Chapter 7 Quantitative High Resolution Mass Spectrometry of Biogenic Amines

129

CHAPTER 7

QUANTITATtVE HIGH RESOLUTION MASS SPECTROMETRY OF BIOGENIC AMINES

BRUCE *A. DAVIS AND DAVID A. DURDEN Psychiatric Research Division, University Hospital, Saskatoon, Saskatchewan S7N OX0 (Canada)

7.1

INTRODUCTION

7.1.1

Historical background and development

The necessity for the determination of low concentrations of biogenic amines in brain and brain regions, blood plasma, cerebrospinal fluid and small populations of cells has stimulated the search over the past twenty years for analytical methods of improved sensitivity and specificity.

The term biogenic amines includes the

trace amines (phenylethylamine, phenylethanolamine, tryptamine, 5 and e-tyramine,

m-

and p-octopamine and

and 1-synephrine), the catecholamines (dopamine, adren-

aline, noradrenaline), and their metabolites (3-methoxytyramine, metanephrine, and normetanephrine), the aliphatic amines (piperidine and histamine) and the polyamines (putrescine, cadaverine, spermidine and spermine). Mass spectrometry in conjunction with chromatographic procedures is one of the

most sensitive and specific methods available for the quantitation of biogenic amines. by

a

In this review, the emphasis will be on the analysis of the trace amines

thin-layer chromatographic-mass spectrometric technique which we have develop-

ed, refined and exploited over the last ten years. The first attempts at quantitating biogenic amines by mass spectrometric measurement of a selected ion were carried out on relatively crude extracts from rat brain [l-31.

It was soon realized, however, that more accurate results could be

obtained if the amines were first derivatized and purified by chromatography. The l-(~,~-dimethylamino)naphthalene-5-sulfonyl (dansyl) derivatives proved to be the

most suitable with regard to both chromatographic and mass spectrometric properties. Dansyl chloride was originally introduced as a fluorescent end-group reagent for proteins and peptides [4], and then as a derivatizing reagent for amino acids [5], [6-141

This was soon extended to include quantitation by fluorescence measurements and confirmation of identity by

mass

spectrometry 114-191, the latter

assisted by the publication of numerous spectra of dansyl amines [20-23].

The

130 partial mass spectrum and structure of bis-dansyl p-tyramine are presented in Figure 7.1. Since 1973 we have employed a high resolution thin-layer chromatographic-high resolution mass spectrometric method for the determination of a number of biogenic amines in tissues and body fluids as their dansyl derivatives, with deuteriumlabelled analogues as internal standards.

These studies have included the identi-

fication and distribution of the trace amines phenylethylamine (PEA), tryptamine (T), and 2- and E-tyramine (mTA and pTA) in rat organs and brain regions 124-281, in rabbit brain [291, in human brain [30], their subcellular distribution in rat brain [31], and their urinary excretion in the rat and human (32-351. Other ainines measured by this method include phenylethanolamine (PEOH) [36], 2- and p-synephrine (mSYN and pSYN) [37,38] and 2- and e-octopamine (&A

and pOA) [381 in various tis-

sues, benzylamine in rats pre-treated with pargyline [391, tetrahydroisoquinoline and tetrahydro-8-carboline alkaloids [401, Y-aminobutyric acid (GABA) in astrocytes [41], adrenaline ( A )

in fowl diencephalon (421, and amphetamine and p-hydroxyam-

phetamine in the rat following amphetamine injection [43-461.

In addition, trace

amine levels in brain [46-561 and in urine 157) after the administration of monoamine oxidase inhibitors, anti-psychotics and other drugs have been measured.

>

i

k In z W

170

I-

z

M

W

603

2 I-

4w

d

U

Figure 7.1

.+...L

263

l l .

x5 370

i

The partial mass spectrum and structure of bis-dansyl 1-tyramine.

A somewhat similar procedure using low resolution mass spectrometry of

the

dansyl amines with the internal standard (either a homologue of the compound under investigation or a compound possessing a similar evaporation profile but different mass) added just prior to quantitation in the mass spectrometer has been used by Seiler et al.

1581

to estimate tissue levels of putrescine [59-611, serotonin

131 (5-hydroxytryptamine, 5-HT) and bufotenin [ 6 2 1 , GABA [591 and piperidine [ 6 3 ] , and by Dolezalova et al. and Stepita-Klauco et al. to measure piperidine [64-671 and cadaverine [68-701 in the brains of snails and mice. 7.1.2

Mass spectrometric quantitative techniques

Mass spectrometric methods for the analysis of biogenic amines have followed two main lines of development:

gas chromatography-mass spectrometry (GC-MS) and thin-

layer chromatography-mass spectrometry (TLC-MS)

.

Each of these has been applied in

a number of ways, depending on the resolution achieved in the chromatography and mass spectrometry.

Packed GC columns, which are capable only of low resolution,

can be employed with mass spectrometry at low or high resolution (GC-LRMS or GCHRMS).

Capillary or high resolution gas chromatography coupled with low or high

resolution mass spectrometry (HRGC-LRMS or HRGC-HRMS) can also be used, although there are few examples in the literature.

The higher the resolution of chromatog-

raphy and mass spectrometry the greater the specificity of the method.

Similarly,

TLC-MS can be either low or high resolution, depending on the number of TLC separations and the resolution of the mass spectrometer (TLC-LRMS and TLC-HRMS).

Our

method involves three TLC separations on three different plates in three different solvent systems, which is roughly equivalent to the high resolution obtained with GC capillary columns, and the mass spectrometer is operated at high resolution (7,000-10,000). In

all of the above procedures, the mass spectrometer is adjusted so that one or

more ions characteristic of the compound under investigation are monitored continuously as an aliquot o € the sample (from the direct probe or outlet o € a GC column) enters the ion source.

This technique has acquired a number of names, including

integrated ion current, mass fragmentography and single, mltiple or selected ion detection, monitoring or recording.

The International Union of Pure and Applied

Chemistry has studied this problem of nomenclature but has made no recommendations (711. Watson et&.

[721 suggest selected ion monitoring (SIM) and it is this term

which shall be used in this review.

The acronym, SIM, can therefore be added to

those given above to complete the description of the procedure.

For example, the

abbreviation for our method, high resolution thin-layer chromatography-high resolution mass spectrometry with selected ion monitoring, would be HRTLC-HRMS-SIM. The choice of internal standard and when in the procedure it is added are of crucial importance.

Isotopically-labelled (usually deuterium) analogues of the

compound to be measured, although expensive to buy or difficult or time-consuming to synthesize, are ideal internal standards for mass spectrometric quantitation. When

added t o the tissue homogenate or biological fluid, they are carried through

all the steps of extraction, derivatization and chromatography in exactly the same way as the endogenous amine, so that losses due to decomposition, adsorption and inefEicient extraction are automatically corrected

for.

All other analytical

methods m s t rely on homologues or other compounds with similar properties for

132 internal

standards, with

the

attendant

requirements

for

calibration curves,

recovery estimates and supplementation studies which increase the number of samples to be analyzed.

7.2

PROCEDURE

7.2.1

Chemical and biochemical preparation of the sample for mass spectrometry

The isolation, derivatization and separation of trace amines as currently em-

ployed in the preparation of samples for high resolution mass spectrometric analysis was first described by Durden et al. for PEA [24] and the d-hydroxyarylalkylamines [37,38] and by Philips %&. for pTA [26], T[27] and mTA [ 2 8 ] .

This proced-

ure, which will be described here only briefly, is outlined schematically in Figure

7.2. An aliquot containing a known amount of the appropriate dideutero- or tetra-

deutero-amine internal standard is added to the tissue homogenate or biological fluid before any processing takes place.

Two procedures are in use depending on

the amount of tissue or fluid to be analyzed.

If more than 100 mg of tissue or 0.5

ml of fluid is to be analyzed, tha amine fraction is first isolated by ion-exchange chromatography and then dansylated.

Otherwise, the homogenate or physiological

fluid is derivatized directly by the addition of sodium carbonate and an acetone solution of dansyl chloride. The derivatization solutions are concentrated under a stream of nitrogen and the dansyl amines extracted into benzene.

If d-hydroxya-

mines are to be quantitated, they may be acetylated at this point provided that the separation of

m

and 1-isomers is not required, otherwise the dansylated isomers

are separated by TLC and then acetylated separately.

The benzene extract or acet-

ylating mixture is dried under nitrogen and the derivatives are re-dissolved in toluene and transferred to silica gel TLC plates which are developed uni-dimensionally on two or three different plates each in a different solvent system.

Follow-

ing each chromatographic separation, the zone containing the dansyl amine of interest is visualized briefly under ultraviolet light, outlined with a spatula, powdered and extracted. After the final chromatographic separation, the powdered zone is transferred to a micro-extraction tube, and extracted by elution with 25-30 v l of ethyl acetate. The micro-extraction tube is then sealed at both ends with hematocrit sealing clay and stored at -17°C until mass spectrometric analysis.

7.2.2

Mass spectrometric procedure for the quantitation of biogenic arnines

The procedure for the mass spectrometric quantitation by SIM of biogenic amines is a modification of earlier procedures developed for other types of compounds

[2,73-751.

In our laboratory in Saskatoon, ions are resolved using an AFJ MS902S

high resolution double-focussing mass spectrometer equipped with a direct insertion probe, operating at 8kV acceleration potential, electron energy of 70 eV and 500 PA emission.

The direct probe has been modified so that it is of fixed length and

133

Tissue

Deuterated Amine

Subcellular Fraction,

Internal Standard

Physiological Fluid

25 ng

Supernatant Triton X-100, adjust OH to 7.0. If tissue

Percolate through Bio-Rad AC

weight is

501J-X2, wash with Na Acetate Elute with

less than

and distilled H20.

100 mg.

Methanol/HCl or Ethanol/NH3.

Thin-laver Chromatography. hidimensionally, two or three systems.

with Selected Ion Monitoring 7,000-10,000 Resolution Figure 7.2. trace amines.

Schematic outline of procedure for the ARTLC-YQMS-SIM

analysis of

134 such t i a t t h e probe t i p (Pyrex o r q u a r t z g l a s s ) e n t e r s t h e s o u r c e t o w i t h i n 1-2 mm of t h e e l e c t r o n beam.

,

Because r e p e a t e d use of a pyrex o r q u a r t z sample h o l d e r in-

c r e a s e s i t s s u r f a c e a c t i v i t y with c o n c u r r e n t decomposition of t h e d a n s y l compounds, t h e probe t i p i s r e p l a c e d f r e q u e n t l y . The S I M procedure i s normally performed with t h e mass s p e c t r o m e t e r tuned t o a r e s o l u t i o n of 7,000 t o 10,000, depending on t h e s u b s t a n c e t o be a n a l y z e d .

The mass

s p e c t r o m e t e r is f i r s t focussed o n t o an i o n c h a r a c t e r i s t i c of t h e compound under i n v e s t i g a t i o n ( p r e f e r a b l y t h e molecular

i o n M)

and

t h e mass r a t i o

of

t h e peak

matching decade box i s set s o t h a t an ion of known e x a c t mass of a mass r e f e r e n c e compound (such a s perfluorotri-2-butylamine

or perfluorokerosene)

t h e o s c i l l o s c o p e i n t h e high mass p o s i t i o n .

Immediately b e f o r e i n t r o d u c t i o n of t h e

i s c e n t e r e d on

sample, t h e r a t i o of t h e decade box i s changed s o t h a t t h e mass of t h e correspondi n g ion of t h e d e u t e r a t e d i n t e r n a l s t a n d a r d o c c u r s i n t h e high mass p o s i t i o n .

An

a l i q u o t of t h e sample e x t r a c t ( u s u a l l y 5 lil) is t r a n s f e r r e d t o t h e probe t i p by means of a s y r i n g e , t h e s o l v e n t i s c a r e f u l l y e v a p o r a t e d and t h e probe i s i n s e r t e d i n t o t h e h o t ion s o u r c e .

As t h e sample e v a p o r a t e s t h e s i g n a l s from t h e endogenous

amine and d e u t e r a t e d i n t e r n a l s t a n d a r d a r e recorded a l t e r n a t e l y .

A f t e r measuring

t h e a r e a s under t h e two p r o f i l e s by p l a n i m e t r y o r by computer [ 3 6 ] ,

t h e amount of

endogenous amine can be c a l c u l a t e d , c o r r e c t i n g f o r i s o t o p i c and chemical i m p u r i t i e s i n t h e d e u t e r a t e d s t a n d a r d and n a t u r a l i s o t o p i c c o n t r i b u t i o n s of mass s i g n a l .

t h e low t o h i g h

A t y p i c a l example of t h e p r o f i l e s o b t a i n e d by t h i s procedure can be

seen i n F i g u r e 7 . 3 f o r dansyl PEA and t h e i n t e r n a l s t a n d a r d d a n s y l PEA-d4.

m/z 354.1402

\

4 TIME Figure 7 . 3 Mass s p e c t r o m e t r i c s e l e c t e d i o n m o n i t o r i n g p r o f i l e s of t h e molecular ions of d a n s y l phenylethylamine and d a n s y l phenylethylamine-d~+.

135 7.3

EVALUATION OF H I G H RESOLUTION MASS SPECTROMETRY VS OTHER ANALYTICAL TECHNIQUES

7.3.1

Specificity Comparison of v a l u e s f o r t r a c e amines measured by d i f f e r e n t a n a l y t i c a l

(i techn

ques. S p e c i f i c i t y i s one of t h e most important c r i t e r i a by which an a n a l y t i N e v e r t h e l e s s , t h e l i t e r a t u r e is r i f e with examples of

c a l t e c h n i q u e can be judged.

a n a l y s e s which were thought t o be s p e c i f i c but which s u b s e q u e n t l y were proven n o t t o be s o .

As a r u l e , as a n a l y t i c a l methods become more advanced and s o p h i s t i c a t e d ,

r e p o r t e d v a l u e s f o r a given compound f a l l , specificity.

i n d i c a t i n g t h a t e a r l i e r methods lacked

I n comparing r e s u l t s from d i f f e r e n t l a b o r a t o r i e s ,

one must b e a r i n

mind, however, t h a t d i f f e r e n c e s i n animal s t r a i n , housing c o n d i t i o n s , d i e t , age and post-mortem h a n d l i n g of t i s s u e s may a f f e c t t h e t i s s u e l e v e l s of some compounds. TABLE 7 . 1 Values f o r phenylethylamine, 1-tyramine and b r a i n a s measured by d i f f e r e n t a n a l y t i c a l t e c h n i q u e s . Authors and Reference F i s c h e r %&. Durden % g , Saavedra W i l l n e r gtt. Suzuki & Yagi Martin & Baker P h i l i p s % &. Karoum % g . Edwards %&. P h i l i p s %&. Tallman &&. Duffield Saavedra & Axelrod Snodgrass & Horn &. Philips P h i l i p s & Boulton

st.

&.

Sloan

Warsh gt &. Artigas & Gelpi

[761 "241 [771 [791 [811 [78] 1301 I861 [801 [261 [841 [851 [871 [911 [271 [491 [881 [891 [901

Method

PEA ( n g / g )

SPF HRTLC-HRMS-SIM REA GC-LRMS-SIM SPF GC-ECD HRTLC-HRMS-SIM GC-LRMS-SIM GC-CI-MS-SIM HRTLC-HRMS-SIM REA GC-CI-MS-SIM REA Radio-dansyl HRTLC-HRMS-SIM HRTLC-HRMS-SIM SPF GC-LRMS-SIM GC-LRMS-SIM

492

t r y p t a m i n e i n whole r a t pTA ( n g / g )

T (ng/g)

1.8

1.5

1.7 5.0 1.1 2.1 8.1 1.0

4.2 2.0 12.9 2.2

22.0 69 .O 0.5

0.35

20.9 not d e t e c t a b l e
Analyses of t h e t r a c e amines PEA, pTA and T i n r a t b r a i n over a period of a few years

using d i f f e r e n t

a n a l y t i c a l methods

s p e c i f i c i t y (Table 7.1).

i l l u s t r a t e p e r f e c t l y t h e importance of

PEA i n rat b r a i n was f i r s t measured by F i s c h e r e t a l .

[76] who o b t a i n e d a v a l u e of 492 ng/g by t h e spectrophotofluorimetric (SPF) method, y e t a year l a t e r Durden e t a l . HRMS-SIM.

[24] had o b t a i n e d a v a l u e of 1 . 8 ng/g u s i n g HRTLC-

S i n c e then o t h e r methods have been employed and confirmed t h e l a t t e r

r e s u l t , f o r example,

t h e radioenzymatic a s s a y (REA) [ 7 7 ] , gas chromatography w i t h

e l e c t r o n c a p t u r e d e t e c t i o n (GC-ECD) c a l i o n i z a t i o n (GC-CI-MS-SIM) Yagi

[81]

were

able

[80].

1781, GC-MS-SIM

[79] and GC-MS-SIM

Using an improved SPF procedure,

t o o b t a i n a v a l u e of

5.0

ng/g.

w i t h chemi-

Suzuki and

A s i m i l a r phenomenon was

136 observed f o r PEA i n r a b b i t b r a i n ,

i n which e a r l y r e s u l t s by Borison e t a l .

[82]

(340 ng/g) u s i n g gas chromatography with flame i o n i z a t i o n d e t e c t i o n (GC-FID) Mosnaim e t a l .

and

[831 (400 ng/g) u s i n g SPF t u r n e d o u t t o be much h i g h e r than t h e

v a l u e o b t a i n e d by Boulton

&.

"291 (0.44 ng/g) u s i n g HRTLC-HRMS-SIM.

developments can be observed i n Table 7 . 1 f o r pTA and T.

Similar

The r a d i o e n z y m a t i c a s s a y

produced a v a l u e of 12.9 ng/g f o r pTA [841 compared t o 2.0 ng/g by HRTLC-HRMS-SIM "261.

GC-CI-MS-SIM

gave a v a l u e of

2 . 2 ng/g

y i e l d e d a somewhat h i g h e r v a l u e of 4 . 2 ng/g

22.0 ng/g

1851, but a n o t h e r GC-MS procedure For T t h e REA gave a v a l u e of

[861.

[871, s i m i l a r t o t h a t r e p o r t e d f o r a SPF method (20.9 ng/g)

much h i g h e r than t h a t o b t a i n e d by HRTLC-HRMS-SIM

(0.50,

0.35 ng/g)

[88],

[27,49].

but One

paper [89] d e s c r i b i n g a GC-LRMS-SIM procedure r e p o r t e d T t o be n o t d e t e c t a b l e , even with t h r e e pooled b r a i n s , and a n o t h e r [901 r e p o r t e d T t o be less than 0.38 ng/g, presumably t h e lower l i m i t of d e t e c t i o n .

g-TA

[791 and 5.8 ng/g by

[921 as measured by t h e REA, 3 . 8 ng/g by GC-LRMS-SIM

GC-CI-MS-SIM ed.

a s s a y gave a v a l u e of 69

PEOH has been r e p o r t e d t o be p r e s e n t i n whole r a t b r a i n a t a l e v e l of

ng/g 1911.

6 . 2 ng/g

A radio-dansyl

[80],

but by HRTLC-HRMS-SIM

1361 a v a l u e of o n l y 0.06 ng/g was o b t a i n -

has been measured i n t i s s u e only by t h e HRTLC-HRMS-SIM

t e c h n i q u e [281 so

a comparison with o t h e r a n a l y t i c a l methods i s n o t p o s s i b l e . w i t h o u t a t r u e i n t e r n a l s t a n d a r d ( t h a t i s , a compound s i m i l a r t o

TLC-LRMS-SIM

t h e one under i n v e s t i g a t i o n is added t o t h e d i r e c t probe) has been used t o measure t h e l e v e l of p i p e r i d i n e i n s n a i l b r a i n . do not a g r e e .

The r e s u l t s of d i f f e r e n t workers, however,

Using d a n s y l d e r i v a t i v e s and d a n s y l p y r r o l i d i n e a s s t a n d a r d (added

t o t h e d i r e c t probe n o t t o t h e homogenate), Dolezalova e t a l . ng/g

and

Seiler

& &.

napthalene-5-sulfonyl

[63],

(bansyl)

using

the

derivatives,

corresponding found

[651 measured 277

l-(N,N-di-n-butylamino)

17 ng/g

piperidine

in

snail

brain. (ii)

a.

E v a l u a t i o n of t h e s p e c i f i c i t y of t h e d i f f e r e n t a n a l y t i c a l t e c h n i q u e s . T h i n - l a y e r chromatography-mass s p e c t r o m e t r y .

HRTLC-HRMS-SIM

It i s i n t e r e s t i n g t o n o t e t h a t

i s t h e only method p r o v i d i n g t h e lowest v a l u e s

t h e r e f o r e , t h e c o r r e c t ones) f o r a l l t h e above-mentioned

(and

amines.

t h i s i s undoubtedly t h e g r e a t e r s p e c i f i c i t y of t h e HRTLC-HRMS-SIM greater

specificity is

due not

only

to

t h e high

resolution

of

presumably,

The r e a s o n f o r procedure. the

This

thin-layer

chromatography ( t h r e e p l a t e s each developed u n i d i m e n s i o n a l l y i n a d i f f e r e n t s o l v e n t system) and t h e mass s p e c t r o m e t r y ( r e s o l u t i o n of 7,000 t o lO,OOO), n a t u r e of t h e d a n s y l d e r i v a t i v e .

but a l s o t o t h e

The low mass r e g i o n of t h e spectrum of a b i o l o g i -

c a l e x t r a c t i s r a t h e r complex due t o t h e p r e s e n c e of molecular i o n s from s u b s t a n c e s o t h e r t h a n t h e one of weight s u b s t a n c e s .

i n t e r e s t and fragment i o n s d e r i v i n g from h i g h e r molecular

The d a n s y l d e r i v a t i v e s of b i o g e n i c amines n o t only p o s s e s s h i g h

enough molecular w e i g h t s so as t o be r e l a t i v e l y f r e e of i n t e r f e r i n g i o n s , b u t a l s o one o r more m a s s - d e f i c i e n t

s u l f u r atoms which

f a c i l i t a t e t h e r e s o l u t i o n of

the

137 characteristic dansyl amine ions from background ions, many of which are non-mass deficient hydrocarbons. b.

Radioenzymatic assay.

Since the specificity of the REA is somewhat depend-

ent on the purity and specificity of the enzyme and even more dependent on the selectivity of the isolation procedure, it may be that the high values obtained for T, pTA and PEOH are due to alternative substrates for the methylating enzyme which were not adequately separated before

OK

after methylation.

Secondly, the REA as

originally utilized was unable to distinguish between positional isomers, for example the 0-, fi- and p-isomers of octopamine[93].

By dansylation of the methyla-

tion product mixture and isolation of the products by two

OK

three TLC separations

it has been shown [941 that separation and quantitation of &A

and @A are pos-

sible, and that these values are lower than those of the original REA [93]. c.

Gas chromatography-mass spectrometry.

The GC-MS-SIM procedure usually

gives values comparable to those obtained by HRTLC-HRMS-SIM, provided that the choices of derivative, internal standard and ion or ions to be recorded are wisely made.

The ideal internal standard, as mentioned above, is the deuterated analogue

of the compound under investigation.

Many investigators, however, employ homo-

logues or other similar compounds as internal standards, accepting the resulting uncertainty which arises because of possible differences in the extraction, chromatographic and mass spectrometric properties of the standard compared to the amine to be analyzed.

A frequently used device to ensure specificity is to monitor two

or more ions of the same compound; if both

(OK

all) are present and the relative

intensities are in the same ratio as for the authentic compound this is taken as proof of the purity of the substance being analyzed.

While it i s probably correct

to assume that the ratio remains constant for periods of a few hours, it has been observed that the ratio of ion abundances can vary significantly from day to day depending on ion source cleanliness, instrument tuning and changes in source temperature [95].

It is of particular importance to bear this in mind when analyzing

biogenic amines as their perfluoroacyl or trimethylsilyl derivatives, the most popular derivatives for GC-MS because of the ability of the corresponding reagents to react readily with the usual functional groups found in the biogenic amines.

Un-

fortunately, the most abundant ions in the spectra of these derivatives ate often common to several different amines (and also their acidic and alcoholic metabolites), so that specificity must rely on chromatographic resolution (which is comparatively low for the packed columns which are most often used) and the ratio of ion abundances (which, as mentioned earlier, can be variable due to instrumental factors).

Furthermore, these common ions are generally of relatively low mss,

which increases the likelihood of interference from background ions.

For example,

m/z 174 for the trimethylsilyl derivatives of primary amines 1961, m/z 58 for dimethyl tertiary amines [ 9 7 , 9 8 ] and m/z 140 for the pentafluoropropionyl derivatives of methyl secondary amines [ 9 9 ] are each the base peak in the mass spectrum

138 and a l l r e s u l t from c l e a v a g e of t h e a , & a l k y l

carbon bond with t h e c h a r g e remaining

on t h e n i t r o g e n fragment which p r o v i d e s no s t r u c t u r a l i n f o r m a t i o n . The dangers non-unique

inherent

i n q u a n t i t a t i n g b i o g e n i c amines by monitoring

low mass,

ions a r e i l l u s t r a t e d by t h e r e s u l t s o b t a i n e d f o r PEA i n r a t b r a i n

i n which t h e masses m/z 91 (C7H7) and 104 (CBHB) were monitored. ed f o r rat b r a i n ( 8 . 1 ng/g) t a i n e d by HRTLC-HRMS-SIM.

The v a l u e o b t a i n -

[861 i s s u b s t a n t i a l l y h i g h e r than t h e 1.8 ng/g (241 obThe m o n i t o r i n g of low mass, non-unique

c i r c u m s t a n c e s be a c c e p t a b l e and HRTLC-HRMS-SIM,

[861

produce

values

i o n s can i n some

comparable t o t h o s e

o b t a i n e d by

as, f o r example, i n t h e measurement of PEA i n human u r i n e [lo01 i n

which t h e v a l u e o b t a i n e d (8.0 Pg/24h) a g r e e s with t h a t o b t a i n e d by HRTLC-HRMS-SIM [32,35], [79],

even though

using

t h e masses monitored were m/z 91 and 104.

pentafluoropropionyl

obtained a value of 1.7

ng/g

derivatives

and

deuterated

i n order

acetyl-2-trimethylsilyl

to differentiate

the

Jacob

Ct. [ l o l l

fragment i o n s of

employ-

t h e !-trifluoro-

d e r i v a t i v e s of c a t e c h o l a m i n e s t o be measured from i n t e r f e r -

A few i n v e s t i g a t o r s a r e u s i n g GC-CI-

ing s u b s t a n c e s having t h e same nominal mass.

MS by which they o b t a i n i n t e n s e pseudo-molecular s p e c i f i c i t y [80,102-1061 d.

standards

f o r PEA i n r a t b r a i n u s i n g masses m/z 93 f o r t h e

i n t e r n a l s t a n d a r d and m/z 104 f o r t h e endogenous amine. ed GC-HRMS-SIM

Willner e t a l .

internal

ions and t h e r e f o r e an i n c r e a s e i n

due t o t h e uniqueness of such i o n s .

Gas chromatography.

GC i s i n h e r e n t l y much less s p e c i f i c than mass spectrom-

e t r i c methods s i n c e t h e s p e c i f i c i t y i s based f o r t h e most p a r t on r e t e n t i o n time and t h e r e s o l v i n g power of t h e column.

The s p e c i f i c i t y can be improved somewhat by

a s u i t a b l e c h o i c e of d e t e c t o r and d e r i v a t i v e . detector w i t h

For example,

the electron capture

f l u o r i n a t e d d e r i v a t i v e s is more s p e c i f i c (and more s e n s i t i v e ) than

t h e flame i o n i z a t i o n d e t e c t o r .

The r e l a t i v e l y low s p e c i f i c i t y of GC i s at l e a s t

p a r t l y r e s p o n s i b l e f o r t h e wide v a r i a t i o n i n v a l u e s r e p o r t e d f o r PEA i n human u r i n e and r a t b r a i n .

I n u r i n e , v a l u e s of 770

[loo],

400 [ 1 0 7 ] , 1 0 . 3 [lo81 and 6 . 8 [lo91

vg/24 h have been measured by GC, whereas v a l u e s determined by MS a r e c o n s i s t e n t l y l e s s than 10 vg/24 h [ 3 2 , 3 5 , 8 6 ] .

S i m i l a r l y , t h e high v a l u e (400 ng/g) f o r PEA i n

r a t b r a i n r e p o r t e d by F i s c h e r e t a l .

[76] has been brought down only a f t e r e x t e n s -

i v e c l e a n u p of t h e sample b e f o r e GC by Martin and Baker (781 t o 1.1 n g / g , which compares f a v o r a b l y with v a l u e s o b t a i n e d by HRTLC-HRMS-SIM there

is a c a r e f u l p r e l i m i n a r y c l e a n u p of

the

[24,30].

Obviously, i f

sample and a s u i t a b l e c h o i c e of

column and i n t e r n a l s t a n d a r d , an a c c u r a t e v a l u e can be o b t a i n e d by GC. e.

High-performance

l i q u i d chromatography.

The s p e c i f i c i t y of HPLC r e s t s on

t h e r e s o l u t i o n of t h e column and t h e unique d e t e c t i o n methods a v a i l a b l e f o r a l i m i t e d number of compounds.

The c a t e c h o l a m i n e s (and o t h e r c a t e c h o l - t y p e

a r e u s u a l l y measured e l e c t r o c h e m i c a l l y

compounds having a low o x i d a t i o n p o t e n t i a l ( t h a t i s

<

0.7 v o l t s ) .

s m a l l amounts p r e s e n t and t h e i r high o x i d a t i o n p o t e n t i a l s , not

been determined by HPLC-EC.

compounds)

(EC), with s p e c i f i c i t y dependent on t h e s e Because of t h e

t h e t r a c e amines have

To measure t h e t r a c e amines by HPLC i t would

p e r h a p s be n e c e s s a r y t o p r e p a r e f l u o r e s c e n t d e r i v a t i v e s and q u a n t i t a t e them f l u o r i metrically.

An a t t e m p t

i n t h i s l a b o r a t o r y t o do t h i s u s i n g t h e d a n s y l d e r i v a t i v e s

h a s s o f a r b e e n u n s u c c e s s f u l b e c a u s e o f t h e enormous number of p e a k s p r e s e n t , t h e r e l a t i v e l y p o o r r e s o l v i n g power o f HPLC columns and t h e s m a l l amounts of t h e a m i n e s present

[110].

A few t y p e s of compounds, s u c h a s t h e i n d o l e s ,

possess a n a t u r a l

f l u o r e s c e n c e and c a n t h e r e f o r e be d e t e c t e d and q u a n t i t a t e d f l u o r i m e t r i c a l l y w i t h o u t t h e need f o r d e r i v a t i z a t i o n .

When HPLC i s compared w i t h MS o r t h e REA, t h e v a l u e s

o b t a i n e d by HPLC a r e sometimes somewhat h i g h e r , p a r t i c u l a r l y i n t h e p i c o g r a m r a n g e , s u g g e s t i n g a n i n f e r i o r s p e c i f i c i t y f o r HPLC [ 1 1 1 , 1 1 2 ] .

ill11 o b s e r v e d t h a t v a l u e s f o r DA measured by HPLC-EC

For e x a m p l e , Warsh were 9% h i g h e r

(p

<

et &. 0.01)

t h a n t h o s e o b t a i n e d by GC-MS. f. their

Spectrophotofluorimetry. specificity relies

SPF methods a r e g e n e r a l l y n o t v e r y s p e c i f i c s i n c e

a l m o s t e n t i r e l y on a c h e m i c a l

s e p a r a t i o n of

compounds.

Even w i t h f a i r l y e x t e n s i v e c l e a n u p p r o c e d u r e s , c o n t a m i n a t i o n by s u b s t a n c e s f o r m i n g similar

fluorophors,

non-linear

difficulties

i n obtaining consistently

r e l a t i o n s h i p b e t w e e n c o n c e n t r a t i o n and

low b l a n k s ,

fluorescence

intensity

q u e n c h i n g ) c o n t r i b u t e t o u n c e r t a i n t y i n t h e a c c u r a c y of t h e r e s u l t s .

and

the

(due t o

Not s u r p r i s -

i n g l y , a l l t h e v a l u e s l i s t e d i n T a b l e 7 . 1 measured by SPF a r e s u b s t a n t i a l l y h i g h e r t h a n t h o s e o b t a i n e d by mass s p e c t r o m e t r i c m e t h o d s .

It i s c l e a r t h e n t h a t , HRMS-SIN

of

i n c o m b i n a t i o n w i t h TLC o r GC w i t h c a p i l l a r y c o l u m n s ,

t h e m o l e c u l a r i o n of

a h i g h m o l e c u l a r w e i g h t amine d e r i v a t i v e o f f e r s

t h e g r e a t e s t s p e c i f i c i t y of t h e methods c u r r e n t l y a v a i l a b l e . 7.3.2

Sensitivity

(i)

Definition.

C o n s i d e r a b l e c a u t i o n must be e x e r c i s e d i n a s s e s s i n g t h e r e l a -

t i v e s e n s i t i v i t i e s of t h e v a r i o u s a n a l y t i c a l t e c h n i q u e s , s i n c e t h e l i m i t s of s e n s i t i v i t y reported t h i s review, (MDQ),

i n t h e l i t e r a t u r e a r e n o t a l w a y s b a s e d on t h e same p r e m i s e s .

In

t h e s e n s i t i v i t y w i l l be e x p r e s s e d a s t h e minimum d e t e c t a b l e q u a n t i t y

which we d e f i n e a s t h e q u a n t i t y o f a s u b s t a n c e i n a b i o l o g i c a l sample g i v i n g

r i s e t o a s i g n a l ( o r number of c o u n t s ) t w i c e t h e s i z e o f t h e b l a n k .

The MDQ r e f e r s

t o t h e amount of t h e s u b s t a n c e i n t h e o r i g i n a l s a m p l e , n o t t h e amount i n t h e a l i quot put i n t o t h e instrument.

The MDQ f o r a number o f b i o g e n i c amines measured by

v a r i o u s t e c h n i q u e s is summarized i n T a b l e 7.2. ( i i ) T r a c e amines. HRMS-SIM.

Most t r a c e amine a n a l y s e s h a v e b e e n c a r r i e d o u t u s i n g HRTLC-

The MDQ r a n g e s from 100-200

pg

[23,113].

The a c t u a l amount i n s e r t e d

i n t o t h e i o n s o u r c e of t h e mass s p e c t r o m e t e r i s u s u a l l y c o n s i d e r a b l y l e s s t h a n t h i s s i n c e o n l y a f r a c t i o n i s p u t on t h e d i r e c t p r o b e and r e c o v e r i e s a r e g e n e r a l l y l e s s t h a n 40%.

However,

TLC s e p a r a t i o n s ,

b e c a u s e t h e s a m p l e s h a v e b e e n i s o l a t e d and p u r i f i e d by t h r e e

t h e y a r e v e r y p u r e s o i t i s p o s s i b l e , when n e c e s s a r y ,

to put t h e

e n t i r e sample i n t o i o n s o u r c e , w h e r e a s i n column c h r o m a t o g r a p h i c t e c h n i q u e s (GC-MS, GC, HPLC) u s u a l l y o n l y a s m a l l f r a c t i o n i s i n j e c t e d o n t o t h e column.

140 TABLE 7 . 2 .

Minimum d e t e c t a b l e q u a n t i t i e s f o r some b i o g e n i c amines ( i n picograms).

Mpthod

T r a c e Amines Catecholamines (PEA, mTA, (A, NA, DA) pTA, OA, SYN) 100-200 1131

HKTLC-MS-SIM

30011 [ 2 3 , 1131

(27,

400 [lo01

GC-MS-SIM

-

HPLC

10-500 [ 124,126,127 1

100 (5HT) [ 121-123,125

-

2500-5000 .[ 114,1201 (5F1T) FID

-

Radiodansyl

The MllQ of

100-200

500 (T) (891 5-10(5HT)[118] 5000 ( T I [ R 7 1

1,000 [781 Ecn

100 I1151

p~

Other Amines (Piperidine, Histamine)

-

1-5 [116,117]

20,000 [82] FID

GC

Polyamines

250 ( T ) [ 2 3 , 1131

100 ~101,111,1191

10-2.50 [77, 84, 941

REA

Indoleamines ( T , 5HT)

1

300 [ 6 3 1

-

100 [ i 3 4 1

2000 (1281 2000 [ i m l

-

in

l000-10,000 [ 132,1331 500[1361

Ecn

-

[i311

100 [1231 3000[129]~cr1

20,000[1351 FIT,

100 [1151 (5HT)

-

-

is by no means t h e lowest t h a t can be a t t a i n e d bv MS.

Each a d n e w i l l have a much lower b a s i c s e n s i t i v i t y depending only on t h e mass s p e c t r o m e t r i c p r o p e r t i e s of t h a t compound.

The h i g h e r p r a c t i c a l MDO a r i s e s because

of l o s s e s of t h e sample d u r i n g i s o l a t i o n and d e r i v a t i z a t i o n and because of c o n t r i butions

t o t h e blank

from o t h e r s u b s t a n c e s i n t h e e x t r a c t and t r a c e i m p u r i t i e s

p r e s e n t i n e x t r a c t i n g s o l v e n t s and chromatographic m a t e r i a l s .

Contributions t o the

blank can a l s o a r i s e from i s o t o p i c i m p u r i t i e s i n t h e i n t e r n a l s t a n d a r d . observed,

for

example,

that

i n using dideutero internal

standards

We have

the

MDO

is

somewhat h i g h e r (200-500 pp) t h a n when u s i n g t e t r a d e u t e r o i n t e r n a l s t a n d a r d s (50150 pg) i n which t h e amount of u n d e u t e r a t e d amine p r e s e n t i s known t o he s m a l l e r . The REA, i n which t h e e n t i r e sample i s c a r r i e d through t h e procedure and counted is c a p a b l e of MDQ a s low a s 10 pg

f o r OA 1941 and 100 pp. f o r PEA

[771.

This

method, however, has seldom been a p p l i e d t o t h e a n a l v s i s of t r a c e amines. GC-MS,

a l t h o u g h p o t e n t i a l l y a very s e n s i t i v e and s p e c i f i c t e c h n i q u e f o r measur-

i n g t r a c e amines, h a s a l s o seldom been used f o r t h i s purpose [ 8 9 , 1 0 0 ] .

The proced-

u r e s commonly employed f o r t h e i s o l a t i o n and d e r i v a t i z a t i o n of samples f o r GC-MS r e s u l t i n high r e c o v e r i e s compared t o HRTLC-HRMS,

h u t u s u a l l y only a s m a l l a l i o u o t

can be i n j e c t e d , o t h e r w i s e o v e r l o a d i n g of t h e column o c c u r s and reduces t h e r e s o l u tion.

The o v e r a l l r e s u l t is a s e n s i t i v i t y s i m i l a r t o t h a t o b t a i n e d bv HRTLC-HRMS.

By i n v e s t i n g more t i m e and e f f o r t i n p u r i f i c a t i o n of t h e sample p r i o r t o i n j e c t i o n i n t o t h e GC, it may he p n s s i h l e t o i n j e c t more c o n c e n t r a t e d s o l u t i o n s o f t h e sample and t h u s o b t a i n an i n c r e a s e i n s e n s i t i v i t y .

141 GC-ECD

i s s i m i l a r t o GC-MS

i n t h a t i t is i n p r i n c i p l e a very s e n s i t i v e method,

but because of t h e p r e s e n c e of numerous o t h e r s u b s t a n c e s i n t h e b i o l o g i c a l e x t r a c t

is

i n p r a c t i c e only moderately s e n s i t i v e .

M a r t i n and Baker

[ 7 8 ] , f o r example,

a p p a r e n t l y can d e t e c t about 4 pg of PEA ( i n j e c t e d amount) u s i n g GC-ECD,

but

the

sample m s t be so d i l u t e d t o reduce t h e c o n c e n t r a t i o n s of o t h e r compounds t h a t a nanogram or more must be p r e s e n t i n t h e o r i g i n a l sample.

GC-FID,

a s one would

e x p e c t , i s even less s e n s i t i v e , with MDQ of 20 ng f o r PEA (821 and 5 ng f o r T [1141 b e i n g r e p o r t e d , n e i t h e r of which i s s e n s i t i v e enough t o measure t h e s e amines i n r a t b r a i n , even i f s e v e r a l a r e pooled. The r a d i o d a n s y l method f o r T is a l s o not s e n s i t i v e enough

f o r measuring t h i s

amine i n most t i s s u e s s i n c e about 10 ng is t h e smallest amount d e t e c t a b l e

[91],

a l t h o u g h t h e method is adequate f o r s e r o t o n i n and t h e c a t e c h o l a m i n e s [1151. (iii)

Catecholamines and s e r o t o n i n .

HRTLC-HRMS-SIM

of t h e d a n s y l d e r i v a t i v e s

of t h e c a t e c h o l a m i n e s with a MDQ of about 3 ng has n o t proven t o be as s e n s i t i v e a s f o r t h e t r a c e amines, mainly because of t h e very s m a l l r e l a t i v e i n t e n s i t y (1% o r l e s s ) of t h e molecular ion and t h e b a s i c c o n d i t i o n s r e q u i r e d f o r d a n s y l a t i o n (which favour oxidation).

I n a d d i t i o n , t h e very high m o l e c u l a r weights of t h e t r i s - d a n s y l

c a t e c h o l a m i n e s ( n e a r l y 1000 amu) a r e a t o r above t h e upper l i m i t of t h e mass range i n which our mass s p e c t r o m e t e r can be o p e r a t e d a t maximum s e n s i t i v i t y .

Acetylation

of t h e s i d e - c h a i n hydroxy group a f t e r d a n s y l a t i o n i n c r e a s e s s l i g h t l y t h e r e l a t i v e i n t e n s i t y of t h e molecular i o n [ 3 7 ] . The REA a p p e a r s t o be t h e most s e n s i t i v e method a v a i l a b l e f o r q u a n t i t a t i n g t h e c a t e c h o l a m i n e s and 5-HT,

t h e most advanced m o d i f i c a t i o n s being c a p a b l e of q u a n t i -

t a t i n g a s l i t t l e a s 1 pg of A and NA, [118].

GC-HRMS-SIM

having MDQ of

5 pg of DA [116,117], and 5-10

[ l o l l and GC-LRMS-SIM

about 100 pg.

GC-FID

pg of 5-HT

[111,119] a r e somewhat less s e n s i t i v e ,

h a s been used t o measure 5-HT

in brain in

amounts of 2.5 ng [1201. I n t h e l a s t f i v e y e a r s , HPLC with e l e c t r o c h e m i c a l o r 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 e c a t e c h o l a m i n e s and indoleamines h a s become i n c r e a s i n g l y p o p u l a r , due t o good s e n s i t i v i t y as w e l l a s t o t h e low c o s t per a s s a y , speed of a n a l y s i s and s i m p l i c i t y

of sample p r e p a r a t i o n .

MDQ of 100 pg [111,121-1271

can be r o u t i n e l y o b t a i n e d , and

i n one procedure [124] i n which t h e e n t i r e sample i s i n j e c t e d o n t o t h e column, a n MDQ of 10 pg i s claimed.

( i v ) O t h e r amines.

S e i l e r [63] has employed t h e TLC-LRMS-SIM

procedure t o meas-

u r e p i p e r i d i n e as i t s b a n s y l d e r i v a t i v e , i n which t h e e n t i r e sample is e v a p o r a t e d on t h e d i r e c t probe and i n s e r t e d i n t o t h e i o n s o u r c e .

300 pg can be measured i n t h i s way,

He r e p o r t s t h a t as l i t t l e a s

P i p e r i d i n e h a s a l s o been measured by GC-LRMS-

[1291, b u t i n t h e s e c a s e s t h e MDQ is 2 o r 3 ng. Histamine h a s been measured by GC-LRMS-SIM [ 1 3 0 ] , M A [1311 and HPLC with f l u o r -

SIM [128] and GC-ECD

i m e t r i c d e t e c t i o n of t h e 2-phthalaldehyde

d e r i v a t i v e [123].

The REA is t h e most

142 s e n s i t i v e method with a NDQ of about 10 pg, HPLC can d e t e c t 100 pg and GC-LRMS-SIM about 2 ng. The MDQ f o r polyamines has not example, h a s used TLC-LRMS-SIW rat

been r e p o r t e d

f o r most methods.

Seiler,

for

t o measure p u t r e s c i n e a s i t s dansyl d e r i v a t i v e i n

[60,611 and f i s h [591 b r a i n i n amounts a s low a s about 300 ng/g, but t h e r e i s

no r e a s o n t o assume t h a t t h i s is t h e MDQ.

S e i l e r has a l s o employed t h e d a n s y l de-

r i v a t i v e s f o r t h e HPLC f l u o r i m e t r i c q u a n t i t a t i o n of a s l i t t l e a s 1 ng o f t h e polyamines

in various tissues

[1321.

c a d a v e r i n e and spermidine as quantities OE

Samejima e t

al.

[1331 r e p o r t

that

putrescine,

t h e i r f l u o r e s c a m i n e d e r i v a t i v e s can be d e t e c t e d

l e s s than 10 ng by HPLC.

s t a n d a r d s o l u t i o n s , used GC-LRMS-SIM

in

Smith and Daves [1341, working only w i t h

and d e u t e r a t e d i n t e r n a l s t a n d a r d s t o measure

down t o 100 pg of p u t r e s c i n e , c a d a v e r i n e and spermidine and 5 ng of spermine, t h e

h i g h v a l u e s f o r t h e l a t t e r being due t o sample r e t e n t i o n on t h e GC column and peak broadening.

About 20 ng is t h e MDQ f o r t h e measurement of polyamines by GC-FID

[1351, whereas a s l i t t l e a s 500 pg can be d e t e c t e d by GC-ECD

of t h e p e n t a f l u o r o -

benzoyl d e r i v a t i v e s 11361. 7.3.3

Convenience and e f f i c i e n c y

I n o r d e r t o a c h i e v e t h e maximum p o s s i b l e s p e c i f i c i t y and s e n s i t i v i t y a l l procedures

r e q u i r e an e x t e n s i v e p u r i f i c a t i o n of

The HRTLC-HLYS-SIX

samples b e f o r e i n s t r u m e n t a l a n a l y s i s .

method i n v o l v e s an e x t r a c t i o n , d e r i v a t i z a t i o n and TLC p u r i f i c a -

t i o n procedure by which about 60-70 samples can be processed i n two days.

However,

an advantage of t h i s method i s t h a t each sample then r e q u i r e s only 5 or 6 minutes o f mass s p e c t r o m e t e r t i m e and because of i t s high p u r i t y t h e e n t i r e sample can, i f n e c e s s a r y , be put on t h e d i r e c t probe and i n s e r t e d i n t o t h e i n s t r u m e n t .

Consider-

i n g t h e high c o s t of purchasing and o p e r a t i n g a mass s p e c t r o m e t e r such e f f i c i e n t u s e of t h e i n s t r u m e n t i s d e s i r a b l e .

I n p r a c t i c e , workup procedures f o r GC-MS-SIW

have been less time-consuming than

t h o s e f o r HRTLC-HRMS-SIM,

although t h i s can sometimes r e s u l t i n reduced s p e c i f i c i t y

due

numerous i n t e r f e r i n g

to

the

presence

of

s u b s t a n c e s not

r e s o l v e d on packed

columns and i n lower s e n s i t i v i t y due t o t h e requirement of g r e a t e r d i l u t i o n of t h e sample t o reduce t h e c o n c e n t r a t i o n of

i n t e r f e r i n g substances.

mass s p e c t r o m e t e r time i s r e q u i r e d than f o r HRTLC-HRMS-SIM

I n a d d i t i o n , more

s i n c e one must wait f o r

t h e compounds of i n t e r e s t t o e l u t e , and then w a i t f u r t h e r while u n i n t e r e s t i n g compounds a r e e l u t e d .

I f an e x t e n s i v e cleanup of t h e sample i s undertaken and t h e GC

c o n d i t i o n s changed t o reduce e l u t i o n time and i f more than one compound can be determined from a s i n g l e i n j e c t i o n , t h e mass s p e c t r o m e t e r t i m e r e q u i r e d per compound analyzed can become c o m p e t i t i v e w i t h t h e HRTLC-HRMS-SIM F o r GC-ECD

o r GC-FID,

procedure [ g o ] .

an e x t e n s i v e and e f f i c i e n t c l e a n u p of

t h e sample is o f

paramount importance s i n c e s p e c i f i c i t y r e l i e s mainly on t h e r e s o l v i n g power

Of

the

column and t h e r e t e n t i o n time of t h e s u b s t a n c e . The h i g h e r t h e p u r i t y of t h e sample t h e less l i k e l y i s t h e p o s s i b i l i t y of an i n t e r f e r i n g s u b s t a n c e c o - e l u t i n g

143 with

the

compound

of

interest.

Furthermore,

a r e l a t i v e l y pure sample p e r m i t s

s m a l l e r volumes of s o l v e n t t o be used and t h e r e f o r e a l a r g e r f r a c t i o n oE t h e sample can be i n j e c t e d . For maximum s p e c i f i c i t y , t h e REA must i n c l u d e chromatographic p u r i f i c a t i o n o f t h e sample i n a d d i t i o n t o s e l e c t i v e e x t r a c t i o n .

Up t o 50 samples per day can be

a n a l y z e d , a l t h o u g h t h e time r e q u i r e d t o p r e p a r e and p u r i f y t h e enzyme i s u s u a l l y i n addition t o this. Twenty t o t h i r t y samples can be analyzed by HPLC i n a day, and f o r a l i m i t e d number of t y p e s of compounds ( c a t e c h o l s and i n d o l e s ) very l i t t l e p r e l i m i n a r y processing is required.

In f a c t , HPLC l e n d s i t s e l f w e l l t o automation f o r t h e analy-

s i s of t h e s e compounds (124,1371.

It i s p a r t i c u l a r l y s u i t e d t o metabolic s t u d i e s

i n which a number of m e t a b o l i c a l l y r e l a t e d compounds i n t h e sample can be determined s i m u l t a n e o u s l y on t h e b a s i s of a common f u n c t i o n a l i t y , a s i n t h e c a s e s of i n d o l e [1381 and c a t e c h o l compounds [123,138,139]. 7.3.4

Other f a c t o r s

A g r e a t s t r e n g t h of t h e mass s p e c t r o m e t r i c t e c h n i q u e s is t h a t v i r t u a l l y any t y p e o f compound w i t h mass l e s s than 2,000-3,000

amu can be analyzed.

The REA, on t h e

o t h e r hand, i s l i m i t e d t o t h o s e s u b s t a n c e s f o r which s u i t a b l e enzymes e x i s t and a r e e a s i l y prepared o r commercially a v a i l a b l e . compounds p o s s e s s i n g r e a d i l y o x i d i z a b l e

i s s t r i c t l y limited t o those

HPLC-EC

functional

groups

(e.g.

5-

c a t e c h o l and

hydroxyindole compounds).

Mass s p e c t r o m e t r y i s becoming i n c r e a s i n g l y important i n t h e a n a l y s i s of metabolites

formed

[33,140-1431.

following

the

ingestion

of

stable

isotope-labelled

precursors

Because of t h e h a z a r d s a s s o c i a t e d with them, r a d i o a c t i v e compounds

a r e no l o n g e r a c c e p t a b l e f o r use i n m e t a b o l i c s t u d i e s on humans. Finally,

t h e q u e s t i o n of

t h e c o s t s of

i n s t r u m e n t must be c o n s i d e r e d .

p u r c h a s i n g and o p e r a t i n g a p a r t i c u l a r

With o r w i t h o u t a d a t a system, a mass s p e c t r o m e t e r

i s t h e most e x p e n s i v e i n s t r u m e n t of t h o s e d i s c u s s e d .

Not only i s t h e i n i t i a l c o s t

h i g h , p a r t i c u l a r l y f o r a h i g h r e s o l u t i o n mass s p e c t r o m e t e r , b u t because of i t s comp l e x i t y and s i z e , maintenance and r e p a i r c o s t s a r e h i g h .

By comparison, GC,

and HPLC a r e r e l a t i v e l y i n e x p e n s i v e t o buy, m a i n t a i n , r e p a i r and o p e r a t e .

SPF

The REA

and r a d i o d a n s y l procedure a r e a l s o r e l a t i v e l y i n e x p e n s i v e i n terms of t h e i n i t i a l c o s t of t h e l i q u i d s c i n t i l l a t i o n c o u n t e r , but t h e c o s t of t h e r a d i o c h e m i c a l s can be e x c e e d i n g l y high

if

a n a l y s e s a r e c a r r i e d out d a i l y .

In addition,

s p e c i a l pre-

c a u t i o n s a r e r e q u i r e d i n t h e h a n d l i n g and d i s p o s a l of t h e r a d i o a c t i v e s u b s t a n c e s .

SUMMARY AND PROMISING DEVELOPMENTS

7.4

A summary of t h e advantages and l i m i t a t i o n s of sented

i n Table

7.3.

Although

e x t e n t on t h e background of

t h e v a r i o u s procedures i s pre-

t h e c h o i c e of method depends t o a c o n s i d e r a b l e

the investigator,

t h e n a t u r e of t h e problem and t h e

p e r s o n n e l and equipment a v a i l a b l e , high r e s o l u t i o n mass s p e c t r o m e t r y is unequalled

144 i n terms of s p e c i f i c i t y and s e n s i t i v i t y .

However, a number of approaches can he

t a k e n t o improve s e n s i t i v i t y and s p e c i f i c i t y s t i l l f u r t h e r .

One approach i s t o use

a d e r i v a t i v e which produces g r e a t e r molecular i o n o r unique fragment ion i n t e n s ities.

The b a n s y l d e r i v a t i v e s e x h i b i t i n t h e i r mass s p e c t r a very l a r g e M-43

( u s u a l l y t h e b a s e peak) as w e l l a s s i g n i f i c a n t molecular i o n s [63,14&-146].

ions

Unfor-

t u n a t e l y t h e t r i s - b a n s y l c a t e c h o l a m i n e s have such h i g h molecular weiKhts t h a t t h e y are o u t of t h e range of maximum s e n s i t i v i t y on most mass s p e c t r o m e t e r s [1461.

How-

e v e r , i f t h e d e r i v a t i z a t i o n procedure i s modified so t h a t only t h e amino Froup i s b a n s y l a t e d and t h e p h e n o l i c and c a t e c h o l i c hydroxy groups a l k y l a t e d , t h e r e s u l t i n g mixed d e r i v a t i v e h a s a more u s e f u l molecular weight (400-700

M-43 ion a s base peak. TABLE 7 . 3 amines.

amu) and r e t a i n s t h e

This i s r e f l e c t e d , a t l e a s t for standard s o l u t i o n s , in a

Advantages and l i m i t a t i o n s of p r o c e d u r e s f o r t h e a n a l y s i s of b i o g e n i c

Procedure

Advantages

Limitations

Very s e n s i t i v e f o r t r a c e amines, h i g h l y s p e c i f i c , can be adapted t o many amines, e f f i c i e n t use can measure 60 of i n s t r u m e n t samples per day.

Not very s e n s i t i v e f o r c a t e cholamines, equipment i s expensive, r e l a t i v e l y high b l a n k s , t e d i o u s sample p r e p a r a t i o n , t e r t i a r y amines cannot be dansylated.

GC-MS-SIM

S e n s i t i v e , very s p e c i f i c for high r e s o l u t i o n MS, c a p i l l a r y column o u t l e t can he i n s e r t e d d i r e c t l y i n t o i o n s o u r c e , can be adapted t o most amines, can measure more t h a n one compound per injection.

Less e f f i c i e n t use of MS time than above, expensive equipment.

REA

Very s e n s i t i v e f o r most hiog e n i c amines, i n e x p e n s i v e equipment, can p r o c e s s up t o 50 samples per day.

Aot always s p e c i f i c , t e d i o u s , r e q u i r e s h a n d l i n g of r a d i o a c t i v e s u b s t a n c e s , expensive reagents.

HPLC

Relatively inexpensive, sensit i v e , f a i r s p e c i f i c i t y , can measure more than one compound p e r i n j e c t i o n , simple sample preparation.

Not always s p e c i f i c ,

GC

I n e x p e n s i v e , can be coupled t o a mass s p e c t r o m e t e r .

Not w e l l developed f o r b i o g e n i c amines, poor s p e c i f i c i t y , e x t e n s i v e sample c l e a n up r e q u i r e d .

Radio-dansyl

F a i r l y s e n s i t i v e , p o t e n t i a l of wide a p p l i c a t i o n t o amines.

S p e c i f i c i t y l i m i t e d , expensive r e a g e n t , r e q u i r e s h a n d l i n g of r a d i o a c t i v e substances. Reaction of d a n s y l c h l o r i d e i s n o t c o n s t a n t a t low amounts.

HRTLC-HRMS-SIM

-

145 much g r e a t e r s e n s i t i v i t y than f o r dansyl d e r i v a t i v e s [ 1 4 6 ] .

U n f o r t u n a t e l y , bansyl

c h l o r i d e i s not comincrcially a v a i l a b l e and i s f a i r l y time-consuming

t o p r e p a r e and

p u r i f y , so t h a t i t s u s e on a r e g u l a r b a s i s i s r e s t r i c t e d . An obvious and promising approach is t o combine h i g h r e s o l u t i o n GC ( i . e .

l a r y columns) with high r e s o l u t i o n mass s p e c t r o m e t r y .

capil-

Such a t e c h n i q u e has not y e t

been a p p l i e d t o t h e a n a l y s i s of b i o g e n i c amines, but has been s u c c e s s f u l l y employed i n t h e d e t e r m i n a t i o n of t h e a c i d m e t a b o l i t e s of some b i o g e n i c amines 1142,143,147-

1491. i s a t e c h n i q u e which i s s e e i n g i n c r e a s e d u s e i n t h e hope of

P o s i t i v e ion CI-MS

iinproving both s p e c i f i c i t y and s e n s i t i v i t y [80,85,103-105].

Although most of t h e

i o n c u r r e n t i s c a r r i e d by t h e molecular ion o r p r o t o n a t e d molecular i o n , t h i s does

not n e c e s s a r i l y l e a d t o i n c r e a s e d s e n s i t i v i t y compared t o e l e c t r o n - i m p a c t

(EII-MS

s i n c e C I u s u a l l y produces a s m a l l e r t o t a l ion s i g n a l due t o a s m a l l e r ion s o u r c e aperture.

Negative ion CI-MS

11501 g r e a t e r than EI-MS have been r e p o r t e d .

i s more r e c e n t , but c l a i m s of s e n s i t i v i t y f o u r times

and t h i r t y times

[1511 g r e a t e r than p o s i t i v e ion CI-MS

Levy and Markey [152] have been a b l e t o d e t e c t 1 pg of mela-

t o n i n i n 1 ml of plasma by n e g a t i v e i o n CI-MS. Finally, (SMPM),

a

new

and

elegant

technique,

selected

is showing g r e a t promise [153-1551.

metastable

peak

monitoring

I n t h i s method, only s e l e c t e d d a u g h t e r

i o n s formed from a s e l e c t e d p a r e n t ion in t h e f i r s t f i e l d - f r e e r e g i o n of a d o u b l e f o c u s s i n g mass s p e c t r o m e t e r a r e r e c o r d e d .

This r e s u l t s i n a d r a m a t i c r e d u c t i o n i n

t h e s i z e and number of background i o n s s i n c e most, i f not a l l , o t h e r fragments der i v i n g from endogenous c o n t a m i n a n t s , column bleed o r s o l v e n t i m p u r i t i e s a r e formed by d i f f e r e n t mechanisms and a r e t h e r e f o r e t r a p p e d i n t h e e l e c t r o s t a t i c s e c t o r of t h e mass s p e c t r o m e t e r .

Although t h i s i n i t s e l f does not u s u a l l y r e s u l t i n an abso-

l u t e i n c r e a s e i n s e n s i t i v i t y , t h e absence of i n t e r f e r i n g background ions allows t h e g a i n of

t h e mass

Spectrometer t o be i n c r e a s e d so t h a t

improvement i n s e n s i t i v i t y .

in practice

there

i s an

Durden [1551 has r e f i n e d t h i s t e c h n i q u e with a r e l a -

t i v e l y i n e x p e n s i v e but s o p h i s t i c a t e d m o d i f i c a t i o n t h a t permits m u l t i p l e m e t a s t a b l e peak m o n i t o r i n g , which

is n e c e s s a r y i f

one

is t o u s e a s

internal standards the

d e u t e r i u m - l a b e l l e d analogues of t h e endogenous compounds oE i n t e r e s t . t i o n of

The a p p l i c a -

t h i s t e c h n i q u e t o t h e d e t e r m i n a t i o n of b i o g e n i c amines h o l d s promise of

improved s p e c i f i c i t y and s e n s i t i v i t y . ACKNOWLEDGEMENTS We wish t o thank our c o l l e a g u e s i n t h e P s y c h i a t r i c Research D i v i s i o n whose work c o n s t i t u t e s a l a r g e p a r t of t h i s review, D r . A.A.

Roulton f o r h i s a d v i c e and c r i t -

i c a l r e a d i n g oE t h e m a n u s c r i p t , and Saskatchewan Health and t h e Medical Research Council of Canada f o r f i n a n c i a l s u p p o r t .

146 RE FERENCE S

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