u r e s 3 and 4 show the c h r o m a t o g r a m s obtained for

a normal p a t i e n t and a p a t i e n t with p h e o c h r o m o c y t o m a

r e s p e c t i v e l y

Method Comparison

R e s u l t s o b t a i n e d by the H P L C a n a l y s i s for

n o r e p i n e p h r i n e and e p i n e p h r i n e d e s c r i b e d above were

c o m p a r e d with those obtained for total catecholamines

employing the continuous flow a d a p t a t i o n of the

method of Von E u l e r and Floding{9) (Figure 5) In

general, r e s u l t s obtained by the HPLC p r o c e d u r e w e r e

significantly lower than those obtained by the

fluorometric total catecholamine method Although the

frequency of false positive and false negative r e s u l t s by

the l a t t e r approach has been well documented (10), in

our s e r i e s of 51 h y p e r t e n s i v e p a t i e n t s t h e r e was only 1

r e s u l t in which the difference was significant, the total

catecholamine p r o c e d u r e yielding a r e s u l t of 77/~g/24 hr

as c o m p a r e d to 14 ~g/24 hr by the H P L C method This

difference was confirmed by the r e p e a t e d analysis of

the d i s c r e p a n t sample

References Ranges Table 4 shows the a g e - r e l a t e d r e f e r e n c e r a n g e s ob-

t a i n e d for our l a b o r a t o r y These r e s u l t s are difficult to compare with those published by Moyer et al (8) and De

S c h a e p d r y v e r et al (11) as our r e f e r e n c e r a n g e s a r e in

t e r m s of ~g/g creatinine and a r e based on r a n d o m urine collections, while the r a n g e s quoted by both the above groups a r e in ~g/24 hr W h e r e we do have this data, i.e., for the h y p e r t e n s i v e population, our r e s u l t s c o m p a r e well with those p r e v i o u s l y published by Moyer e t al (8) The 95th p e r c e n t i l e value for the total catecholamine

p r o c e d u r e is 85 ~g/24 hr

Excretion of Urinary Catecholamines and Metabolites in Patients with Neural Crest Tumors

Tables 5 and 6 show the u r i n a r y e x c r e t i o n p a t t e r n s in

p a t i e n t s with p h e o c h r o m o c y t o m a and n e u r o b l a s t o m a

r e s p e c t i v e l y In the p h e o c h r o m o c y t o m a group, u r i n a r y

n o r e p i n e p h r i n e and e p i n e p h r i n e w e r e m a r k e d l y

e l e v a t e d in 6 of the 7 p a t i e n t s , while u r i n a r y d o p a m i n e was m a r k e d l y e l e v a t e d in only 4 of the 7 p a t i e n t s In

s h a r p c o n t r a s t , u r i n a r y VMA and total catecholamines

w e r e significantly i n c r e a s e d in all these p a t i e n t s (upper limit of normal for VMA in our l a b o r a t o r y was 6.0 mg/g creatinine) U r i n a r y HVA excretion, on the o t h e r hand, was normal t h r o u g h o u t the p h e o c h r o m o c y t o m a group

A n a l y s i s of d a t a from the n e u r o b l a s t o m a group is also

i n t e r e s t i n g U r i n a r y dopamine, n o r e p i n e p h r i n e , and

e p i n e p h r i n e w e r e e l e v a t e d in 5, 3 and 1 of the 7 p a t i e n t s

r e s p e c t i v e l y This b a t t e r y of t e s t s would have allowed for the diagnosis of 6 of the 7 p a t i e n t s in this group

U r i n a r y total catecholamines provided the diagnosis in

2 of t h e s e p a t i e n t s In c o n t r a s t , u r i n a r y VMA and HVA

w e r e e l e v a t e d in all p a t i e n t s Of the 6 l a b o r a t o r y t e s t s employed above, it would a p p e a r t h a t the H P L C analysis of u r i n a r y VMA is the most r e l i a b l e for the

d e t e c t i o n of a neural c r e s t tumor U r i n a r y HVA allows

t h e d i f f e r e n t i a t i o n of t h o s e p a t i e n t s w i t h

p h e o c h r o m o c y t o m a f r o m t h o s e p a t i e n t s w i t h

n e u r o b l a s t o m a

ACKNOWLEDGEMENTS The a u t h o r s are grateful for the technical a s s i s t a n c e

of Miss Sylvia Drop (total catecholamines)

REFERENCES

1 Von Fraenkel, F Ein foil yon doppelseitigem, v611ig latent

verlaufnen nebennierentumor und gleichzeitiger

nephritis mit ver~inderungen am circulationapparat und

retinis Arch Path Anat 103, 244 (1886)

2 Armstrong, M.D., McMiUan, A., and Shaw, K.N.F

3-Methoxy-4-hydroxy-D-mandelic acid, a urinary

metabolite of norepinephrine Biochim Biophys A c t a 25,

442 (1957}

3 Bravo, E.L., Tarazi, R.C., Gifford, R.W., and Stewart, B.H

Circulating and urinary catecholamines in pheochromo-

cytoma N e w EngL J Me& 301,682-686 (1979)

4 Gitlow, S.E., Mendlowitz, M., and Bertain, L The

biochemical techniques for detecting and establishing the

presence of a pheochromocytoma Am J CardioL 26,

270-279 {1970)

5 Galen, R.S and Gambino, S.R Beyond Normality: The

Predictive Value and Efficiency of Medical Diagnosis

John Wiley and Sons, New York, NY (1975), 62-74

6 Soldin, S.J and Hill, J.G Simultaneous liquid-

chromatographic analysis for 4-hydroxy-3-methoxyman- delic acid and 4-hydroxy-3-methoxyphenylacetic acid in

urine Cli~ Chem 26, 291-294 (1980)

7 Riggin, R.M and Kissinger, P.T Determination of catecholamines in urine by reverse-phase liquid

chromatography with electrochemical detection AnaL Chem 49, 2109-2110 (1977)

8 Moyer, T.P., Jiang, N.-S., Tyce, G.M., and Sheps, S.G Analysis for urinary catecholamines by liquid

c h r o m a t o g r a p h y with a m p e r o m e t r i c d e t e c t i o n :

methodology and clinical interpretation of results Clin Chem 25, 256-263 (1979)

9 Von Euler, U.S and Floding, I Fluorometric estimation of

noradrenaline and adrenaline in urine A c t a PhysioL Scand~ 33, Suppl 118, 57 (1955)

10 Amery, A and Conway, J A critical review of diagnostic

tests for pheochromocytoma Am Heart J 73, 129-133

(1966)

11 De Schaepdryver, A.F., Hooft, C., Delbeke, M.-J., and Vande Noortgaete, M Urinary catecholamines and

metabolites in children J Pediatrics 93° 266-268 (1978)