Joud of Chvmatography, 143 (X977) 221-246 Ehnedied .!%ppliclmlms o Elsevier §cientifii Publishing Company, Amsterdam

CHROMBIO.

-Printed

‘. in The Netherlands

038

REVIEW CHROMA’FQGRAPMY OF BIOGENEC AMINES. APPLKX.BLE SEPARATION AND DETECTION NIKOLAUS

SELLER*

Mzx-Plan&-Institu (Received

I. GENERALLY METHODS

te for Bmin

December

ith,

Research,

Unit for _Neuracfiemihtry.

Frunkfh-t/Cf

(G. F. R.)

1976)

CONTENTS l.introduction _.._____... ______.___.______._._.___.____~__~.._ 2.Generally applicablemethods. ___ . . _. __ . . __ . . . _ _. . __ . _ __ . . . _ . . . _ . . k Paper and thk-layer chromato~phy and paper and thin-layer electropharesis offreeamines............................................. B. Ionerchange chromatography of tines _. _. _____. _. . _ __. . _ . . ___ . . _ C. Coloured derivatives of amines for thin-layer chromatographic separation. - . . . . D. Fiuoresce,at derivatives of amines for thin-layer and column chromatographic separation .____.___._._._.._ _.__ __.__.__._..___ _ ._._ __._. a 4Ct?Ioro-7-nitrobenzo[c~-~.2.5-o~adiazoIe ______ . __ . ___ . . ____. - __. b. Fluorescent sulphonyi chlorides _ _ _ _ _ _ . . _ _ . . _ . . _ . _ . . . . _ _ . _ . - _ _ . c. Isotkioc-yanates . . __ . ___. ________ . __. _. . _____ . _. ___ _. __ . . _ d.Fluoreseamine . . . . . _ . . . . . . _.._._..__ ..__ ___ .._. _ _.._.__ __ e. eridor.4 and pyridoxaf+phosphate . . . . . _. . . . . . . . . . . _. . . . _. . - - _ f. w-Fomyl-o-hydroxyacetuphencne and benzo-r-pyrone. . . . . . . . . -. -- . . . 3.Summary......_........................._...______.~~..-__. ~fer~ces...................................................

221 223 224 226 229 231 231 232 238 239 239 240 240 240

p&bway of tryptophan, COlljUgWZS (SUJP~tes, a=bkS, li$~~~O~d% etc.) of f&e tryptophan metabohtes and peptides. A redent trend in analytical bio&em&$ry was therefore the development of methods that allow the; determination of fmz&ionaUy and metabolically related compounds from the same sample. However, although in certain instances interest may be focused exdeveIy on a single amine, one must devise methods that allow the determination of the individual amine in the presence of many other related compounds of comparable concentration. In methods of this type chromatography is particularly useful. The concentration of biogenic amines in tissues is, with few exceptions, low, i.e., generally lower than 10 mole per gram of wet tissue. The necessity for the determination of low concentrations of biogenic amines in discrete areas of the brain, in small cell populations and in single cells stimulated the improvement of the sensitivity of the methods. The possibility of achieving the quanti&tive determination of picomoh or even femtomole amounts of amines, amino acids and peptides is a major criterion for the suitability of a method in neurochemistry. Less sensitive methods may be applied successfUJ.lyto urine analysis. Many naturally occurring amines and amino acids have been identified and quantitated in tissues and body fluids in th’e iast 20 years. The application of more advanced methods to their assay frequently revealed, however, that their actual concentrations were sometimes lower than. was originally thought by several orders of magnitude. &Hydroxy-r-aminobutyric acid ]1,2], choline and acetylchohne 131, piperidine [ 4] and putrescine [ 5,6] in the brain are examples of compounds for which revisions of their concentration in tissues were made of the basis of advanced methods. Improvements in the specificity of the methods was as important as an increase in sensitivity. The still increasing application of mass spectrometry in combination with other separation methods if analy+kal biochemistry is one of the latest trends aimed at improving specificity. The exploitation of the specificity of enzymic reactions, mostly combined with the use of radioactive substrates, and immunological methods are alternatives to separation methods for increasing sensitivity and specificity [7,8 1. Radioenzymic assays have been established for catecholamineS 19-121, serotonin [ 13,141, fi-phenylethylamines [X5,16] , histamine [17-191, choline and acetylcholine [20--251 and putiescine IS], among others, and radioimmunoassays are available for a few important biogenic : amines [ 26-291. Further insight into the motecular events associated with mekbolic aberenormously increased the demands for routine assays of an increasing number of amines and amino acids for diagnostic p-zoses, and for the manitiring of therapeutic measures. Autumated methods had to be developed to meet the special requirem ents of clinical analysis. Automated methods, with few exceptions, do not include separation steps, but ut&ze specific sfmcturap features of the amines for determination. They are normally of Emif& specifieity and applicable only under thoroughiy defined ckcm~_ in addition to autimation and theincreaseinsensitti~ and specificity,% furtherQemi became apparent.After two decades of nearly exclrtatve consideration of a small number or'physioiogieaEy a& ph~~x~&&~ imwi tant amines, the catecholamines, -tonin, hisanb, ~tykholine, in:--

eee.

=tions

West was extended to other amines of biological origin for which the physiological significance is less apparent or less wea established. Methods for the determination of these amines are being developed. The term “biogenic amine” has gradually seemed to regain its literal sense, which was origin&y ~4, for instance, by Guggenheim in his nom c&sic book “Die biagenen Amine” [30]. Comprehensive reviews of analytical procedures for the assay of biogenic amines do not seem to exist, if we neglect the short chapters in standard books on thin-layer chromatography (TLC) and gtiiquid chroma@raphy (GLC), and the autfior's short reviews 131,321. The reason for this is clew: most

papers reviewing analytical methods for biogenic amines are devoted either to a special method or to a special approach [V&33451 or 8 certaincompound [46--S?]. Furthermore, establish&, generally accepted and gene-y applecable chromatographic methods for the determination of zrnims are, with few exceptions, not avaiiab!e. Any given &ple

of biological origin poses different

problems, depending on its complexity and the concentration of the amines in the sample. It is not possibte, by using a single method, to establish a complete profile of all important amines in a given biological sample. Most methods are in principle not suitable for such an analysis: with the exception of the completely non-specific detection methods used mainly in GLC, the flameionization detector and the more specific nitrogen-sensitivealkali detitor, and the mass spectrometer, virtually no sensitive method exis& for the determination of tertiary amines. They are normally not recognized, provided that the moIecule has specific features for sensitive detection, as is the case, for instance, with bufotenin. Colour and fluorescent reagents for terizuy amines do not meet the requirements of the sensitivity needed for tissue analysis. Disregarding these limitations, methods exist that are sufficiently versatile to be adapted to a given andytical problem. Existing methods have been improved successwly during recent years by the appk&iOn of refined chromatographic procedures or by increasing the sensitivity of detection by suitable derivative formation. it is the purpose of this paper to review these methods. .Z.GEMERALLYAPPLiCABLE ~XETHODS Alfphatic mono-, di- and polyamines do not exhibit structural features that

permit their sensitive or specific detection. Interference refractimetry was suggesti for the determination of amines after elution from thin-layer chromat~g~ms, but its low sensitivity (pg amounts of amphetamine 158)) is one of several reasms for the restricted application of this method. Disregarding detection with non-specific flame-ionization detectors, in all methods currently in use for the asay of primary and secondary amines- the amiao groups are utiiized for the formation of derivativessuitable for sensitive determination sad/or improvement of separation. While the sensitivity of detection depends on the derivatization reaction, the specificity is limited solely by the quality of the sepzmtion procedure. En practice, nearly alI detection reactionshave been combined with aElseparationprocedures.

A. P&er and fizinn_Cayerchivmatogmphy phore~is of five amines

and

paper

and tfrin-layer e&&m-

.A large numher of solvent and buffer systems suitable for the separation of nonderivatized amines on paper [59-741 and thin layers hy chromatography ]80-1061 or electrophoresis ]63--65,38,107-1x41 have been proposed. In addition to the usual layers (ceUulose, alumina and silica gel), ion-exchange paper and thin layers f&S, 1151 have been considered, and also separations by l&and exchange 11163. The references cited in this paragraph mainly describe sepa&ions of aliphatic amines. Although the reference list is incomplete, it demonstrates the wide application of amine separations in analytical biochemistry. Ch~om&ographic separations of underivatized @-phenyiethylamines, catecholamines, histamine, indoleamines and acetytcholine will be discussed in a subsequent paper. For the detection and determination of separated primary and secondary amines, ninhydrin has most commonly been used, and Dragendorff reagent for tertiary. amines ]117]. A number of other generally applicable detection reactions are available [105]_ Colour reactions of primary amines with 2,5-d& methoxytetrahydrofuran plus p-diiethyiaminobenzaldehyde ]llS] , and the more generally applicable reactions with 2,Sdichloroquinone 4chloroimide [I193 or potassium permanganate [77] ,orthe fluorimetric procedure of Segura and Gotto [ 1201 for the detection of organiccompounds on thin-layer chromatograms, may prove us&d in certain amine analyses, as well as the reaction of tertiary amines with cr,y-anhydroaconitic acid to give coloured products [121] _ Considerable progress in the detection of primary amines was made, however, by using fluorescamine as spray reagent [122-1241, which can reveal picomole amounts. The reaction of fluorescamine with primary amines is illustrated in Fig. 1. Ahhough there are certain difficulties, quantitative evaluation of chromatograms sprayed with or dipped in Buorescamine is possible. An a&ernative method is the application of the o-phthaldialdehyde reaction in the presence of thiolcontainmg compounds (Zmercaptoethanol) Cl251 for the fluorescence staining in TLC, thin-layer electrophoresis (TLE), paper chromatography (PC) or paper electrophoresis (PE). Tine sensitivity of this method 11261 in OUT hands was not as good as that of the reaction with fluorescamine, but was useful.

Fig. I. Reaction

of fhm-escarnine

with B primary

amine.

Despite considerable experience, there is no generally accepted approach to the analysis of the free amines in a bioZogic$ sample. Disregarding~ffie complexity of the mixture and the varying concentrations of its eomponen&. there is an additional difficulty inherent in their struekral feature: the S&S arid the free bases of many natural aiiihatic an&es qd 8-phenylethykmines are

readiiy soluble in m&er. Elomogeuization with acids [0,2-0.4 M HClO, ; IQ% ti&kmwz&i~ acid; a2etone-O.l M H4=E (95: 5)] is effective for theirextraction from tissues, but it is diMcult ta concentrate huge volumes of the acidic solutions without loof trace amounts of the mines. Some of the lowmoIecular-weightarnineszuevolatile. On the other hand, certain &mjug&es (for instance acetates) may be hydroiyzed dur&agevaporation, and evaporation of neutr&ized solutions causes heavy losses. In fact, water-vapour distillation of alkaline solutions was used uutil recentay for the separation of volatile from non-volatile mines and amino acids [62,63,127J28]. Aromatic mines and some &pheuylethykunines can be extracted from &lkaline soh&ions with ethyl acetate, diethyl ether and sinGI& solvents. The polyamides spermidiue and spermine were mostly extracted _tith n-butauol [86,IOs] before chromatographic separation. A generally app&zble solventextraction procedure for amines does not exist and, moreover, some biologicahIy important amines aze unstzble In alkaliuesolutions. Not only catecholmines and related eompo~undsbut even simple aliphaticd&nines may c3’e paxtiallylost due to decomposition in ail&in@ solutions_ A method known as ion-pair extraction may form a basis for-future development. Armnonium compounds (choline, acetylcholine, etc.), primary, secondary and tertiary amines aud an&m acids form iou p&s with anions. The ion pais with tetrapheuylborate [129]. Hg& 2- f 130], anthmcene-2.sulphonate 11311 and di-(Zethylhexyl)phosphate [ 132 J , among others [133--1371, can be extracted with organic sofvenb. Ion-pair extraction has proved useful in drug analysis [13’7,138] and the determination of acetylcholine 11391 and of enzymes involved iu acetyleholine metabolism [X29, 130,140]. Ion pairs are suitable for separation by partition chmmatographic methods. Column chromaiographicseparationsutillzing this principle for the assay of some biogenic ami.neshave recently been published [X41-1431. Among the alipbatic arnines,a group of di- and polyamines (Fig. 2) deserves specie! mention. These erminesare ubiquitous in the natural world and probably p&y ba& roles in ceil biology [ 144,145]. Moreover, they might be useful as markers of neoplastic growth and indicators of the effectiveness of cancer chemotherapy [146--1481. These mines can sewe as a typical example of the present situation in the analysis of amines in tissues and body fluids, and to 0

HzWCHzh-NM

@

ii+-(CH&-NHZ

@

ctrN-W-fzk-W

@

lt$ex2~s-Nit-~~ti;-w2

@

~-~l~-NH-CCH~~

Ed_

2;

Smct~a

2 = puQe&ne

S=qhmaine.

-f&cn;l, fgm&ae

-2 of

the

~~~klialni~b~tane);

usfnraf

di- and gU??gamineS-1 = %3-~~Wropan9;

3 = ca~yd

(r,s_diamino~tane);4

=

spenaidine;

the various strategies applied to the solution of a mod impotit dyticd problem. me theore&zd and practical importance of the polyamides has stimuiated the e&abhshment of a large number of an&&cd methods. %chrc,ch [x45] summa&ed about 30 solvents for PC, 11 solvents for TLC and 12 different buffers for PE of the nonderivatized potyamines. Most workers used extraction of alkaline solutions with rt-butanol in order to at~u.~~~&~ti the po)yamines and to separate them from amino acids. It should be emphasized that the chromatographic systems used for the separation of diphatic emines are also generally suitable for ammo acid separations. Ninhydrin was nearEy dways used for detection and determination. Some laboratories preferred, however, to determine spermidine and spermine by staining with amid0 black [IOS] . Most of these methods never gamed much attention, but certain versions of PE [107,108] found wide appheation u&ii recently. It turned out, however, that the urinary spermine concentrations, as measured with the PE method, were higher than those found with other procedures ]148] _ Prrkescine concentrations in .&sues were even higher by an order of magnitude than the results obtained with more advanced methods [5,6,149]. Therefore, virtueBy all of these methods have been abandoned as far as tissue, blood and urine analysis are concerned. For the. estabhshment of metabolite patterns of radioactive pmcur~m, PE and preferably TLE [X50, 1511 are, however, the most suitable techniques. For this purpose, it is advisahb to apply trichkxtxcetic acid or neutrahzeci perchloric acid extracts (neutralized with KHCQ~, in order to remove perchlorate) instead of n-butanol extracts on the chromatographic plates. Putrescine, and probabiy other d&nines, might form degradation pmducts in alkaline soMions, as was mentioned before. A recentversion ofTLC,nvnely separation on si.licageistitered-glassplates combined wi*& in situ fluorimetry after reaction with fluorescamine, was suggested as an improvement of the determination of polyammes on a microscale 11231. N-3-Ammopmpylheptane-l,7d.iamine was used as the internal standard. The sensitivity of detection with this method is of the order of 100 pmole. Its specificity and apphcabihty in tissue and body-fluid analysis have not yet been established. However, this example shows that the TLC of free amines is still under development.

&nxm.stra~

B_ ion-exchange chrwmatogmpizy of amines The ion-exchange cohunn chromatographic pm-separation of complex biologica? mixtures, and sub_equent determination of amines in the fmctions by using specific methods, has a long history. This approach is still one of the most important in the chemical analysis of biogenic amines. The construction of automated devices for ammo acid analysis following the work of Spa&man et d. ]l52] suggested the utilization of these devices for amine analysis in the seme way es for s.mho acid ansly& The p7ork of Ferry and co-workers ]67-72,153] is exemplary in this respect_ This group, and others [XX--1601, uss almost exciusivefy cohunns packed with sdphonated polystyrene resins and either pH or salt -g.ra&entss,or com5in# pH and salt “; grzdients, for the succe_tive ehrtion of the different amines. As aromatic amines

in particular show considerable interactions with the polymer matrix, the elution patterns do arot follow exactly the ion competition equilibria. The complete &so&ion of a compkx mixture of biogenic amines cannot be expected with this method, but starting from large tissue samples it was possible to identify a number of aliphatic amines and phenylethylamines in the brain if additional separation methods (PC, FE) were applied [72,153,154]. The same approach was successful for the detection of amines in urine [68,71,155,16L] and cerebrospind fitid [69]. On of the drawbacks of this approach Is that identification of the amines is based exclusively on chromatographic criteria, which are insufficient in principle and may read to erroneous conclusions. Technical improvements to commercial amino acid analyzers have been considerable during the last decade. The development of spherical resins and polymercoated glass spheres with a narrow range of diameters of 10 pm and less increased the resolution and sensitivity. Even with ninhydrin as the detection reagent, the sensitivity is now in the nanomole range. An improvement in sensitivity came from the application of continuous fluorescence monitoring, using fluorescamine [162,163] or o-phthaldialdehyde [164,165] as reagents. Under favourable conditions, these methods ahow the measurement of picomole amounts of amines eIuted from columns with diameters of l-3 mm. Nevertheless, the routine measurement of ahphatic amines or of &phenylethylamines in tissues and body fluids plays only a negligible role, and even the c2inicaUy important catechol- and indoleamirres and their metabohtes are norm-. ally not determined by automated ionexchange methods. However, as will be discussed in a subsequent paper, some procedures meet the requirements of routine clinical analysis_ The interest in .polyamines as possible markers of malignancy induced methodical developments, which are briefly summarised here. They illustrate the general trends of the current development of ion-exchange column chromatography. Ion exchangers for the separation of di- and polyamines from amino acids and other amines have been in use for 20 years 11661 and almost all types of commercid resins have been appliecl~L45] _ Elaborate separations were achieved using cellulose phosphate columns [X67]. The amines were eIuted by salt or pH gradients, and were determined in the cotfected fractions using dinitrophenylation [l66,l68,169~, enzymatic methods [l69,170] or condensation with o-phthaldia.Fdehyde [167,169,171] _ The t&iotEsness of these procedures was snrmounted by the automated modifications. Commercial amino acid analyzers were adapted in several laboratories for polyamine separations-using, with one exception fl72], reaction with ninhydrim and cotorimetry at 570 nm for puantit&ion 1x73~lSla]i . Separations are achieved by two-or multi-step ‘gradient elution using pM and/or sodium chloride gradients. An example is shown in Fig. 3. Recently, an attempt was made to apply hgandexchange’ chromatography to the separation of poayamines [IS21 _ Cdiukxe ion errchangers and different polystyrene sufphonated n&ns were loaded with 0~‘: &I*’ and Ni*.l , but the results were not adequate to meet current standards. The sensiti&y of detection was increased from about 560 to 0.2 nmole when ~fluorezam ine was used [X72]. The standard deviation of the procedure ii of the order of f 5% and the recavery is better than 90% [17’7-1791,

Fig. 3. El&on pattern of amines from a sulpbonated polystyrene ion-exchange column using a two-step buffer system with an exponential pH and NaCl edient in the second step. For details, see ref. 176. Buffer flow-rate. 70 ml/h- 1 = Ammonia; 2 = monoacetyi-1.3-dia4 = monacarbamyl-2.,4diaminobutane; 5 minopropane; 3 = monoacetyi-1,4diaminobutane; = arginiue; 6 = 1,4diamino-2-bydroxybutaue; 7 = diaminopropane; 8 = N’ -monoacetyispermidine; 3 = Ns-monoacetylsperrnidie; 10 = 1,4_biaminobutzure (putrescine); 11 = 1,5diaminopeatane (eadaverine); 12 = N-(3aminopropyl)-1,3diaminopropene; 13 = spermidine; 14 = agmatine; 15 = N, N’-bis-(3-aminopropyl)-1,3diaminopropane; 16 = spermine (20 nmol of each amine). According to Tabor et al- [176].

Fig. 4. Separations of serum and cerebrospinal fluid polyamines using an automated higbperformance liquid chmmatigraphic technique in combination with the re~.~ti~n of amines with o-pbtbaldialdebyde:-(After acid hydrolysis the equivalents of I.25 ml of serum and cerebrospiti fluid were separated). According to Mar-ton and Lee [ 183I_

C.. CWtxmd

derivativesafamines

far H&z-layer

cAmmatagraphic

sepat-atim

A reagent ,tifzble for the analysis of small amounts of amines (and amino acids) &odd ftdfiH the f&owing criteria: (a) Rapid quantiMive reaction under mZd conditions in water or water-cont9ining media; (b) specificity her primary or secondary amid group; (c) high sensitivity of detection; (d) favourable

chromatu~Mc

pmpeties of the derimtives; and (e) few polarity of the rem

tion prochcts in order to pemit the acmulation

of the reaction products by solvent extraction. Several compounds are available that react either with primary amino groups or with primw and secondamino groups, but no reagent is known to be specific for secondary amino groups, nor is there a derivative-forming reaction known for tertiary amines that is suitable for quantitative am3lysi.s. The reagents summarized in Fig. 5 react, with one exception, with primary and secondzy amines in weak alkaline soWions to give coloured deriuatives in high yields. The derivatives are stable, can be extracted from the reaction mixture with organic solvents and are suitable for chromatographic separation and specizophotometric determination in the nanomole range. Hence they meet at least partially the above-mentioned criteria. Solvent systems for the TLC (and PC) of these derivatives have been formulated for only a few amines. BnEy 2,4&itrofiuorobenzene (Dnp-F), the well known end-group reagent of Sanger [PS4,185], and the more speciEcaL ly reacting 2,d-dinitrobetienesulphonic acid [X86], which leads to the szme derivatives, have been used extensively, both for the determination of certain urinary and tissue constituents. PC! [1$7,188] and ‘FIX [X36,189-1941 were used for separation, and it was shown that the derivatives are also suitable for ionexchange column and GLC separation [190]. As was mentioned before, dinitrophenyI&ion was utilized for the determination of amines in column elates [195]. The mass spectra of the Dnp derivatives have been studied to

s.., F

-$a

+q;N& F

COQ

ET% COct

.*te 8usefulnes of this method for the unambiguous identifkation of amine.31191:. Restriction of the sensitSy of detection to the naaaomob rmge is the main reason for the 1imSed use of coloured deriwtives for the an&y& of mines in tissues, md for the preference for fluorescent reagents of similar reactivity znd separation chm&eristics. In order to make use of existing experience, ami to increase the sensiWity of detection, tritiated Dnp-F mmsintroduced for tissue mine &lysis [X281. With this reagent, a few picomoles of a compound cm he detected; however, these amounts zxe invisible and it is difficult, therefore, to control the quality of the separations on the thin kyers. IT +&e specificity of the separations is not adequately controlled, erroneous results might be obtained. Increase of the sensitivity of detection by utilization of radioactive reagents is not new. [13’1 ]p-Iodobenzenesulphonyi chloride hzs been known for 30 years [I%], but it was never gener&y applied, for the above reasons. show

&

El

N

N-c-s

/

N-C-5

N-c-s

In Fig. 6, reagents currently used for the fluorescence IaheJ.lingof amines (and amino acids) are summarized. The same reactive groups (activat&d halogen, dphonyl chloride) are used both for fluorescence and colour labelling. fsothiocyanates and aldehydes are used only for special purposes. The specificity of the reactions and the formation of side-products during derivative formation are, in prinkple, the same for coloured and fluorescent derivatives. The obvious advantage of flrrorescence Iabehing is the increased sensitivity ofdetection. Another advantage is the wide range of iinearity between amount of substance and fluorescence intensity (i.e., photometer response), which simplifies and improves the in situ evaluation of TLC separated fluorescent compounds [2052081 and the contirmous monitoring of column effluents_ The use of coloured and fluorescent Feeagentsinvolves the same strategies: either the total tissue extracts or body fluids are made to react first, and separation procedures are applied exclusively to the derivatives, or a certain compound OF a group of related compounds is pre-separated by solvent extraction, ion-pair extraction, cohnnn chromatography, etc., and tne detection reaction is applied to the pre-separated compounds. In no case is a step involving purification of the derivatives dispensable, because normally sideproducts are formed dcring derivative formation; hydrolytic cleavage of the reagent is the most common side-reaction. The information available does not permit a thorough com@xison of the advantages of the different reagents. The amounts of information about the various reagents differ considerably and for some reagents is only fmgmenky. Fluorescent reagents are briefly survey& in the following sections; for a more detailed detiription, see ref. 209.

As an aryl halogenide with activated halogen (see Fig. 6, No. 5), 4chloro-7nitrobenzo[c] -X,2,5-oxadiazole (Nbd-Cl) is closely related to Dnp-F. Et reacts readily in aqueous sofutions [210,211] or in organic solvents [2X2-214] with primary and secondary amines, and less readily with phenols and thiols at pH 8. Thiolcontaining compounds, however, react rapidly at pH 7 [215;. Usually I-20 rg of amine dissolved in 25-500 ~1 of solution is mised with four volumes of a 0.05% solution of Nbd-Cl in methanol, and 5O---LOO ~.llof 0 1 M N&CO, solution are added. Completion of the reaction is achieved by heating at 55” for 60 rain, and the yields are mostly > 95% [2IOJ. Separation of the reaction produet from excess of reagent is trsu&y achieved by silica-gel column chromatography_ A thorough study of the chemicd and physical properties of Nbd derivatives has not been published. They afe stable in solution and on thin-layer plates. if protected from mdiation. fn eonfxast with most other ffuorescent lab&Is, the absor@ttoP maxima of Nbd derivatives are in the visible region (kma 96%). After 30 min, the Schiff bases are reduced to the corresponding pyridoxyl derivatives (Fig. 12). The excess of NaBH, is removed by acidification 1263, 264]. Ion-exchange column chmmatographic systems have heen used exclusiveiy for the separation of pyridoxyl derivatives, which were monitored in the effluent by absorptiometry (ahsorption maxima at 255 and 328 nm) or by fluorescene measurement (fluorescence emission maximum at 400 nm). Amounts of EG--l60 pmol of an amino acid. are detectable.

Fig. 12. Derivative-forming reaction of a primaryamine

with

pyridox&

One of the advantages of the reagent is the possibility of radioactive 1abtzUing the application of sodium borotritide (N&P=) as reducing agent. This cc£ is commereiaEy available with high specific activity.

by

f; .~-Fonnyl-o-hydmxyaceropttenone and beam-y-pymne w-Formyio-hydroxyacetophenone and bensoy-pyrone react with primary and secondary aliphatic and aromatic amines to form &uninovinyl o-hydroxyphenyl ketones (Fig. 13). Kostka 12651 utilized this reaction for &e&a&e formation with amines, and for their sensitive detection on thin4ayer chromatograms. The chromatographic characteristics of 54 enamine derivatives were studied using ethyl acetate-benzene (I:@, chloroform-xylene (4:1) and acetone-qlene (1:9) and sllicz gel G Iayers_ The &aminovinyl o-hydroxyphenyl ketones of .aliphatic amines are yellowish, while the derivatives of aromatic amines are orange; they fluoresce in the W region, Amounts of O.I1 gg of the derivatives can be detected on a thin-layer plate.

Fig. 13. Formation of @-aminovinyl acetophtinone and benzcj-y-pyrone.

o-hydroxyphenyl

ketones

from

w-formyi-o-hydroxy-

As the formation of the enamine derivatives is rapid and specific for primary and secondary amines, this derivatization procedure deserves more attention than it seems to have received hitherto. 3. SUMMARY

This first part of the review on “Chromatography of Biogenic Amines” is devoted to the description of generally applicable separation and detection methods. Gas chromatographic and gas chromatographie-mass spectrometric methods, and applications of chromatographic methods to specific amines or groups of related amines and their metabolites, will be covered in Part II. Trends in the development of separation methods (paper and thin-layer chromatography, paper and thin-layer electrophoresis and ion-exchange methods) are described, using mostly aliphatic amines as examples as they do not exhibit features that permit their specific determination. Reagents suggested for the formation of coloured and fluorescent derivatives of amines are reviewed and their applications are described. Within the limitations of the mostly inadequate information that is available, the relative usefulness of the different derivative-forming reactions are compared. REFERENCES 1 N. Setierand hi. Wiechmann.Boppe-Seyler’sZ. Physic&Chem., 350 (L969) 1433. Y. Yoshino, F-V. DeFeudisand K.A.C. Elliott. Canad. J. B&&em.. 48 (1970) 147. W-B. Stavinoha, ST. Weintrzub and AT. Mod&, J_ Neurochem.. 23 (iS74) 885.

2 3 4 5 6 7

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Chromatography of biogenic amines. I. Generally applicable separation and detection methods.

Joud of Chvmatography, 143 (X977) 221-246 Ehnedied .!%ppliclmlms o Elsevier §cientifii Publishing Company, Amsterdam CHROMBIO. -Printed ‘. in The N...
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