Analysis of Catecholamines by HPLC

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Analysis of Catecholaminesby HPLCStig Allenmark aa Clinical Research Center, University ofLinköping S-581 85 Linköping, SwedenPublished online: 24 Feb 2007.

To cite this article: Stig Allenmark (1982) Analysis of Catecholamines by HPLC,Journal of Liquid Chromatography, 5:s1, 1-41

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JOURNAL OF LIQUID CHROMATOGRAPHY, 5 ( S u p p l . l ) , 1-41 ( 1 9 8 2 )

,1PtALYSIS OF CATECHOLAMINES BY HPLC

S t i g A l l en ina rk

C l i n i c a l Research Cen te r , U n i v e r s i t y o f L i n k o p i n g S-581 85 L i n k o p i n g , Sweden

INTRODUCTION

The c a t e c h o l a m i n e s (CAI c o n s t i t u t e a c l a s s o f b i o g e n i c

amines w h i c h have l o n g been known t o possess hormonal a c t i v i -

t y and t o be compounds b i o s y n t h e s i z e d v i a t h e t y r o s i n e n ie tabo l -

i c pathway. E p i n e p h r i n e ( a d r e n a l i n e ) ( E ) , t h e f i r s t hornione

was, i n f a c t , i s o l a t e d b y Takaniine a s e a r l y as 1901 f r o m t h e

a d r e n a l m e d u l l a ( 1 ) . The chemica l s t r u c t u r e s were a l s o e l u c i -

i - ( 3 .4-d 1 hydroxy- R - ( - ) - 1 - ( 3 , 4 - d i h y d r o x y - pheny 1 ) e t hy 1 ani i ne ; pheny 1 ) - 7 - ( m e t hy 1 a,ij i no ) - dopail l ine ( D A ) i iorep 1 ne ph r i ne ( NF ) e t h a n o l , euinephrine ( t )

K - ( - ) - I - ( 3,4-d 1 hyc!roxypheny 1 ) - 2 - a n i i noe t ha no1 ;

SCHEME 1. Chemical s t r u c t u r e s o f t h e c a t e c h o l a m i n e s

1

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ALLENMARK 2

da ted e a r l y and a l l shown t o be d e r i v a t i v e s o f c a t e c h o l . The

a b s o l u t e c o n f i g u r a t i o n o f t h e b i o l o g i c a l l y a c t i v e for i i is o f NE

and E was l a t e r shown t o be R (2 ,3 ) .

From t h e chemica l p o i n t o f v iew, t h e c a t e c h o l r i n g s t r u c -

t u r e , because o f i t s easy o x i d a t i v e c o n v e r s i o n , makes t h e s e

compounds r a t h e r s e n s i t i v e t o a i r and l i g h t , e s p e c i a l l y i n

a l k a l i n e s o l u t i o n where t h e y a r e r a p i d l y o x i d i z e d spontane-

o u s l y . The ampho te r i c p r o p e r t i e s o f t h e CA's, due t o t h e p r e s -

ence o f amino- as w e l l as p h e n o l i c groups, iiiake c o n v e n t i o n a l

e x t r a c t i o n p rocedures , i . e . f r o m an a c i d phase, d i f f i c u l t be-

cause o f t h e wa te r s o l u b i l i t y o f t h e i o n i c s p e c i e s o b t a i n e d .

I o n p a i r o r ca t i on -exchange e x t r a c t i o n t e c h n i q u e s niay be used,

b u t f o r t u n a t e l y more s e l e c t i v e e x t r a c t i o n r e t h o d s e x i s t , wh ich

a r e based on t h e a b i l i t y o f t h e 3 ,4 -d ihyd roxypheny l m o i e t y t o

f o r m c y c l i c complexes w i t h b o r i c a c i d o r a lum ina , and w h i c h

w i l l be t r e a t e d i n more d e t a i l l a t e r .

The c a t e c h o l r i n g g i v e s r i s e t o UV a b s o r p t i o n maxima a t

200-220 nin and 280-300 nm, r e s p e c t i v e l y . I r r a d i a t i o n a t e i t h e r

o f t hese maxima g i v e s r i s e t o weak n a t i v e f l u o r e s c e n c e (Aeflliss =

310-340 nm).

The p r i m a r y anlino g roup i n DA and NE i s an e x c e l l e n t n u c l e o p h i l e

i n r e a c t i o n s p r o d u c i n g h i g h l y f l u o r e s c e n t d e r i v a t i v e s by t h e use o f

reagen ts such as f l u o r e s c a m i n e , dansy l c h l o r i d e o r o - p h t a l a l d e h y d e .

O x i d a t i o n o f t h e c a t e c h o l r i n g , e.g. b y t h e a c t i o n o f po tass iu i x

f e r r i c y a n i d e and a1 k a l i n e a s c o r b a t e , produces t r i h y d r o x y i n d o l e ( T H I )

d e r i v a t i v e s w i t h h i g h f l u o r e s c e n c e quantum y i e l d s ; a r e a c t i o n t h a t

has been w i d e l y used f o r a n a l y t i c a l purposes, b u t wh ich i s n o t w e l l

a p p l i c a b l e t o DA.

P r i o r t o t h e i n t r o d u c t i o n o f t h e HPLC-methodology i n t h e e a r l y

1 9 7 0 ' s most methods used o r a t t e m p t e d f o r t h e e s t i m a t i o n o f CA's i n

b i o l o g i c a l m a t e r i a l were based on e x t r a c t i o n o n t o a lum ina f o l l o w e d

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ANALYSIS OF CATECHOLAMINES 3

by e l u t i o n and convers ion t o T H I - d e r i v a t i v e s and s e l e c t i v e f l u o r e s -

cence measurements (4 ,5) , column chromatographic s e p a r a t i o n p r i o r

t o a n a l y s i s (6-111, paper- or t h i n - l a y e r chromatography (TLC) w i t h

pre- o r post -chromatographic d e r i v a t i z a t i o n (12,131 and, l a s t b u t

n o t l e a s t , gas chromatographic (GLC) s e p a r a t i o n methods i n v o l v i n g

d i f f e r e n t t ypes o f d e r i v a t i z a t i o n r e a c t i o n s f o r t h e use o f f lame

i o n i z a t i o n (14,151, e l e c t r o n cap tu re (16-21) o r masspectrometr ic

(20-23) d e t e c t o r systems. None o f these methods can be s a i d t o

f u l f i l l a l l requi rements f o r a good a n a l y t i c a l procedure because

t h e y a r e e i t h e r t o o complex and l a b o r i o u s g i v i n g l o w r e c o v e r i e s ,

o r l a c k s e l e c t i v i t y o r s e n s i t i v i t y .

Some GLC-resul ts f rom s t u d i e s o f b i o l o g i c a l m a t e r i a l (24) a r e

a l s o , i n t h e l i g h t o f l a t e r i n v e s t i g a t i o n s , comp le te l y er roneous

and have r e c e n t l y been s e v e r e l y c r i t i c i z e d (25 ) . The GC-MS metti-

odology, however, has been shown d u r i n g t h e l a s t yea rs t o p r o v i d e

t h e s e l e c t i v i t y and s e n s i t i v i t y necessary f o r CA-analyses o f b i o -

l o g i c a l specimens, and a h i g h - r e s o l u t i o n mass f ragmentographic

technique was r e p o r t e d r e c e n t l y t o be a success fu l t o o l f o r quan-

t i t a t i o n o f CA's i n plasma ( 2 6 ) .

I n 1970 a rad ioenzymat ic method, based on t h e enzyme-catalyzed 3 i n t r o d u c t i o n o f a r a d i o l a b e l ( H o r 14C) v i a 3 -0 -me thy la t i on o f t h e

CA's and concomi tant TLC-separation and use o f l i q u i d s c i n t i l l a t i o n

c o u n t i n g f o r q u a n t i t a t i o n was i n t r o d u c e d (27 ) . T h i s method, which

l a t e r has been f u r t h e r developed and improved (28,291,

g r e a t importance due t o i t s extreme s e n s i t i v i t y d e s p i t e t h e d i s -

advantages w i t h rega rd t o comp lex i t y , l a b o u r and c o s t .

i s o f

A r e c e n t l y pub1 ished rev iew by K r s t u l o v i c (30 ) cove rs the

methods o t h e r than HPLC i n use f o r t h e a n a l y s i s o f CA's i n more

d e t a i l t han what has been p o s s i b l e o r a p p r o p r i a t e t o i n c l u d e i n

t h i s i n t r o d u c t o r y s e c t i o n .

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li ALLENMARK

Since HPLC-methods, g e n e r a l l y speaking, a r e f a v o u r a b l e f o r

t he separa t i on and a n a l y s i s o f p o l a r , h y d r o p h i l i c compounds, i t

i s n o t s u r p r i s i n g t h a t HPLC has made a s t r o n g impact on CA-analy-

s i s as seen f rom the e x p l o s i v e i nc rease i n t h e number o f papers

pub l i shed i n t h i s f i e l d . I n t h e f o l l o w i n g , t h e v a r i o u s f a c t o r s

o f importance i n CA-analysis by means o f HPLC w i l l be t r e a t e d and

a survey o f e x i s t i n g HPLC-methodology, b iomed ica l and r e l a t e d

a p p l i c a t i o n s as w e l l as p o s s i b l e f u t u r e t r e n d s w i l l be g i ven .

CHROMATOGRAPHIC PROPERTIES OF CATECHOLAMINES. COLUMN AND MOBILE

PHASE SELECTION

Numerous i n v e s t

o f b i o g e n i c amines

t o t h e p o l a r n a t u r e

p o s s i b l e t o u t i l i z e

g a t i o n s have d e a l t w i t h t h e r e t e n t i o n behaviour

n v a r i o u s l i q u i d chromatographic systems. Due

o f these compounds, p a r t i c u l a r l y t h e CA's, i t i s

q u i t e d i f f e r e n t r e t e n t i o n mechanisms f o r separa-

t i o n , i n most cases w i t h t h e use o f aqueous b u f f e r systems as t h e

mob i l e phase. The va r ious c o n d i t i o n s t h a t have been used a r e

summarized i n Table 1.

The d i v i s i o n i n t o d i f f e r e n t t ypes o f r e t e n t i o n mechanisms g i v e n

i n Table 1 should n o t be taken t o o c a t e g o r i c a l l y and deserves a

comment. I t i s always d i f f i c u l t t o know whether a s i n g l e t ype o f

e q u i l i b r i u m between t h e s t a t i o n a r y and mob i l e phases i n a column

i s t h e s o l e f a c t o r govern ing r e t e n t i o n o f a g i v e n compound. F u r t h e r ,

many d i f f e r e n t models f o r t h e r e t e n t i o n process i n i o n - p a i r chromato-

g raph ic systems have been proposed and t h e b o r d e r l i n e s between what

has been c a l l e d here hydrophobic i n t e r a c t i o n , dynamic ion-exchange

and i o n - p a i r reversed-phase p a r t i t i o n a re , i n f a c t , n o t very c l e a r -

c u t . I n f a c t , because chromatography measures o n l y e q u i l i b r i a and

n o t k i n e t i c p r o p e r t i e s , i t i s u n f o r t u n a t e l y imposs ib le t o deduce any r e a l mechanism f rom chromatographic data. Never the less.

t h i s c l a s s i f i c a t i o n should be u s e f u l i n t h e f o l l o w i n g d i s c u s s i o n

o f t he r e t e n t i o n behav iou r o f C A ' s under v a r i o u s chromatographic

c o n d i t i o n s and w i l l be e x p l a i n e d i n f u r t h e r d e t a i l below.

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6 ALLENMARK

Cation exchange HPLC represents the most obvious and s t r a igh t - forward method. Retention should be - w t r o l l e d only by d i f fe rences in net charge a t the buffer pH and ,Lrength used. The compounds elute therefore in order of ir , L , i n g bas i c i ty . I t a l s o has the advantage t h a t ac id i c and neutral compounds should e l u t e in the void volume. I t has the drawbacks, however, t h a t i t i s often d i f f i -

c u l t t o obtain a reasonably high operating column ef f ic iency (say H < 0.05 m m ) , peak t a i l i n g i s a frequent problem and the adsorbent may be prone t o chemical degradation, espec ia l ly i f a s l i g h t l y elevated column temperature i s used. Another problem i s

due t o the f a c t t h a t the batch-to-batch var ia t ion in the proper- t i e s of the ion exchanger i s so g r e a t , t h a t chromatographic re ten- t ion parameters under otherwise ident ica l conditions a r e not comparable unless material from one s ing le batch i s used throughout.

Most frequently strong cation-exchange mater ia l s (containing sulphonic acid groups) have been used together with purely aqueous buffer systems a t pH-values between 2 and 6 . Much work has been performed with the use of l o n g g l a s s columns (mostly in com- bi nat i o n w i t h electrochemical de t ec t ion 1 dry-packed w i t h r e s ins such a s Zipax SCX (DuPont) (31,321, Vydac SCX (The Separations Group, Hesperia, Ca l i f . ) (33-35) and Corasil CX (Waters) (36) . Although adequate separation f o r the given purpose can gener- a l l y be achieved by a proper adjustment of the mobile phase, the column ef f ic iency obtained was f o r the most par t very low. De- s p i t e the problems mentioned e a r l i e r , s ign i f i can t ly b e t t e r column e f f i c i enc ie s a re obtained with the use of slurry-packed s t ee l co l - umns and small p a r t i c l e diameter (5-1011) cation-exchange mater ia l s such a s Par t i s i l -10 SCX ( 3 3 ) , Nucleosil SA 1011 (37) or Nucleosil SA 511 (38,39).

The re ten t ion behaviour of the CA's and t h e i r metabolites on oc tadecyl -s i l ica s ta t ionary phases on e lu t ion with purely aqueous buffers of varying pH and ionic strength was thoroughly inves t i - gated by Molnar a n d Horvath ( 4 0 ) . Under these conditions acid

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ca techo ls a r e r e t a i n e d as w e l l and r e t e n t i o n i s e f f e c t e d by hydro-

phobic i n t e r a c t i o n s between t h e s t a t i o n a r y phase and t h e hydrophobic

p a r t o f t h e compound i n ques t i on . The a u t h o r s used phosphate b u f f e r s

throughout t h e i r s t u d i e s . It has been suggested t h a t i o n - p a i r i n g o f

t h e amines i s t h e reason f o r t h e i nc rease i n r e t e n t i o n , peak symmetry

and column e f f i c i e n c y observed (41) on t h e a d d i t i o n o f c e r t a i n a c i d s

t o t h e rnobi le phase. I r r e s p e c t i v e o f t h e mechanism r e s p o n s i b l e f o r

t h i s phenomenon, t h e e f f e c t on t h e r e t e n t i o n can be s u b s t a n t i a l as

shown i n Table 2. I t i s r e a d i l y seen t h a t r e t e n t i o n o f t h e amines

i nc reases w i t h i n c r e a s i n g h y d r o p h o b i c i t y o f t h e added coun te r ion .

E s p e c i a l l y s t r i k i n g i s t h e e f f e c t produced by t h e presence o f an

a l k y l s u l p h o n a t e i on . Th is method o f r e g u l a t i n g r e t e n t i o n , i n t r o d u c e d

by Knox and Jurand (43) and termed "soap chromatography", was r a p i d l y

adopted as a power fu l new technique f o r use i n HPLC o f b i o g e n i c amines.

The chromatographic r e t e n t i o n mechanism can be though t o f acco rd ing t o

two extreme models, one i n which an a l k y l s u l p h o n a t e i o n - p a i r o f t h e

amine i s r e t a i n e d by t h e hydrophobic s t a t i o n a r y phase, and ano the r

i n which t h e a l k y l s u l p h o n a t e covers t h e s t a t i o n a r y phase by hydrophobic

i n t e r a c t i o n and conver t s i t t o a cat ion-exchanger on which r e t e n t i o n

o f t h e amine can take p lace - t h e r e f o r e t h e te rm "dynamic ion-exchange".

The methcd has been s u c c e s s f u l l y a p p l i e d t o HPLC-separations o f CA's i n

many l a b o r a t o r i e s i n r e c e n t years. A d e t a i l e d i n v e s t i g a t i o n on t h e

e f f e c t o f d i f f e r e n t m o d i f i e r s (sodium oc tadecy l - as w e l l as o c t y l

su lpha te ) , s o l v e n t systems and s t a t i o n a r y (C-18) phases appeared

r e c e n t l y ( 4 5 ) . The e f f e c t o f sodium dodecyl su lpha te on t h e r e t e n -

t i o n o f t h e CA's and o t h e r b i o g e n i c amines i n t h e presence o f some a c i d

m e t a b o l i t e s has a l s o been tho rough ly i n v e s t i g a t e d ( 4 6 ) .

A r e l a t e d technique, i o n - p a i r reve rsed phase p a r t i t i o n , was i n t r o -

duced by S c h i l l (47,48) and a p p l i e d t o a l k y l - s i l i c a suppor t s f o r t h e

s e p a r a t i o n o f b i o g e n i c amines (48,49). Here, t h e column i s coa ted w i t h

an o rgan ic s o l v e n t such as 1-pentanol o r methylene c h l o r i d e , p r e s e n t

i n a few p e r c e n t i n t h e aqueous mob i l e phase c o n t a i n i n g t h e a l k y l s u l - phonate o r o t h e r s u i t a b l e c o u n t e r i o n . R e t e n t i o n o f t h e amines i s sup-

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TABLE 2

ALLENMARK

E f f e c t o f Various Acids on the Re ten t ion Behaviour of Catechol - amines and Re la ted Compounds. Cond i t i ons : Column: 3.9 x 300 mm VBondapak C-18 (Waters Assoc.), 0.1 M a c i d i n water , pH 3.0. (Abbrev ia t i ons : MNE:a-methyl no rep inephr ine , DHBA: 3 ,4-d ihydroxy- benzylamine, DOPA: L-3,4-dihydroxyphenylalanine, MD:a-methyl-DOPA, CD: carb idopa (2-hydrazino-3-(3,4-di hydroxyphenyl )-.?-methyl p r o p i o n i c a c i d ) , MDA:u-methyl dopamine, DOPAC: 3,4-di hydroxyphenyl a c e t i c a c i d ) .

(Reproduced f rom r e f . 41 w i t h t h e pe rm iss ion o f t h e a u t h o r s and t h e pub1 i s h e r )

Compound Capaci ty r a t i o s , k'

HN03 H2S04 CH3C02H

NE 0.4 0.3

MNE 0.8 0.6

E 1 .o 0.7

DHBA 1.2 0.8

DOPA 1.4 1.3

DA 2.2 1.5

MD 3.7 3.3

CD 5.3 4.7

MDA 6.2 4.2

DOPAC 11.2 11.2 __________- - -_______- - - -

0.2

0.3

0.4

0.5

0.9

0.9

1.8

2.6

2.4

7.4

H3P04

0.3

0.6

0.7

0.8

1.2

1.6

3.1

4.4

4.3

11.2

TCA

1.8

3.2

413

5.1

3.7

9.5

11.3

-

27.7

10.3

os*

2.5

4.7

5.7

7.9

3.7

15.3

- 16.3

-

8.4

* 0.05 mM sodium o c t y l su lpha te i n 0.1 M n i t r i c a c i d

posed t o be governed by means o f i o n - p a i r p a r t i t i o n i n g between a

s t a t i o n a r y l i q u i d o rgan ic phase and t h e aqueous mob i l e phase.

I o n - p a i r p a r t i t i o n chromatography o f CA's has a l s o been per formed

i n the s t ra igh t -phase mode (50 ) . I n t h i s case a s i l i c a g e l column

impregnated w i t h p e r c h l o r i c ac id /sodium p e r c h l o r a t e served as t h e

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s t a t i o n a r y phase. E l u t i o n w i t h o r g a n i c mob i l e phases such as tr i-

b u t y l phosphate - hexane (65:35) o r bu tano l - methylene c h l o r i d e

(40:60) gave t h e e l u t i o n o rde rs shown i n Table 1.

I n summary, t h e r e a r e many success fu l ways t o ach ieve a good

HPLC-separation o f CA's and t h e b e s t cho ice f o r a g i v e n a n a l y t i c a l

purpose w i l l depend on f a c t o r s such as t h e d e t e c t i o n method used,

i n t e r f e r i n g compounds i n the sample e t c . I n genera l , however, due

t o the o f t e n ex t reme ly l ow c o n c e n t r a t i o n s t h a t have t o be analyzed,

chromatographic systems based on p u r e l y aqueous systems, i s o c r a t i c

e l u t i o n and h i g h column e f f i c i e n c y and s e l e c t i v i t y shou ld be

advantageous.

METHODS FOR THE DETECTION OF CATECHOLAMINES I N COLUMN EFFLUENTS

A. UV-detect ion

The use o f UV-detect ion f o r t h e a n a l y s i s o f CA's i s s t r o n g l y

hampered by i t s r e l a t i v e i n s e n s i t i v i t y . The p r a c t i c a l l i m i t o f

d e t e c t i o n under op t ima l c o n d i t i o n s can be r o u g h l y es t ima ted t o

0.01 nmol (S /N = 2 ) . For many b i o l o g i c a l samples o f i n t e r e s t t h i s

i s f a r f rom s u f f i c i e n t .

B. F l u o r i m e t r i c d e t e c t i o n

F l u o r i m e t r i c d e t e c t i o n has been used b o t h wi th and w i t h o u t

d e r i v a t i z a t i o n o f t he CA's. The use o f f l u o r i m e t r i c r e c o r d i n g o f

CA's and o t h e r b i o g e n i c amines u t i l i z i n g t h e i r n a t i v e f l u o r e s -

cence i s desc r ibed i n a r e c e n t paper ( 5 1 ) . W i th a s topped- f l ow scanning

techn ique

used f o r i d e n t i f i c a t i o n purposes. SchUsler-van Hees e t a1 c l a i m

t h a t a d e t e c t i o n l i m i t o f 10 nM ( = ca 20 pg) can be o b t a i n e d on

d e t e c t i o n o f n a t i v e f l uo rescence making use o f X e x c = 284 and

by e x c i t a t i o n a t 285 nm and m o n i t o r i n g t h e 340 nm emiss ion

spec t ra o f t h e v a r i o u s peaks c o u l d a l s o be reco rded and

Xe,iss = 316 nm ( 5 2 ) . Th i s h i g h s e n s i t i v i t y seems ques t i onab le ,

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c 0

- m

b

UV

( 2

80

nrn

) u

v(2

54

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) F

LUO

RE

SC

EN

CE

(2

85

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0n

m

- 0- -

v

n Y

r -

I

ME

SO

H

Ta

p

5-H

T

c - TRP

c -

5-O

H

IAA

C

- AA

c c

- IA

- ILA

- IA

A

C

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however, i n view o f o t h e r r e p o r t s . Thus, Jackman e t a1 (53) found

op t ima l d e t e c t i o n c o n d i t i o n s on e x c i t a t i o n a t 200 nm and r e p o r t

t h e d e t e c t i o n l i m i t s NE = 100 p g and DA = 300 pg.

A h i g h e r s e n s i t i v i t y can be achieved, however, i f t h e amines a r e

d e r i v a t i z e d b e f o r e t h e i r f l u o r i m e t r i c a n a l y s i s . Th i s may be done

e i t h e r b e f o r e t h e HPLC-separation as a precolumn d e r i v a t i z a t i o n o r

i n a postcolumn r e a c t o r j u s t b e f o r e t h e d e t e c t o r i n l e t . The main

disadvantage o f a l l precolumn d e r i v a t i z a t i o n methods i s t h e l a b o -

r i o u s p re t rea tmen t o f t he sample i n v o l v i n g e x t r a c t i o n w i t h an o r -

gan ic so l ven t , a procedure which seems d i f f i c u l t t o au tomat i ze

and t h e e f f e c t i v e n e s s o f which has t o be r i g o r o u s l y checked by

i n t e r n a l s t a n d a r d i z a t i o n procedures. Some common d e r i v a t i z a t i o n

procedures a r e summarized i n Scheme 2.

The reagents most commonly used f o r precolumn d e r i v a t i z a t i o n

o f t h e b i o g e n i c amines a r e o -ph ta la ldehyde (OPA) , d a n s y l c h l o r i d e

(DNS-C1) and f l uo rescamine (FA). OPA-der ivat ives have been r e -

p o r t e d (54,55) t o possess e x c e l l e n t s t a b i l i t y and t o be w e l l sep-

a r a t e d on reve rsed phase HPLC under i s o c r a t i c c o n d i t i o n s . The HPLC

separa t i on o f DNS-der ivat ives has been s t u d i e d by Schwedt (56-58) and

by F r e i ( 5 9 ) . The use o f f l uo rescamine f o r precolumn d e r i v a t i z a t i o n

p r i o r t o HPLC has been e x t e n s i v e l y i n v e s t i g a t e d by Imai (60-62) .

I n a l l these d e r i v a t i z a t i o n methods a p r imary amino group i n t h e

s u b s t r a t e i s r e q u i r e d and t h e y a r e t h e r e f o r e a p p l i c a b l e o n l y t o iJE and DA as f a r as t h e C A ' s a r e concerned. Ima i found t h a t t h e very p o l a r

n a t u r e o f t he d e r i v a t i v e s ob ta ined w i t h f l uo rescamine made them m i - g r a t e t o o f a s t on o r d i n a r y reversed-phase columns and t h e r e f o r e a g l y -

c o l t ype ge l (TSK LS 160 g e l , 5 o r 10 u ) was used. Schwedt, u s i n g a

s t ra igh t -phase HPLC-system, ob ta ined two peaks co r respond ing t o NE

(63 ) .

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12 ALLENMARK

R-NH, + QcHo struc!uce of the fluotescent reaction product unknown

CHO

NHRl

OH OH

THI OH R1-H orCH3

SCHENE 2. Commonly employed reactions for the transformation of catecholamines into fluorescent derivatives

In postcolumn fluorogenic derivatization methods a reactor, placed between the column outlet and the fluorimetric detector, will convert the amines to highly fluorescent reaction products. To fully utilize this principle, the reactor must have a low dead-volume t o

prevent band broadening but also allow for an intimate mixing of the reaction partners and a sufficiently long reaction time. Ex- tensive studies have been carried out on three different post-column reactions, viz. the THI-, the OPA- and the FA-reactions. The last

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r e a c t i o n , however, which has been s u c c e s s f u l l y a p p l i e d t o a n a l y s i s

o f amino ac ids , as w e l l as b o t h p r imary and secondary amines,

has n o t been used f o r post-column d e t e c t i o n o f CA's

o f my knowledge.

t o t h e b e s t

The f i r s t a p p l i c a t i o n o f t h e THI-method subsequent t o HPLC sepa-

r a t i o n o f CA's was c a r r i e d o u t by Mor i and r e p o r t e d i n a s e r i e s

o f papers (64,65). The method was f u r t h e r s t u d i e d by Schwedt (66) and

o t h e r s (67,GB) whereby d i f f e r e n t modes o f HPLC-separation were

t r i e d . Q u i t e r e c e n t l y Yui e t a1 (69-71) r e p o r t e d on a c a t i o n -

exchange HPLC system a l l o w i n g t h e d e t e r m i n a t i o n o f NE and E i n

amounts down t o a few picograms. U n f o r t u n a t e l y , t h e a u t h o r s have

n o t i n c o r p o r a t e d DA i n t h e i r i n v e s t i g a t i o n s . Okanioto e t a1 , however,

have shown t h a t a l l t h r e e CA's as w e l l as DOPA can be analyzed

s imu l taneous ly by the THI method o f d e t e c t i o n (67) .

Post-column d e r i v a t i z a t i o n w i t h OPA i s an a1 t e r n a t i v e h i g h l y

s e n s i t i v e d e t e c t i o n method which has t h e advantage t h a t b i o g e n i c

p r imary amines i n genera l can be d e t e c t e d b u t t h e d isadvantage w i t h

r e s p e c t t o CA-analysis t h a t E w i l l be exc luded. Another f a c t o r o f

importance i s t h a t t h e r e a c t i o n t ime r e q u i r e d i s much s h o r t e r

(20 sec as compared t o 4 min f o r t h e THI-method under op t ima l

c o n d i t i o n s ( 6 7 ) ) .

C. E lec t rochemica l d e t e c t i o n

Since the f i r s t r e p o r t i n 1973 by Adams and coworkers ( 7 2 ) on

t h e success fu l use o f an e lec t rochemica l f l o w c e l l as a d e t e c t o r

component f o r l i q u i d chromatography systems, t h e r e has been an

e x p l o s i v e development w i t h i n t h i s area o f research . The d e t e c t i o n

p r i n c i p l e i s i l l u s t r a t e d i n F i g . 2 . The column e f f l u e n t i s passed

ove r t h e anode su r face o f t h e d e t e c t o r f l o w c e l l t o which any

compound which i s e l e c t r o o x i d i z a b l e a t t h e p o t e n t i a l chosen can

l o s e one o r more e l e c t r o n s and become t rans fo rmed ( r e v e r s i b l y o r i r r e -

v e r s i b l y ) i n t o an o x i d i z e d r e a c t i o n p roduc t . Fo r t h e CA's t h i s e l e c t r o -

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14 ALLENMARK

fro cot

electrode

from colann I

I I

reference, > electrode exit

counter electrode exit

working electrode

I

FIGURE 2 . The pr inc ip le f o r electrochemical de tec t ion in a flow c e l l . Thin-layer type ( l e f t ) , wa l l - j e t type ( r i g h t ) .

chemical oxidation primarily means a two e lec t ron reaction to y ie ld an o-quinoid s t ruc ture . I t i s important t o keep in mind t h a t a l l electrochemical de tec tors work by converting (chemically and e l ec - trochemically) a par t of the compound to be analyzed to a product and consequently should be regarded a s a kind of post-column reac tors highly dependent upon the reaction conditions in the c e l l . Thus, not only the anode po ten t i a l , b u t a l s o the mobile phase composition, pH,

ionic strength e t c . a r e f ac to r s t h a t g rea t ly a f f e c t the de tec tor response. Such influences on the electrochemical de tec t ion of CA's were thoroughly studied by Moyer a n d Jiang ( 7 3 ) and an i l l u s t r a t i o n of t h e i r r e s u l t s i s given i n Fig. 3.

A widely used ce l l type, o r ig ina l ly introduced by Adams e t a1 ( 7 2 ) , i s the thin-layer flow c e l l housing a graphite paste anode in the bottom ce l l half and connected to an Ag/AgCl reference and an auxi l ia ry e lec t rode in a th ree e lec t rode configuration. This system has been explored and fu r the r developed by Kissinger and h is co l labora tors in a s e r i e s of papers ( 7 4 - 7 7 ) . A modification of which i s more r e s i s t a n t t o organic solvents.

the ce l l i s equipped with a glassy carbon e lec t rode material

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PH [Po4 '1, M

FIGURE 3. Influence o f a ) pH and b ) ionic s t rength o n the amperometric de tec tor response a t constant anode potential (+0.5 V vs. Ag/AgCl). (Reproduced from r e f . 73 with the permission of the authors and the pub1 i sher . 1

Another pr inc ip le i s used in the wa l l - j e t type of de tec tor c e l l

(78), anode surface. These types of c e l l s have been assumed to give a higher coulometric y ie ld in genera l , due t o the hydrodynamic proper- t i e s and a r e usually equipped with a glassy carbon e lec t rode . Detailed descr ip t ions of the construction of such c e l l s have recently appeared (78,79).

Here the column e f f l u e n t i s d i rec ted perpendicularly t o the

Lankelma and Poppe (80) have thoroughly investigated the 6 requirements necessary f o r a high l i n e a r dynamic range ( 10 1,

high coulometric y ie ld and low noise level of a de tec tor c e l l . Using a l a rge v i t reous carbon anode separated by a 50 p spacer from the counter e lec t rode they achieved a 97% coulometric y ie ld a t a flow r a t e of 20 ml/h.

Detector c e l l s of the wal l - je t type with a ro t a t ing d isc carbon paste o r vitreous carbon e l ec t rode were recently introduced by Brunt

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16 ALLENMARK

(81-83). Desp i te t h e l a r g e r c e l l volume i n t h i s arrangement, t h e cou lomet r i c y i e l d i s h i g h l y i nc reased i n these c e l l s due t o t h e r o t a t i o n

o f t he e l e c t r o d e . Th is reduces t h e t h i c k n e s s o f t h e d i f f u s i o n l a y e r ,

r e s u l t i n g i n a s i g n i f i c a n t l y i nc reased d e t e c t o r response.

A comparison o f t h e performance o f EC-detectors used w i t h

HPLC f o r t he d e t e r m i n a t i o n o f CA's and some r e l a t e d compounds

r e c e n t l y appeared (84).

D. Other methods o f d e t e c t i o n

A v a r i a n t o f t h e rad ioenzymat i c method o f CA-analys is (27-29)

makes use o f HPLCsepara t i on o f t h e e n z y m a t i c a l l y generated, r a d i o -

l a b e l l e d 3-0-methyl - d e r i v a t i v e s t h a t a re subsequent ly determined

q u a n t i t a t i v e l y by l i q u i d s c i n t i l l a t i o n c o u n t i n g . Thus, t h e techn ique

can be regarded as a precoluinn d e r i v a t i z a t i o n p r i o r t o HPLC.

K l a n i e c k i e t a1 (85) used a cat ion-exchange column w i t h an am-

monium phosphate b u f f e r , pH 8.5, c o n t a i n i n g 1% a c e t o n i t r i l e as

the mob i l e phase f o r t h e separa t i on and ob ta ined an o r d e r o f

i n c r e a s i n g r e t e n t i o n : NMN < MOT < MN . The l i m i t o f d e t e c t i o n

was g i v e n as 9, 17 and 19 pg/ml f o r NE, E and DA, r e s p e c t i v e l y .

The HPLC-separation has a l s o been c a r r i e d o u t w i t h t h e use o f

reversed-phase systems (86-89). A l i m i t o f d e t e c t i o n o f ca 1 pg has been c la imed ( 8 8 ) .

*

A study o f t h e d e t e c t i o n l i m i t s f o r t h e v a r i o u s methods o f

a n a l y z i n g C A ' s i n e f f l u e n t s f rom HPLC columns was p u b l i s h e d

r e c e n t l y (90) and t h e r e s u l t s a r e shown i n Table 3.

* NMN = normetanephrine, MOT = 3-niethoxytyramine, MN = meta- n e p h r i ne

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ANALYSIS OF CATECHOLAMINES

TABLE 3

1 7

An Est imate o f t h e D e t e c t i o n L i m i t s f o r CA's Obta ined so f a r i n HPLC w i t h D i f f e r e n t Methods f o r Column E f f l u e n t A n a l y s i s

(Reproduced f rom r e f . 90 w i t h t h e pe rm iss ion o f t h e a u t h o r s and t h e pub1 i s h e r . )

Method L i m i t s o f d e t e c t i o n (pg)

OA NE E

1. E lec t rochemica l 2 5 25 25

2. U l t r a v i o l e t 1250 1400 1200

3. Na tu ra l f l uo rescence 300 300 300

4. OPA post-column d e r i v a t i za t i on 130 75 -

5. T H I 800 1 1

TECHNIOUES FOR THE ISOLATION OF CATECHOLAMINES FROM A BIOLOGICAL M A T R I X

The p r e p a r a t i o n o f a sample f rom a b i o l o g i c a l source as a homogeneous

s o l u t i o n o f t he species o f i n t e r e s t and s u i t a b l e f o r a n a l y s i s by HPLC,

rep resen ts an advanced b i o a n a l y t i c a l problem o f g r e a t chemical com-

p l e x i t y a t l e a s t o f q u a n t i t a t i v e es t ima tes a r e t o be made. Each

b i o l o g i c a l m a t r i x o f f e r s d i f f e r e n t problems and a v a r i e t y o f methods

f o r p re t rea tmen t o f t h e b i o l o g i c a l m a t e r i a l has been worked ou t .

Therefore, t he s e l e c t i o n o f a s u i t a b l e method f o r p rocess ing t h e

o r i g i n a l m a t e r i a l f o r CA-analysis by HPLC w i l l be e n t i r e l y dependent

on t h e n a t u r e o f t he b i o l o g i c a l source. A genera l rep resen ta -

t i o n o f t h e i s o l a t i o n techniques used i s g i ven below.

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18

Biological material

Pretreatment involving b i ol og ica 1 separation techniques

Extractive removal of the CA's from the l i qu id phase

I I

Liquid phase

Extract f o r HPLC-analysis

ALLENMARK

The complexity of t h i s sample preparation procedure will a l s o depend on the s e l e c t i v i t y of the HPLC system used f o r ana lys i s . For example, i t was shown recently t h a t r a t brain endogenous biogenic amines could be analyzed, in v i m , by HPLC w i t h a C-18 reversed- phase column and electrochemical detection without any "work-up" a t a l l of the perfusate from the brain (91) . Usually, however, an HPLC method f o r quant i ta t ive ana lys i s of CA's cannot be applied, unless an extensive pre-purification of the biological material has been ca r r i ed out. In the following some d i f f e r e n t methods used t o achieve t h i s will be b r i e f ly discussed.

While the pretreatment of the biological material only has the purpose of giving a l i qu id phase su i t ab le f o r an e f f e c t i v e l iqu id- so l id ex t rac t ion of the CA's, the purpose of t he ex t rac t ion s tep i s two-fold: 1 ) t o i s o l a t e the CA's a s s e l ec t ive ly a s poss ib le , a n d

2 ) t o obtain a preconcentration to a small volume su i t ab le f o r in- j ec t ion in to the chromatograph.

Of the l iqu id-so l id ex t rac t ion methods, v iz . the use of a ca t ion- exchanger, e i t h e r in the column (92) or in the batch (93) mode, the use of acid-washed alumina (36) according t o the method o r ig ina l ly developed by Anton and Sayre (4,5), o r the adsorption t o a boric acid a f f i n i t y gel (94-96), only the l a t t e r two techniques a r e

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selective for the CA's in the sense that adsorption to the solid phase is governed by the presence of the o-dihydroxyphenyl functional group. of cyclic boronates or alurninates, i s outlined below. Accordingly, it is essential that acidic alumina (i.e. aluminium oxide that has been treated with a strong mineral acid) i s used for this process.

The adsorption process, based on the reversible formation

In their exhaustive investigation on CA isolation via adsorption onto alumina Anton and Sayre found an optimal recovery at pH 8.6 (41, which was interpreted as the result of a competition between an increased stability at lower pH and a better adsorption at higher pH. Correct pH-adjustment is therefore an important factor in the alumina extraction method. In most cases it is also essential to add an antioxidant (usually sodium metabisulphite or ascorbic acid) and EDTA to protect the CA's during the work at this relatively high pH. Elution from the adsorbent has been effected by means of various mineral and organic acids (hydrochloric, perchloric or acetic acid). Acetic acid has the advantage that the extract may be easily concentrated by evaporation. A detailed description of the alumina extraction method as applied to brain tissue and urine with the aid o f a centrifugal microfiltration technique giving almost 100% recovery with as little as 100 111 of eluent has recently been pub1 ished (97).

The boric acid gel method for CA isolation has been applied to urine (96) as well as plasma (98) with excellent recoveries (8125%) on elution from a column with 1.3 M acetic acid in methanol (98). It

SCHEHE 3. Chemistry of the reversible adsorption o f catecholamines to a boric acid affinity gel

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was suggested t h a t t h i s i so l a t ion technique should be superior because pH adjustment before adsorption seems t o be much l e s s c r i t i c a l and a lower pH ( 7 . 5 ) can be used. A boronic acid subs t i tu ted micropar t icu la te s i l i c a adsorbent f o r potential use in high performance l i qu id a f f i n i t y chromatography ( H P L A C ) of nucleosides, nucleotides and carbohydrates has recently been synthesized by Mosbach and co l labora tors ( 9 9 ) .

With the rapid development of column switching techniques in H P L C i t seems very l i k e l y t h a t ex t rac t ion and preconcentration of CA's from biological material can be performed "on l i n e " with the use of an ex- t r a c t i v e HPLC-precolumn of the boronic acid type pr ior t o the ana ly t ica l column, using a column switching technique s imi la r t o the one described by Davis a n d Kissinger ( 1 0 0 ) .

QUANTITATIVE DETERMINA BY HPLC

for the determination of serotonin in plasma

ION OF CATECHOLAMINES I N BIOLOGICAL MATERIAL

A p rerequis i te f o r a quan t i t a t ive ana lys i s i s the knowledge of two f ac to r s f o r each individual compound to be quant i ta ted : a ) the recovery from the biological source and b ) the de tec tor response f ac to r . This l a t t e r f ac to r i s most often readi ly determined from in jec t ions of mix- tu res of the compounds in varying known concentrations and p lo t t ing the de tec tor response in a r b i t r a r y uni t s a s a function of the concentration of each individual compound. A good de tec tor system should give a l i n e a r concentration dependence which does not change with time. The recovery problem i s usually solved by use of the in te rna l standard technique and

i s often based on the assumption t h a t the recovery of the in te rna l standard a n d o f a l l the compounds t o be quant i ta ted i s the same. Neglection of differences in recovery may be acceptable only i f the molecular s t ruc tu res and chemical behaviour a r e very s i m i l a r , a f a c t which in HPLC may often cause problems with respect t o reso lu t ion .

Recovery data f o r the individual CA's and the in te rna l s t a n d a r d used a r e , however, readily obtained experimentally from ext rac t ions

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of samples of known concentration. In many cases when samples of low concentration a r e analyzed, inter-assay recovery va r i a t ions a re found t o be grea te r t h a n the intra-assay d i f fe rences between the individual compounds. Very usefu l , non-endogenous in te rna l standards have been found in 3,4-dihydroxybenzylamine ( D H B A ) , a-methyldopamine ( M D A ) and i soproterenol (N-isopropyl norepinephrine) ( IP) .

The va l ida t ion of a method used f o r quan t i t a t ive ana lys i s i s usually ca r r i ed out in terms of r e l a t i v e standard deviation (RSD) o r coe f f i c i en t of var ia t ion ( C V ) f o r a s e r i e s of repeated analyses on the same objec t which will give the over-all p rec is ion of the method. Large systematic e r r o r s may produce inaccurate r e s u l t s , however, even though the precision of the method i s high. Therefore, i t i s very valuable i f the same material can be repeatedly analyzed by means of two or more qu i t e d i f f e r e n t methods.

A high s e n s i t i v i t y of a method, i . e . a low l i m i t of de tec t ion , does not imply a high precision or accuracy. A l e s s s e n s i t i v e method may give more precise and accurate r e s u l t s over a broad concentration range, provided t h i s l i e s f a r enough away from the de tec t ion l i m i t . The s i tua t ion may be roughly visualized a s depicted in Fig. 4.

I t i s de f in i t e ly t rue t h a t HPLC in combination with a s u f f i c i e n t - l y s ens i t i ve and s t ab le de tec tor system i s the tool of choice f o r quant i ta t ion of small amounts of CA's from biological material even compared t o GC/MS. However, some aspects of the chromatographic system design f o r work a t the "quan t i t a t ive t r ace ana lys i s " level should be noted.

A common problem in t r ace ana lys i s i s t o reduce- the background noise level as much a s possib1.e in order t o reduce the de tec t ion l i m i t a t a given signal-to-noise r a t i o . A high background in i t s e l f can be acceptable a s long a s i t i s s t ab le . I n genera l , however, lowering o f

the background will a l so reduce the noise l eve l . Therefore, in order t o make f u l l use o f the most s ens i t i ve de tec tor systems f o r HPLC,

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cv % I I

I--- lo9 106 lo3 conc..M

FIGURE 4. The coefficient of variation as a function o f the analyzed concentration for two different methods A and B.

the fluorimetric and the electrochemical, very great demands are placed on the chromatographic system with respect to solvent purity, constant flow rate, stability o f the stationary phase, etc. Therefore, from our own experience, it is advisable to use specially purified water (in case of electrochemical detection prepared by distillation from alkaline permanganate) for all aqueous solutions, including the mobile phase, to remove flow rate fluctuations from the pump by an external pulse dampening device, to use a mains voltage stabilizer, if an electrochemical detector is used, to reduce external field influences by means of a Faraday cage, and finally, to stabilize the whole system by continuous operation, i.e. being kept in a recycling mode with the detector on over-night.

SOME APPLICATIONS OF THE HPLC ANALYSIS METHOD TO PROBLEMS WITHIN BIOLOGY AND MEDICINE

A. Determination of catecholamine concentrations in plasma or serum

During the last few years considerable progress has been made in the field of plasma CA analysis and the "normal" levels in

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humans have been more d e f i n i t e l y asce r ta ined , p a r t l y due t o t h e

demonstrat ion o f a good c o r r e l a t i o n between HPLC methods and

rad ioenzymat i c methods f o r N E and E (37,39,101). G o l d s t e i n e t

a1 (101) found the f o l l o w i n g normal plasma l e v e l s : N E = 311*53

(300265) pg/ml and E = 57218) (52238) pg/ml, where t h e va lues

w i t h i n parentheses a r e those ob ta ined by t h e rad ioenzymat i c method.

The r e s u l t agrees ve ry n i c e l y w i t h t h e v a l u e f o r N E = 342230

r e p o r t e d by Davis and K i s s i n g e r (92 ) . The l a t t e r , l i k e w i s e u s i n g

C-18 reversed-phase columns m o d i f i e d by t h e use o f a n i o n i c su r -

f a c t a n t s , found d i f f i c u l t i e s i n chromatographic o v e r l a p w i t h

u r i c a c i d which c o - e x t r a c t s w i t h t h e CA's i n t h e i s o l a t i o n

procedure,

r e p o r t e d by Hjemdahl

(39 ) , b o t h w i t h the use o f cat ion-exchange HPLC and amperometric

d e t e c t i o n , a r e o n l y s l i g h t l y d i f f e r e n t : N E = 252 (254) and E =

75 (73 )

The agreement w i t h o t h e r HPLC-methods i s , however, poo re r . The

p r e s e n t s i t u a t i o n i s summarized i n Table 4. F i g s . 5 and 6 a r e

recen t , i l l u s t r a t i v e examples o f t h e appearance o f chromatograms

ob ta ined w i t h f l u o r i m e t r i c and e l e c t r o c h e m i c a l d e t e c t i o n techniques,

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

a problem n o t mentioned by G o l d s t e i n e t a l . The va lues

e t a1 (37) and ob ta ined i n o u r l a b o r a t o r y

pg/ml ( va lues w i t h i n parentheses a r e f rom r e f . 3 ? ) .

B. Stud ies on b r a i n t i s s u e

Because t h e two p r imary amines DA and NE a c t as n e u r o t r a n s m i t t o r s ,

HPLC has come t o p l a y a dominant r o l e as an a n a l y t i c a l t o o l f o r b r a i n

t i s s u e i n v e s t i g a t i o n s . ECD i s i d e a l f o r t h i s purpose and t h e m a j o r i -

t y o f papers p u b l i s h e d i n t h i s f i e l d d u r i n g t h e l a s t few yea rs a r e

based on t h e Adams-Kissinger technique f i r s t p u b l i s h e d i n 1974 i n -

v o l v i n g a lumina e x t r a c t i o n (31) . I n 1978 K i s s i n g e r and coworkers

improved t h e r e s o l u t i o n and o v e r - a l l s e n s i t i v i t y o f t h e method

by t h e a p p l i c a t i o n o f "dynamic ion-exchange" on a 10 p C-18 reve rsed -

phase column (103) and t h i s techn ique has s i n c e been w i d e l y adopted

i n neurochemical research (104) and f u r t h e r m o d i f i e d t o p e r m i t

t h e simultaneous d e t e r m i n a t i o n o f E and DOPAC (3,4-d ihydroxyphenyl -

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TABLE 4

A Comparison o f "Normal" Plasma Catecholamine Leve ls Found by D i f f e r e n t Methods. Abbrev ia t i ons : ECD= e lec t rochemica l d e t e c t i o n , GC-MS = gas chromatography - mass spect rometry , FD = f l u o r i m e t r i c de tec - t i o n , RE = rad ioenzymat ic precolumn d e r i v a t i z a t i o n

Method CA c o n c e n t r a t i o n (pg/ml )

NE E DA Ref.

HPLC-ECD 31 1 57 - 101

- - HPLC-ECD 342 92

HPLC-ECD 252 75 34 37

HPLC-ECD 292 8 1 29 102

HPLC-ECD 254 73 60 39

- - GC-MS 297 98

HPLC-FD 185 32 - 70

RE-HPLC 355 61 - 89

RE-HPLC 182 87 33 85

a c e t i c a c i d ) as w e l l (105,106). Recent papers desc r ibe a technique

i n which t h e alumina e x t r a c t i o n s tep has been o m i t t e d (91,107,108).

A ve ry good chromatographic r e s o l u t i o n and a h i g h s e n s i t i v i t y

(109 ) who were a b l e t o separate and was ob ta ined by Wagner e t a1

q u a n t i t a t e the CA's and DOPA, DOPAC, as w e l l as two a romat i c amino

a c i d decarboxylase i n h i b i t o r s a-monofluoro- and a - d i f l u o r o m e t h y l -

DOPA,in b r a i n and o t h e r t i s s u e . The au tho rs used a C-18 column w i t h

an 0.02 M c i t r a t e - p h o s p h a t e b u f f e r pH 3.2 m o d i f i e d w i t h ca. 13% o f

methanol and 2.5 mM octane-sulphonic a c i d . D e t e c t i o n was achieved

amperomet r i ca l l y by a wax-impregnated carbon pas te anode a t a work ing

p o t e n t i a l o f +0.30 V.

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ANALYSIS OF CATECHOLAMINES 2 5

NE

NE

1 1Q 0 'Q min min

FIGURE 5. Chromatograms o f CA s tandards (200 pg each o f NE and E) ( l e f t ) , and an e x t r a c t ob ta ined f r o m 1 m l o f a human plasma sample ( r i g h t ) . C o n d i t i o n s : g l a s s column 2 .1~1000 mm w i t h Zipax-SCX, mob i l e phase 0.03 W Na2HP04 c o n t a i n i n g 6% ( b ~ / v ) o f a c e t o n i t r i l e , f l o w r a t e 0.8 ml/min, post-column THI - reac t i on , f l u o r i m e t r i c d e t e c t i o n 403/510 nm.

(Reproduced f rom r e f . 70 w i t h t h e pe rm iss ion o f t h e au tho rs and t h e pub1 i s h e r . )

A m i n i r e v i e w cove r ing a p p l i c a t i o n s o f l i q u i d chromatographic-

f l u o r i m e t r i c systems i n neurochemist ry appeared r e c e n t l y ( 1 1 0 ) .

C. Enzyme k i n e t i c s t u d i e s v i a catecholamine a n a l y s i s by HPLC

H i g h l y s e n s i t i v e HPLC-methods o f CA-analysis a r e i d e a l l y s u i t e d

f o r t h e m o n i t o r i n g o f c e r t a i n enzyme a c t i v i t i e s r e l a t e d t o CA p r o d u c t i o n o r t r a n s f o r m a t i o n r e a c t i o n s . Thus, such methods have g r e a t l y f a c i l i t a t e d t h e s tudy o f enzymes i n v o l v e d i n t h e t y r o s i n e

me tabo l i c pathway and c o n t i n u e t o grow i n importance. As examples

may be g i v e n t h e assay o f t y r o s i n e hyd roxy lase (TH) by t h e use o f

ECD desc r ibed by Blank and P i k e ( 1 1 1 ) and by Nagatsu e t a1 (1121,

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UL

ALLENMARK

DOPA

DHBA

f 01 nA

L -, I I I

8 16 0 0 16

FIGURE 6 . Chromatograms of an e x t r a c t o b t a i n e d from 3 m l o f a human plasma ( l e f t ) and a r e a g e n t blank ( r i g h t ) . Condi t ions: steel column 4 . 6 ~ 2 5 0 mm with Biophase ODS 5p, mobile phase 0.15 M rnonochloroacetate b u f f e r pH 3.10 with 1.0 mM EDTA and 25 m g / l i t of sodium o c t y l s u l p h a t e , f low r a t e 1.3 ml/min, amperometric d e t e c t i o n a t +0.75 V , carbon p a s t e e l e c t r o d e . Plasma c o n c e n t r a t i o n s : NE 415, E 201 pg/ml . (Reproduced from Anal. Chem. 53, 156 (1981 l ( r e f . 9 2 ) with the permission of the a u m o r s and the copyr ight owner, the American Chemical S o c i e t y . )

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15DAYS

1 0

I S

21 DAYS

DA n-

1 5 nA

IS

30 DAY5

1

15"& I S I

- 1 1 'I 8 12 0 4 8 1 2 0 4 8 12

MINUTES

FIGURE 7. Illustration of the increase in neurotransmittor (NE,DA) concentrations with the development of the rat brain hypothalamus. Conditions: steel column 3.9~300 mm with Bonda- pak C-18, mobile phase 0.1 M citrate/phosphate buffer, 0.3 mM in sodium octyl sulphate, flow rate 1.0 ml/min, amperometric detection at +0.72 V, carbon paste electrode. (Reproduced from J. Neurochem. 31, 1461 (1978)(ref. 103) with the permission of the authors and the copyright owner, the International Society for Neurochemistry.)

assays of aromatic amino acid decarboxylase involving U V (113) or amperometric (114,115) detection, the monitoring of tyrosine decarboxylase activity as an enzyme kinetic method for the determination of pyridoxal-5'-phosphate in plasma (116,117), studies of catechol 0-methyl - (COMT) and N-methyl -transferase (phenylethanol- amine-N-methyl transferase, PNMT) activities (118-120) and assays for dopamine-beta-hydroxylase (121,122).

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ECD i s p a r t i c u l a r l y f a v o u r a b l e f o r r e a c t i o n s i n which the

e n z y m a t i c a l l y generated p r o d u c t i s more prone t o e l e c t r o o x i d a -

t i o n t h a n t h e s u b s t r a t e i t s e l f . T h i s s i t u a t i o n i s p r e s e n t i n t h e

assay o f TH. The d e t e r m i n a t i o n o f est rogen-2-hydroxy lase a c t i v i t y

by HPLC-ECD i s ano the r r e c e n t l y p u b l i s h e d example o f an a p p l i c a t i o n

o f t h i s technique t o a r e a c t i o n by which a ca techo l s t r u c t u r e i s

e n z y m a t i c a l l y generated (123) .

D. A p p l i c a t i o n s t o problems w i t h i n c l i n i c a l diagnoses and r e l a t e d

areas

Me tabo l i c d i s o r d e r s , e i t h e r i n h e r i t e d o r produced by acu te

disease, a r e always i n some way r e f l e c t e d i n an abnormal m e t a b o l i t e

p a t t e r n , i f an a p p r o p r i a t e body f l u i d (such as u r i n e ) can be ana-

l y z e d f o r t h a t purpose. Because CA's a r e i n v o l v e d i n many m e t a b o l i c

t rans fo rma t ions , t h e i r a n a l y s i s i n body f l u i d s i s o f g r e a t va lue f o r

t h e c l i n i c a l d iagnos is o f a v a r i e t y o f d isease s t a t e s . P a r t i c u l a r l y ,

n e u r a l c r e s t tumors such as neuroblastoma, pheochromocytoma and

gang1 ioneuroma a r e a l l a s s o c i a t e d w i t h an abnormal s e c r e t i o n o f

CA's. Thus, h i g h l y i nc reased l e v e l s o f plasma CA's, e s p e c i a l l y NE,

may be d i r e c t evidence f o r a suspected pheochromocytoma as i l l u s -

t r a t e d i n F ig . 8. A r a p i d HPLC-method whereby t h e e x t r a c t i o n s tep

has been omi t ted , s u i t a b l e f o r f a s t d i a g n o s i s o f t h e d isease has

been desc r ibed r e c e n t l y (124).

The increased CA p r o d u c t i o n i s accompanied by an i nc reased c a t a -

b o l i c d e a c t i v a t i o n v i a COMT-mediated 3 -0 -me thy la t i on and subsequent

o x i d a t i o n . The r e s p e c t i v e end-products, homovan i l l i c a c i d (HVA),

v a n i l l y l m a n d e l i c a c i d (VMA) and 3-methoxy-4-hydroxyphenylethylene- g l y c o l (MHPG) , a r e conjugated and e x c r e t e d by t h e u r i n e . Therefore , t h e p r o f i l i n g o f these a c i d m e t a b o l i t e s i n u r i n e i s a l s o o f g r e a t

d i a g n o s t i c va lue. Recent ly , K r s t u l o v i c e t a1 (125) demonstrated

t h e c l i n i c a l use o f t h e a n a l y s i s o f u r i n a r y l e v e l s o f VMA and MHPG

by means o f reversed-phase HPLC wi th ECD o r UV-detect ion.

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DHBA I

h

T

DHBA

I I 1 ' 1 '

0 10 20 30 min 1 . 1 1 . 1 '

0 10 20 30 min

FIGURE 8. Chromatograms showing t h e d i f f e r e n c e i n human plasma CA c o n c e n t r a t i o n s o f a normal c o n t r o l s u b j e c t ( l e f t ) and a p a t i e n t w i th a pheochromocytoma ( r i g h t ) . Cond i t i ons : s t e e l column 4 . 6 ~ 1 5 0 mm w i t h N u c l e o s i l SA 5 p , mob i le phase 0.04 M/0.02 M a c e t a t e / c i t r a t e b u f f e r pH 5.2, f l o w r a t e 0.60 ml/min, amperometric d e t e c t i o n a t +0.65 V , carbon pas te e l e c t r o d e . ( R e s u l t s f rom t h e a u t h o r ' s l a b o r a t o r y )

A semi-automated HPLC-method w i t h f l u o r i m e t r i c d e t e c t i o n o f

T H I ' s d e r i v e d f r o m NE and E was used by Nelson and C a r r u t h e r s f o r

tumor diagnoses v i a r o u t i n e u r i n e analyses (68) .

I n t h e serum o f p a t i e n t s w i t h a ma l ignan t melanoma, S - c y s t e i n y l - L-DOPA compounds, e s p e c i a l l y 5-S-cysteinyl-L-DOPA which i s t h e most

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important pheomelanin precursor, are present in highly increased amounts and the reversed-phase HPLC separation and quantitation of these L-DOPA metabolites from serum and urine as well as their diagnostic value have been studied in detail by Hansson et a1 (126,127). It was found that the amount of 5-S-cysteinyl-L-DOPA correlated well with the degree of dissemination of the tumor.

A recent paper describes a study of the metabolization of the hypotensive drug a-methyl-DOPA (MD) to a-methylnorepinephrine (MNE) (128). While the basal plasma concentration of MNE in patients on MD therapy was too low to be determined, it rose to levels in the range 90 - 350 pg/ml after exercise. The authors contribute this to a release of the metabolite from the sympathetic nerve endings.

The two existent sulfoconjugates of DA, the 3-0- and 4-0- sulphate, respectively, were analyzed successfully in urine from Parkinsonian patients undergoing L-DOPA therapy (129). In this case the urine sample was pretreated by means of successive passage through small columns of cation and anion exchange resins, in order to isolate and concentrate the conjugat.es before injection onto a silica gel column. The mobile phase consisted of acetonitrile - 3% aqueous ammonia (85:15) of pH 10.0. UV-detection at 277 nM was used. The time course o f the excretion of these conjugates, as well as the 3-0/4-0-ratio, was readily followed by this method.

E . Miscellaneous biomedical applications

The application of HPLC as an analytical tool for CA studies has attracted interest in many different biomedical areas. The technique is being rapidly explored, especially for investigations on bio- synthesis and metabolism. The possible biotransformation of CA's into tetrahydroisoquinol ine alkaloids via a Pictet-Spengler reaction as a cause of alcohol addicti,on has been studied by different groups (33,

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130,131). S t o u t e t a1

d e t e c t i o n f o r t h e i n v e s t i g a t i o n o f dopamine m e t a b o l i t e s ob ta ined i n e x t r a c t s f r o m c u l t i v a t e d neuroblastoma c e l l s (132). Hypothalamic CA

b i o s y n t h e s i s i n v i t r o has been s t u d i e d r e c e n t l y (133) u s i n g HPLC

and e l e c t r o c h e m i c a l d e t e c t i o n . P l o t s k y e t a l , i n connec t ion w i t h

research on i n h i b i t i o n o f p r o l a c t i n i n h i b i t i o n , determined DA i n

hypophysia l s t a l k b l o o d (134) w i t h t h e same technique. Amperometric

d e t e c t i o n has a l s o been used f o r t h e a n a l y s i s o f a-methyl-DOPA and

i t s p r i n c i p a l b i o t r a n s f o r r n a t i o n p roduc ts i n serum (135). I d e n t i f i c a -

t i o n o f 4-0-methyldopamine i n t i s s u e as an L-DOPA m e t a b o l i t e was

e l e g a n t l y c a r r i e d o u t by B i d a r d and Cronenberger (136) u s i n g HPLC

and d e t e c t i o n by UV, f l u o r i m e t r y and t r i t i u m coun t ing . A v e r y n i c e

example o f how drug e f f e c t s on plasma CA l e v e l s can be v i s u a l i z e d

by HPLC i s g i v e n i n a r e c e n t paper by Watson (137) and shown i n

F i g . 9. The e f f e c t o f t h e a ad renerg i c a n t a g o n i s t c l o n i d i n e , a hypo-

t e n s i v e drug, on t h e CA c o n c e n t r a t i o n s i n plasma i s e v i d e n t f r o m t h e

chromatograms and q u i t e dramat ic .

used HPLC combined w i t h UV and r a d i o - i s o t o p e

FUTURE TRENDS: POSSIBLE FURTHER IMPROVEMENTS AND NEW APPLICATIONS

A t present , t h e m a j o r i t y o f CA-analyses c a r r i e d o u t by HPLC, i n - and time-COnSUming p r e - p u r i f i c a t i o n f rom t h e b i o - c lude a l a b o r i o u s

l o g i c a l m a t e r i a l . However, u s i n g a dual column techn ique i t should

be q u i t e p o s s i b l e t o adsorb t h e CA's on a h i g h a f f i n i t y precolumn,

wash o u t most o t h e r m a t e r i a l and subsequent ly r e l e a s e t h e CA's by

a s o l v e n t change w i t h t r a n s f e r t o t h e a n a l y t i c a l column, by means

o f a s w i t c h i n g va l ve . Such mu l t i co lumn i s o l a t i o n and p reconcen t ra -

t i o n techniques have r e c e n t l y come i n t o use f o r s i m i l a r a n a l y t i c a l

purposes (100) and have t h e advantage o f making t h e a n a l y t i c a l p ro -

cess f a s t e r and e a s i e r t o c o n t r o l by a microcomputer (138) . One

shou ld a l s o , as p o i n t e d o u t by K i s s i n g e r (1391, expec t an in i -

provement i n t h e performance of t h e v a r i o u s HPLC so rben ts a v a i l -

ab le , towards h i g h e r r e p r o d u c i b i l i t y and column e f f i c i e n c y .

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32

2 a

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ALLENMARK

+ 0 W

i -

1 5 ’ 0

MINUTES 0

FIGURE 9. The effect of the hypotensive drug clonidine on dog plasma CA levels. Chromatograms of a dog plasma extract immediately before (left) and 15 min after the intravenous administration of clonidine (20 mg/kg)(right). Conditions: steel column 4.6~250 mm with Partisil SCX lh, mobile phase 0.013 PV0.008 M acetate/citrate buffer pH 5.2 with 10 pM EDTA, flow rate 1.0 ml/min, amperometric detection at +0.50 V, carbon paste electrode. (Reproduced from ref. 137 with the permission of the author and the pub1 isher. )

As far as detector systems are concerned, developments in the construction of electrochemical flow-cells, resulting in a still higher sensitivity, are to be expected. Aniperometric detection with rotating disc electrodes has recently given very promising results for brain tissue analysis (140). Further, experiments with dual working electrodes (34) have demonstrated the possi- bility of distinguishing between chromatographic peaks origi-

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nating from (quasi-)reversible (shown by catechol derivatives) and irreversible electrochemical reactions, respectively.

SUMMARY AND CONCLUSIONS

Very great progress has been made during the last ten years in the field of analysis of catecholamines by HPLC. The improvements in column efficiency, resulting from better bonded- phase packing materials and general column technology, have aided in increased resolution and detectability. Reversed-phase or cation-exchange microparticulate (10 or 5u) columns with aqueous mobile phases and isocratic elution are at present the most successful chromatographic systems; the column type pre- ferred being dependent on the purity of the sample to be in- jected and the type of detector system used. Very low detection limits are obtained with the use of electrochemical detectors or of fluorimetric detectors combined with postcolumn transformation of the catecholamines to trihydroxyindole (THI) or o-phtal- a1 dehyde (OPA) derivatives. However, only EC detectors are capable o f being almost equally sensitive to all catecholamines; by the THI-fluorimetric method DA is severely discriminated, whereas the OPA-technique totally excludes E. For the determination of catecholamines in biological material containing only trace a- mounts, such as plasma or serum, detection limits (S/N = 2) of 1 - 5 pg have been obtained recently with the use of fluorimetric (THI) and electrochemical detection, respectively. Such highly sensitive HPLC-systems are increasingly being used within biology and medicine for catecholamine analysis of diverse biological material and are successfully competing with the radioenzymatic procedures for plasma and for serum. Further developments in the field would probably be possible by the application of a column- switching technique permitting pre-column extraction and sample enrichment as well as by improvements in detector constructions, particularly the electrochemical f 1 ow-cel 1 s.

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34

ACKNOWLEDGEMENTS

ALLENMARK

The va luab le c o n t r i b u t i o n s o f Mrs. L. Hednian t o t h e r e s u l t s

f rom o u r l a b o r a t o r y presented i n t h i s paper and o f P r o f . D.H.

Lewis t o t h e f i n a l l i n g u i s t i c e d i t i n g a r e g r a t e f u l l y acknowl-

edged. The au tho r i s a l s o i ndeb ted t o L i n k o p i n g U n i v e r s i t y f o r

f i n a n c i a l suppor t .

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ANKYSIS OF CATECHOLAMINES 39

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Analysis of Catecholamines by HPLC

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