Tables I and I1 indicate that olefins of the cis configurat’ion formerly had retention values smaller than, those of the trans isomers of the same carbon number. Moreover, the sequence of the hydrocarbons may easily be changed by modifying the quantity of separating liquid impregnating the solid. The vapor pressure of the stationary phase is negligible if only minor quantities of the separating liquid are applied This stationary phase is outstmandingfor its suitability in t,emperature-programmed gas chromatography. Finally, the retention times of the hydrocarbons can deliberately be shifted by adding columns with strongly polar phases such as aluminum oxide. I n this manner it should always be possible to
move the retention time of a trace compound outside the range of the main component and thus to facilitate essentially its quantitative determination. LITERATURE CITED
(1) Breshchenko, E. M., Khim i. Teknol, Topliva i Masel 32 (1954). 12) , , Bruderreck. H.. Schneider. W.. HalBsz, I., ANAL.CHEM.36, 461 (1964).’ ( 3 ) Desty, I). H., Haresnape, J. H., Whyman, B. H. F., Ihid., 32, 302 (1959).
(1962). (6) Halhsz, I., Horvhth, C., ANAL.CHEM. 36, 1178 (1964). ( 7 ) HalAsz, I., HorvBth, C., Ibid., 36, 499 (1964).
(8) HalBsz, I., HorvBth, C., S a t u r e 197, 71 (1963). ( 9 ) HalBsz, I., Kabaker, G., unpublished results. (10) Heine, E., Ph.D. thesis, Vniversitat Frankfurt am Main, West Germany, 1963. (11) Horvhth, C., Ph.D. thesis, Universitat Frankfurt am Main, West Germany, 1963. (12) Lange, S . A., “Handbook of Chemistrv.” Handbook Publishers. Inc., Sand;sky, Ohio, 1956. (13) Simmons, RI. C., Snyder, L. R., ~ L N A L CHEM. . 30,32 (1958).
RECEIVEDfor review hlarch 26, 1964. Accepted May 21, 1964. Presented at 2nd International Symposium on Advances in Gas Chromatography, University of Htuston, Houston, Texas, March 23-26, 1984.
Gas Chromatographic Studies of Catecholamines, Tryptamines, and Other Biological Amines Part I.
Catecholamines and Related Compounds
C. J. W. BROOKS and E. C. HORNING Department o f Biochemistry, Baylor University College o f Medicine, Houston, Texas
b Conditions are described for the gas chromatographic separation of the catecholamines and of 31 other amines of the phenylalkylamine, imidazole, and indole types, through the use of fully acetylated derivatives. A two-component mixed stationary phase of moderate polarity, comprised of 7% F-60 silicone oil with 1% Polymer EGSS-Z, was employed. In general, the peaks obtained by this method were symmetrical and suitable for quantitative evaluation; the reproducibility of gas chromatographic work was aided by the stability of the acetyl derivatives. Regularities noted in retention factors assignable to substituents are of potential value in qualitative analysis. Other compounds, notably methyl esters of Nacetylamino acids and of aromatic acids representing catecholamine metabolites, were conveniently analyzed with the same column. Studies were made of the recovery of amines from dilute aqueous solution by acetylation, extraction, and reacetylation under anhydrous conditions. Gas chromatographic estimation indicated satisfactory retrieval of P-hydroxyphenylethylamine and tyramine, but substantial loss of dihydroxyamines.
T
of microanalytical procedures applicable to organic amines is warranted by the physiological and pharmacological importance of HE DEVELOPMENT
1540
ANALYTICAL CHEMISTRY
many of these compounds (19). Particular interest attaches to the catecholamines, to their natural and synthetic congeners, and to amines of the histamine and tryptamine groups. The analytical problem demands satisfactory means of isolating amines in the small amounts frequently encountered, and convenient chromatographic procedures suitable for separation and estimation of the individual compounds. Considerable progress has been made in the isolation of amines and their partial separation by ion exchange chromatography (9, 12). For the identification and estimation of individual compounds, gas chromatography is likely to be the most useful of the presently available techniques ( I ) . The first significant gas chromatographic studies on biological amines were reported by Fales and Pisano ( 5 ) who used columns coated with SE-30 for the separation of various amines, including serotonin. I3rochmann-Hanssen and Svendsen (2) showed that the products (presumably Schiff’s bases or osazolidines) from reaction of ephedrine and related amines with acetone were satisfactory derivatives for gas chromatography. They also studied acetylated amines, reporting poor results n i t h triacetylepinephrine but stating that the trimethylsilyl ether of the triacetyl derivative gave a single symmetrical peak. Limited data have been presented by othei investigators (11,20).
This paper describes conditions under which the natural amines of the phenylalkylamine, imidazole, and indole types, and many of their synthetic analogues, are separable by the use of their fully acetylated derivatives. In general, these afford symmetrical peaks suitable for quantitative evaluation, while regularities in retention behavior assist characterization. Since the completion of this work, Sen and McGeer (16) have described the gas chromatography of trimethylsilyl derivatives of catecholamines; these compounds showed excellent gas chromatographic properties but unlike the acetyl derivatives did not distinguish epinephrine from norepinephrine, or metanephrine from normetanephrine. EXPERIMENTAL
Gas Chromatography. A i l l separations were conducted isothermally with a Barber-Colman Y o d e l 10 or a n E.1 R. instrument. argon ionization detectornere employed. Column packing. \\ere made n i t h Gas Chrom P (.lpplied Science Laboratories Inc ) Rhich had been hie~edto 80- t o 100- or 100-to 120-meih, acid-m ashed and treated n i t h dichlorodimethylsilane 1 Present address, Department of Chemistrj, The Universitv, Glasgou, W 2 , Scotland On leave of absente, from the Atheroma Research Unit of the Medical Research Council, Glasgoa, Scotland, Mal-September 1963
before the stationary phase was applied by a slurry filtration process. The procedures were carried out as described by Horning, Vanden Heuvel, and Creech (8). Two types of packing were used : 10% neopentyl glycol succinate polyester (KGS) and a t'wo-component ilhase comprising 7TGof silicone oil F-60 ( 1 1 0 ~Corning Corp.) with 1% of ethylene glycol succinate-phenylmethylsiloxane caopolymer EGSS-Z (.Applied Science Laboratories). Freshly prepared columns were preconditioned a t 210" to 225" C. in a stream of argon, for 12 to 24 hours, or until a satisfactory baseline was obtained. Columns were operated a t 170°, 198" and 216" C. Flash heater and detect'or cell temperatures were maintained 30" to 50" C. above column temperature. Glass Utube columns, 6 feet long X 4-mm. i.d. were used. Samples were dissolved in ethyl acet.ate (or other suitable solvent) and injected with a Hamilton syringe. The sample volumes were generally in the range 0.1 to 2 kl., and the amounts of sample injected were of the order of 0.1 pg. for rapidly eluted compounds and 1 pg. for those with long retention times. These quant'ities gave peaks of convenient magnitude when the instruments were used at moderate sensitivity (detector voltage 800, amplifier sensitivity setting nominally 1 X lo-' ma. for full-scale recorder deflection). The limit of mass detection was not determined. Retention data were determined relative to anthracene as a reference standard. Quantitative comparisons were based on peak areas est'imated by triangulation, and in some cases on the product of peak height and retention tirne (4). Reference Compounds and Reagents. Amines were obtained, mostly as salts, from commercial sources. Where necessary, t h e free bases were liberated by titration with sodium hydroxide solution (less t h a n t h e required a m o u n t was used with t h e cat,echolamines): on a n analytical scale (10 to 1000 pg.) it was convenient to evaporate the resulting solutions to dryness in a vacuum desiccator and to take up t h e residual amines in a suitable solvent or reagent. Acetylation was effected quantitatively by treatment of the amine with redistilled acetic anhydride (10 to 20 PI.) and pyridine (distilled over potassium hydroxide) (10 to 20 pl.)) followed by evaporation of the excess reagents in a vacuum desiccator. Acetylated amines could be stored in solution in ethyl acetate a t 5" C. for several weeks without appreciable change. -imino acid methyl esters were prepared by refluxing the acid in methanolic hydrogen chloride (ca. 5% HCl; prepared from acetyl chloride and methanol) for 4 hours, evaporating to dryness, and liberating the free base with alkali. In the case of tryptophan the met'hylation was effected by overnight treatment a t room temperature. N Acetylamino methyl esters were obtained by treatment with acetic anhydride and pyridine, followed by evaporation of the reagents. Methyl esters
of other acids were prepared with diaeomethane in ether. The majority of the amines and amino acids examined in this work were racemic forms or L-enantiomers; their absolute configuration had no bearing on the results and has accordingly not been specified in the tables. Recovery of Amines from Aqueous Solution. A s t a n d a r d aqueous solution containing t h e hydrochlorides of P-hydroxyphenylethylamine, (20 pg., as free amine) ; tyramine (30 pg.) ; octopamine (50 pg.) and normetanephrine (60 pg.) was employed. hliquots were taken (a) for titration with sodium hydroxide, evaporation, and acetylation of the dry residue with acetic anhydride and pyridine; (b) for addition to 100 ml. of water; and (c) for addition to 100 ml. of urine which had been thoroughly extracted with ethyl acetate at uH 1 to remove acidic and neutral material. The solutions from (b) and (e) were buffered bv the addition of solid uotassium carbonate and treated 4 t h acetic anhydride, added in portions of 10, 5, and 5 ml. over a period of 2 hours. The p H was kept above 8 by the addition of solid potassium carbonate as required. The reaction mixtures were left overnight and were then extracted with a total of 300 ml. of ethyl acetate. The extracts were washed with aqueous sodium acetate, dried over sodium sulfate, evaporated to dryness, and the residues were treated with acetic anhydride (30 pl.) and pyridine (20 ~ 1 . to ) effect complete acetylation. The surplus reagents were evaporated in vacuo and the residues from (a), (b), and (c) were examined. The recovery of the amines from aqueous media was compared with that found for the directly acetylated mixture. RESULTS AND DISCUSSION
Acetone Condensation Products. Although gas chromatography of free amines is possible (3, 5, 20) the peaks obtained are frequently marred by tailing because of the polar nature of the amino group. Studies were accordingly made, following previous work ( 7 ) , of the chromatographic behavior of the products formed by dissolving primary amines in dry acetone. I n the case of P-hydroxyamines, oxazolidines ( I S ) may be formed under these conditions. I t is clear from the retention data in Table I that if the P-hydroxyl group is preserved during chromatography, its polarity is masked either by oxazolidine formation or by hydrogen bonding to the azomethine group of the corresponding Schiff's base. Thus, with a strongly polar neopentyl glycol succinate (NGS) column, introduction of a p-hydroxyl group leads to the expected large (15-fold) increase in retention for the acetone derivative; the corresponding factors for introduction of a p-hydroxyl group into tyramine and homovanillylamine are 2.9 and 2.6, respectively.
Table I. Retention Data" for AmineAcetone Condensation Products
Stationary phase
7 7 G F-60,'-
1y0z
10% XGS
Relative retention of derivative 1650b 183OC 2150d
Column temp., "C. Amine p-Phenylethylamine 0 . 1 1 0 . 1 2 p-Hydroxyphenylethylamine 0.18 0.20 Tyramine 0.49 0.46 1.00 0.88 Octopamine Homovanillylamine 0.73 0.68 Normetanephrine 1.28 1.15 Amphetamine 0.09 0.11 . . . 1.8 Tryptamine 5-Methoxytryptamine . . . 4.5 a Anthracene = 1.00. * Anthracene, ca. 15 minutes. Anthracene, ca. 6.5 minutes. Anthracene, ca. 9 minutes.
...
0.22 1.14 3.3 0.86 2.24 0.06 3.4 ...
Similarly, with a less polar F-60-Z column a t 183' C., the retention factors associated with the p-hydroxyl group (4.2, 4.6) greatly exceed those found for the introduction of a P-hydroxyl group (1.8, 1.9, 1.7). The chemical basis of these correlations requires further study, but the data strongly suggest t h a t the compounds either survive chromatography unchanged or undergo specific transformations so t h a t their retention values remain highly characteristic. The different properties of the two stationary phases are well illustrated by the retention changes accompanying the introduction of a 3-methoxyl group into tyramine or octopamine. The ensuing intramolecular hydrogen bonding, involving the phenolic 4-hydroxyl group, greatly reduces its interaction with a n NGS phase; the retention factors were found to be 0.76 and 0.67 for this column. With a n F-60-Z column there is much less selective retention due to polar groups, and the addition of the 3-methoxyl group causes a n increase in retention approximately commensurate with the increased molecular weight. Acetylated Amines. T h e catecholamines gave evidence of substantial decomposition when their acetone solutions were submitted to gas chromatography, suggesting t h a t a general procedure for biological amines would require ether or ester formation for t h e functional groups. Trimethylsilyl derivatives of t h e simpler amines were examined briefly; these compounds showed satisfactory chromatographic properties. Prior attention, however, was directed to acyl derivatives, because of their potential utility in the isolation of amines from dilute aqueous solution, together with their resistance to hydrolysis and their general stability. Representative naVOL. 36, NO. 8, JULY 1964
1541
r HYDROXYPHENYLETUYLAMINE Table 111. Retention Factors for /?Phenylethylamine Derivatives Derived from Data in Table II for 7% F-60/1 Z a t 198" C.
yo
HOMOVANlLLYLAMlNE OCTOPAMINE
Formal transformation 8-H --c @-AcO
.
Figure 1 Separation of acetylated 198' C. with an F-60-Z phase
phenylethylamines at
4-H
Examples Factors 1-2 2 77 3-4 2 68 5+6 2 48 7+8 2 59 12 + 13 2 59 17 + 18 2 28 1+3 5 64 2-4 5 47
+4-hO
3,4-diH + 3,4-diAc0 tural and synthetic amines were accordingly examined as their fully acetylated derivatives, obtained for this purpose by treatment with acetic anhydride and pyridine. The acetylated di- and trihydroxy amines showed very long retention times with a n YGS column, and most of the work was therefore carried out with the two-component phase, 7% F-60 with 1% EGSS-Z,which had been devised by Holmstedt, Vanden Heuvel, Gardiner, and Horning ( 7 ) . PHENYLALKYLAMINES. Acetylated phenylalkylamines gave well defined symmetrical peaks. Figure 1 depicts typical results obtained at 198' C. with an amine mixture including phydrosyphenylethylamine and five representative amines; the different acetyl derivatives are completely separated, and the resolution of metanephrine from normetanephrine is notable. Epinephrine and norepinephrine were
Table II. Relative Retention Data" for Acetylated Amines on 7% F-60/1% Z
Relative retention Column temp., "C. 1980b 216OC S o . Parent amine 1 0-Phenylethylamine 0 39 2 @-Hydroxyphenylethylamine 1 08 1 77 2 20 3 Tyramine 4 Octopaniine 5 91 4 39 D Homovanillvlamine 4 44 3 43 7 43 6 NornietaneGhrine 11.0 .5 44 7 Dopamine 7 53 12 0; 8 Korepinephrine 19.5 9 Homoveratrylamine 2 . 3 5 2.00 10 Metanephrine 9.81 6.80 11 Epinephrine 17.3 11.2 12 Amphetamine 0 37 13 Xorephedrine 0 96 0 84 2 75 14 Vanillylaniine 3 45 15 Mescaline 4.34 16 3,4-])ihydroxynorephedrine 14 1 9 18 17 Deoxyephedrine 0,413 1 05 18 Ephedrine 1 12 1 0 Synephrine 5.3*5 4 17 20 Phenylephrine 4 71 3 36 21 Hordenine 0 36 22 4-Meth~isy-p-phenylethylaniine 1.20 a Anthracene = 1.00. Anthracene, ca. 5 minutes. .4nthraoene, ca. 2.8 minutes
also found to be fully separated a t this temperature, but the retention times (62 and 87 minutes, respectively) were long, and it was more suitable to operate a t 216' C. and accept a less complete separation illustrated in Figure 2. Retention data collected in Table I1 are means of several determinations. The values were highly reproducible ('t to 12%) over periods of one or two weeks, but they diminished gradually over longer periods of time. Retention factors attributable to the introduction of substituents, or to other transformations, may be calculated from these data and some eyamples are given in Table 111. The values of the factors strongly suggest that the various substituents, with the possible exception of the p-acetoxyl groupl which merits further investigation, are preserved intact during chromatography. Moreover, although these data are quite limited, they show that it is feasible to apply group retention factors to predict approximate retention times. Thus, the relative retention time for tetraacetylnorepinephrine a t 198' C. could have been predicted from the value for triacetyldopamine and the mean value for the @-acetoxyl group retention factor, computed from five other examples, to be 7.53 X 2.56-i.e., 19.3. Similarly, one would expect the diacetyl derivative of p-hydroxy-amethylphenylethylamine (p-hydroxyamphetamine) to have a relative reten-
1
ANALYTICAL CHEMISTRY
4-H + 4-Me0 3.4-diH + 3,4-diMeO 3,4,5-triH + 3 4 5-triMe0 3,4-ldiH 3-Me0. 4-Ac0
19 3 18 1
14 6 3 0
6.0
1+ 9
1 + 15
11.1
-
1 01 23
-f
--?;HAC
-+
21 -+56
--h'MeAc 4 6
--NHAc
+
19 10
-
+
8 + 11 3 21
--?jMen
0 0 0 0
91
89 89 16
tion value in the region of 0.37 X 5.5Le., about 2.0. This compound is not yet available for trial.
HISTAMINE
TRYPTAMINE
AND
DERIVATIVES..icetyl derivatives of histamine and 3'-methylhistamine gave peaks with evidence of slight tailing. ,Icetylated tryptamines gave symmetrical peaks. Figure 3 shows a separation of tryptamine, melatonin and serotonin, as acetyl derivatives. Figure 4 indicates that tryptamine, histamine and S-methylhistamine are distinguishable in the presence of various phenylethylamine derivatives. Relative retention data for acetylated heterocyclic amines are given in Table IV. Certain regularities are discernible, and factors derived for some transformations are given in Table V. The introduction of the 5-acetolyl or 5-methosyl group in tryptamines causes the retention to change by a factor similar to that found for the corresponding 4-substituents in phenylethylaniines.
TYR
SEROTONIN
n 10
20
ab
io
'
40
MINUTES
Figure 2.
1542
1+7 2+8 13- 16 1 + 22
Separation of acetylated amines at 2 1 6 " C. with an F-60-Z phase
11
!I B Y
0
XP
Y E L PTO N IN
N- A C E N L R I P I A Y I N E
I
Ir
I
DIACETILSEROTONIN
I
1
The agreement between retention factors which serve to distinguish primary, secondary, and tertiary amines, of all three nuclear types, is very satisfactory and the factors are evidently applicable to the calculation of retention times. Reproducibility of Retention Data.
T h e principle features observed during t h e course of t h e work are illustrated by t h e results in Table VI, for columns operated a t 198" C. T h e relative retention values for column KO. 1 were measured aft'er this column had been in use for several weeks and are closely matched by those recorded a t a similar stage for column S o . 2. A gradual decrease in retention values, noted over a period of three weeks, is attributable to the slow loss of EGSSZ from the stationary phase; deterioration of the column in this respect was accelerated when it was used a t 216" C. Table VI includes figures representing the quantitative response observed, which show that for the injection of acetylated amines in amounts ranging from 0.2 to 0.6 p g . , reproducibility in terms of a n internal standard was achieved even with different columns. Runs carried out with a single column over periods not exceeding a few weeks showed substantial replication of actual peak heights; this was contingent upon
the preservation of the detection system during this time. The relative response computed on a molar basis i b given in the last column of Table VI. The three diacetoxy,Y-acetyl derivatives gave approximately SOY0 of the response observed for the monoacetoxy-S-acetyl derivatives. I n separate experiments these effects were found to be reproducible, and comparative values for derivatives of the following amines Were obtained: vanillylamine, 85%; phenylephrine, 82%, 3,4-dihydrouynorephedrine, 41%. Recovery of Amines from Aqueous Solution. These methods permit t h e
qualitative and substantially quantitative analysk of amines where mixtures containing a few micrograms are available. There remains t h e problem of isolating amines from a natural source such as urine, where their concentrations may be only of the order of 1 pg. per 100 ml. (9) and where the separation from gross amounts of other components is required. The task of isolating amines as a group is complicated by the marked differences in propertiez between individual compounds and by their hydrophilic character. These features have, however, been turned to advan-
Table IV. Relative Retention Data" for Heterocyclic Amines and Their Acetylated Derivatives on 7% F-60/-
1%
z Relative retention
Column temp., "C. . 198" No. Amine 23 S-Methyltryptamine 2.34 24 S,S-F)imethyltryptamine 1.00 25 5-Hydroxy-.V,.Ydimethyltryptamine (bufotenin) ... 26 5-Methoxytryptaniine 2 . 8 4 27 5-;LIethoxy-,Y,Sdimethyltryptamine 2 . 6 0 28 S-Acetyltryptamine 8.73 29 .V-Acetyl-,V-methyltryptamine 7.43 30 .\'-Acetyl-5-acetoxytryptamine (Diacetylserotonin)
. .
216'
... 0.98 3.71 ...
2.22 6.35 5.80 36.0
31 5-Acetoxy-A(-,S-
dimethyltryptamine
32 .V-i2cetyl-5-
5 70
methoxytryptamine 22 5
4 16 15 0
33 .Y-ilcetvl- V-methvl-
histamine 1.23 34 S,T-Diacetylhistamine 2 . 3 1
1.01 1.94
Anthracene = 1 . 0 0 . Retention time of anthracene at 198" C, ca. 5 minutes; at 216' C., ca. 2 . 8 minutes.
Table V. Retention Factors for Derivatives of Tryptamine and Histamine, derived from Data in Table IV for 7% F-60/1y0 Z, a t Two Column Temperatures
Formal transformation
OCTOP
A
5-H NORMET
5-H
-P
5-Me0 5-AcO
5-OH -+ 5-AcO --SHAc + -K Meilc --SHAc + --?; Me2 MINUTES
Figure 4. Separation of amines including histamine and N-dimethylhistamine at 1 9 8 " C. with an F-60-Z phase
Examples Temp., "C.
28 24
--
198" 216"
Factors
2 4 - 31 25 -+ 31
2 58 2 36 2 60 2 27 5.67 5 70 4 16 1 12
28629
0 83
28-+24 32 27 30 31
0 11
28 ~-
--L
--
32 27 30
0 91
0 15 0 12 0 15 0 12
VOL. 36, NO. 8, JULY 1964
1543
Table VI.
Reproducibility" of Retention Data for Separately Prepared Duplicate Acetylated Standard Mixtures of Amines on Two Different Columns
(77, F-60/1Y0 Z) at 198" C . Mole ratios in mixtures 1 and 2
~
Mixture Relative retentionb (i)
1-column No. 1 Peak heightc) Relative mm. response'
Mixture 2-column Yo. 2 ~ _ Peak height, Relatived mm. Relative Relative retention* _ _ _ responsec _ _ _ response (i) (iii) (iv) (ii) (iii) (iv) (ii) (iii) (iv) per mole
~
Parent amine 8-Hvdroxvnhenvle
VOL. 36, NO. 8, JULY 1964
1545
