Part II.
Tryptamine-Related Amines and Catecholamines
E. C. HORNING,' M. G. HORNING,' W. J. A. VANDEN HEUVEL,' K. 1. KNOX,' B. HOLMSTEDT,2 and C. J. W. BROOKS3 Department o f Biochemistry, Baylor University College o f Medicine, Houston, Texas
b Tryptamine-related indole bases and catecholamines were studied with respect to separation patterns and identification procedures. It is not possible to use the same conditions for the study of all organic bases, but for each group of compounds brought under study it was possible to find suitable derivatives and phases. These results, together with information from previous studies, suggest that gas liquid chromatographic methods should b e widely applicable in work with these compounds.
T
with which gas chromatographic methods have been applied in problems involving amines in biology and medicine is largely due to the fact that technological improvements in the preparation and use of columns, and in the preparation of suitable derivatives, have not reached the stage necessary for general scientific acceptance. The present study, based on previous experience with thinfilm columns, was undertaken in order to explore the usefulness of gas chromatography in studies of tryptaminerelated indole bases, and in studies of catecholamines. Biological amines of both types are of considerable interest in many areas in biology and medicine. HE RELATIVE SLOWKESS
EXPERIMENTAL
Gas Chromatography. The gas chromatographs used in this work were Barber-Colman and E.I.R. Instruments (Model 10, 5000 and AC-8). Argon ionization (radium source) and hydrogen flame ionization detection systems were used. 411 columns were glass U-tubes, 6 feet long X 4-mm. i.d. Column packing9 were prepared by the usual methodi: of this laboratory with 80- to 100- or 100- to 120-mesh Gas Chrom P. The phases were (1) F-60-Z : a mixture of 7y0 F-60 (methyl-pchlorophenylsiloxane polymer, Dow Corning Corp.) and 1% EGSS-Z (a copolymer of ethylene glycol, succinic acid and a methylphenylsiloxane monomer, Applied Science Laboratories, Inc.); (2) S G S : 10% neopentyl glycol succinate polyester (Applied 1
Lipld Research Center, Ikpartment
of Biochemistry, Baylor University College of Medicine, Houston, Telas Department of Pharmacology, Karo-
linska Institute, Storkholm, Sueden 1)epartment of Chemistry, The Universit) . Glasgow, Scotland 1546
ANALYTICAL CHEMISTRY
was heated to reflux temperature. Science Laboratories, Inc.) ; ( 3 ) SE-30: The progress of the acetylation reaction 1.25y0 SE-30 (methylsiloxane polymer, was followed by gas chromatography; General Electric Co.); (4) DC-710: 3y0 DC-710 (methylsiloxane polymer, the reaction mixture was injected directly into a system equipped with a hyDow Corning Corp.); (5) JXRdrogen flame ionization detection CHDMS: 0.6% J X R (methylsiloxane system. polymer, Applied Science Laboratories, PROPIOKYL DERIVATIVES.Propionyl Inc.) and 0.2Yc CHDMS(cyc1ohexanedimethanol succinate polyester, Apderivatives were prepared by reaction of the amine or amine hydrochloride plied Science Laboratories, Inc.). The with propionic anhydride in the presvaporizing zone heater was kept 30" to ence of pyridine; acetonitrile was used 40" C. above the column temperature as the solvent and the general proin isothermal runs, and about 30" C. cedure was the same as that used for above the final column temperature in the preparation of acetyl derivatives. temperature programmed runs. The Xixtures were allowed to stand overdetection, cell compartment was kept night before gas chromatographic exa t 240' to 250' C . Samples were injected by Hamilton amination. PENTAFLUOROPROPIONYL DERIVAsyringe in solution in tetrahydrofuran, TIVES. Pentafluoropropionyl derivatives acetone, or ethyl acetate. Derivative Formation. TRIMETHYL- were prepared by reaction of the amine or amine hydrochloride according to SILYL ETHERS.T r i m e t h y l d y l ethers the same method used for acetyl dewere prepared in tetrahydrofuran or acetone solution by reaction with hcxarivatives. However, the direct injection of reaction mixtures gave erratic results methyldisilazane ( 7 ) . with a variety of columns; this beENEAMINES (SCHIFFBASES). Conhavior may have been caused by the densation products of amines with acepresence of pentafluoropropionic acid tone were not isolated; these subsince the isolated derivatives proved to stances are formed readily and they have entirely satisfactory gas chroare best used in gas chromatographic matographic properties. The isolation work without isolation. The comprocedure was carried out by adding pleteness of the reaction can be followed sodium bicarbonate solution to the rein each instance by disappearance of action mixture. The aqueous solution the free amine. ACETYL DERIVATIVES. Acetyl dewas extracted with ethyl acetate, and the ethyl acetate solution was washed rivatives were prepared by reaction with sodium bicarbonate solution and with acetic anhydride and pyridine in with mater. The organic solution was acetonitrile according to the following dried with anhydrous magnesium sulfate general method. .4 10-mg. quantity and reduced to appropriate volume by of the amine, or an equivalent amount of the amine hydrochloride, was placed evaporation of the ethyl acetate (2% solutions were used in the gas chroin 0.3 ml. of acetonitrile, and 0.1 ml. matographic work). of acetic anhydride and 0.1 ml. of An organir base fraction containing pyridine were added. The solution
Figure 1 . Separation of N,N-dimethyltryptamine (DMT), N,N-diethyltryptamine (DET), 5-trimethylsilyloxy-N,N-dimethyltryptamine (5-OH-DMT-TMSi) and 5-trimethylsilyloxy-N,N-diethyltryptamine (5-OH-DET-TMSi) ' C., 19 Conditions: 10% N G S on 100- to 120.mesh Gas Chrom P, 21 6 p.s.i.; argon ionization detection system
I!l 7 t rae.~rr. 182.C
Figure 3.
Figure 2. Separation of eneamines or Schiff bases (acetone condensat;on products) of 5-methoxytryptamine ( 5 - M e 0 Trypt-SB) and 5-trimethylsilyloxytryptamine (5-OH-TryptTMSi-SB)
%
EGSS-2, on 100- to 120-mesh Gas Chrom Conditions: 7% F-60, 1 1 8 2 ' C.; 1 9 p.5.i.; argon ionization detection system
phenolic amines was obtained from epenu by a procedure previously developed for the separation of phenolic amines with an indole structure ( 3 ) .
Separation of tryptamine-related indole bases
The compounds a r e N,N-dimethyltryptamine (DMT), eneamine (acetone condensation product) from tryptamine (TRYPT-SB), 7-trimethylsilyloxyN,N-dimethyltryptamine (7-OH-DMT-TMSi), 4-trimethylsilyloxy-N,Ndimethyltryptomine (4-OH-DMT-TMSi), 5-trimethylsilyloxy-N,N-dimethyltryptamine (5-OH-DMT-TMSi), 6-trimethylsilyloxy-N,N-dimethyltryptomine (6-OH-DMT-TMSi1, and the eneamine (ocetone condensation product) from 5-trimethylsilyloxytryptamine (5-OH-TRYPT-TMSi-SBI. Conditions: 770 F-60, 1 % EGSS-Z, on 100- to 120-mesh Gas Chrom P, 1 8 2 " C.; 1 8 p.5.i.; argon ionization detection system
P;
in
RESULTS AND DISCUSSION
This paper describes work on the separation and identification of tryptamine-related indole bases and on an extension of the previous work of Brooks and Horning with catecholamines. The reviews by Brochmann-Hanssen ( 1 ) and Horning and Vanden Heuvel (6, 6 ) ) and the paper by Vanden Heuvel, Gardiner and Horning ( 8 ) and Part I of this paper contain references to previous work. Most biological amines have several funct'ional groups, and it is usually necessary to prepare suitable derivatives before attempting a GLC separation. In the present work, a series of phenolic amines in the indole series were studied by techniques involving trimethylsilyl ether formation and eneamine formation. Phenolic amines in the catecholamine series were studied through the formation of acetyl derivatives, propionyl derivatives and pentafluoropropionyl derivatives.
EXTRACT E
DMT
1
w7 %
J 0
5-McO- DMT
10
30
20
40
50
60
MINUTES
Figure 4. Comparison of gas chromatographic separation of bases for: upper panel, extract (E) from epena, and, lower panel, mixture of reference samples of N,Ndimethyltryptamine (DMT) and 5-methoxy-N,N-dimethyltryptamine ( 5 - M e 0 - D M T ) Conditions: some as for Figure 1
diethyltryptamine is also shown in Figure 1. I t is evident from the illustration that 5-hydroxy-N,,VV-dimethyltryptamine and N,N-dimethylTryptamine-Related Indole Bases. tryptamine may also be separated readTertiary amines derived from tryptily by conversion of the phenolic amine amine may be carried through G L C to the corresponding trimethylsilyl separation conditions without diffiether. culty. Selective or nonselective Treatment of an acetone solution of phases may be used, and a separation 5-hydroxytryptamine (serotonin) with may be observed on the basis of hexamethyldisilazane resulted in molecular size and shape. This effect the formation of the eneamine of 5is illustrated in Figure 1 for h',iYtrimethylsilyloxytryptamine. h methyl dimethyltryptamine and .V,it'-diethylether group will not be affected by these tryptamine with an NGS column. If conditions, and it is therefore possible a phenolic hydroxyl group is present, to identify 5-methoxytryptamine in the the amine may be carried through a presence of &hydroxytryptamine by reaction with hexamethyldisilazane in subjection of the mixture to reaction tetrahydrofuran or acetone solution with hexamethydisilazane in acetone. to yield the corresponding trimethylsilyl Eneamines will be formed from both (TMSi) ether. Tertiary amine funccompounds, and the 5-hydroxy group t'ional groups are not affected by these will be converted into a trimethylreaction conditions. h separation of silyloxy group; the resulting compounds 5-trimethylsilyloxy-A~,~V-dimethyltrypt- may be separated readily. This is amine and 5-trimethylsilyloxy-Ar,~V- illustrated in Figure 2.
The biological hydroxylation of indoles under a variety of conditions is known to yield phenolic substances; the positions of substitution which have been recognized up to the present time are the 4-, 5-, and 6-positions of the indole nucleus. To separate and identify amines in the indole series it is necessary to devise conditions under which all four possible phenolic isomers (4-, 5 , 6- and 7-substituted) will be differentiated. I t would also be desirable to distinguish tryptamine and N,N-dimethyltryptamine a t the same time. A solution to this problem is shown in Figure 3. If an acetone solution of a mixture of phenolic amines derived from N,Ar-dimethyltryptamine is treated with hexamethyldisilazane, the phenolic compounds are converted into the corresponding trimethylsilyl ethers. Tryptamine will be converted into the corresponding eneamine, while N,N-dimethyltryptamine will be unchanged. A phenolic amine VOL. 3 6 , NO. 8, JULY 1 9 6 4
1547
~-
Table I.
Relative Retention Times Compound
for Indole Bases Related to Tryptamine
Anthracene S,S-Dimethyltryptamine N,AV-Diethyltrjptamine 4-Trimethylsilyloxy-~\,.\'-dimethj ltryptamine 5-Trimethj lsilyloxy-.~,.1'-dimethyltryptamine 6-Trinieth) lsilyloxy-.Y,.1'-dime thyltryp tamine 7-Trimethylsilj loxy-.V,.\ -dimethyltryptamine 5-Trimethyl~ilyloy~-,\',~\'-diethyltryptamine
Tryptamine ilcetone condensation product of trl ptamine 5-Methoxj t r j ptamine Acetone condensation product of 5-methoxytryptamine 5-Me thoxy-S,.Y-dimethyltryptamine Serotonin Acetone condensation product of serotonin Trimethylsilyl ether of acetone condensation product of serotonin
F-60-Z,a 182' C. S G S , b 216' C. 1 .00c 1 OOd 1 05 1 68 2 14 1.71 2.89 3 21 3.19 3 74 3.70 2.23 1.72
4 50 2 69 3 10 8 74
9 50 5 10 0
227" c 7 22 15 0 15 9 11 9
4-Hydroxy-SjS-dimethyltryptamine 3 46 5-Hydroxy-S,S-dimethyltryptamine 5 77 6-Hydro~y-S~S-dimethyltryptamine 5 92 'i-Hydroxy-.~,~V-dimethyltryptamine 4 41 5-H)-droxy-.~l.1'-diethyltryptamine 8 11 a Conditions: 6-feet X 4-mm. i d glass U-tube; 770 F-60 and 1% EGSS-Z; 182' C.;
18 p.e.i. Conditions: 6-feet X 4-mm. i.d glass U-tube; 10% NGS; 216" C., 19 p.s.i. e Anthracene time, 7.0 minutes. ilnthracene time, 6.6 minutes. Anthracene time, 4.9 minutes; 21 p.s.i. 8
with a primary amino group will be converted into the corresponding eneamine-trimethylsilyl ether. Figure 3 shows the order of elution for a number of these substances with an F-60-Z column. .\',.\'-Dimethyltryptamine and tryptamine may be distinguished readily from each other and from nuclearsubstituted indoles. The 4-trimethylsilyloxy ether was relatively unstable, but nevertheless the elution of this substance is clearly shown in the illustration. &Hydroxytryptamine (se-
i\
O
IO
20 MINUTES
Figure 5. Upper panel: separation of acetylation products from treatment of adrenalin with acetic anhydride; lower panel: separation of acetylation products from treatment of adrenalin with acetic anhydride and pyridine Conditions: 3% D C - 7 1 0 on 100- to 120-mesh Gas Chrom P; 2 2 1 ' C.; 20 p.s.i. nitrogen; hydrogen Rome ionization detection system
1548
ANALYTICAL CHEMISTRY
5
10
MINUTES
5 29
rotonin) is converted under these reaction conditions to the corresponding eneamine-5-trimethylsilyl ether. Compounds in this group are eluted considerably later than the corresponding N,S-dimethylamines. With t,hese derivatives, and with the separation conditions illustrated in the figure, it is possible to separate and identify many closely related tryptamine derivatives. These methods were used in a study of the indole amines present in epend, a South rlmerican snuff reported to produce hallucinations. This snuff was obtained and studied by Holmstedt (4). When an isolation procedure suitable for the separation of phenolic amines of the indole series (3) was applied to the snuff, the basic fraction was found to give a very strong positive Ehrlich reaction. Paper chromat'ographic separation of the mixed bases established t'he fact' that the major component was 5-hydroxy-A',,V-dimethyltryptnot amine (bufotenin) or ,V,n'-dimethyltryptamine. Both of these indoles were reported as components of a different hallucinogenic snuff (parica) used for many years in South America and in the Carribean area ( 3 ) . Sufficient material for a classical study of the amines was not available. The problem was therefore invedgated through the use of gas chromatographic techniques. The basic fraction contained two principal components; the major component contained a tert,iary amine group and a substituent group which was not altered by reaction with
Figure 6. Separation of acetyl derivatives of adrenalin and noradrenalin with SE-30 Conditions: 1.25% SE-30 on 1 0 0 - to 120-mesh Gas Chrom P; 1 9 2 ' C.; 19 p.5.i.; argon ionization detection system
hexamethyldisilazane. The retention properties of this compound suggested that the most likely structure was that of 5-methoxy-,l:,iV-dimet~hyltryptamine. (-in authent'ic sample of this substance was generously provided by Dr. Irvine Page.) Figure 4 shows a comparison of the gas chromatographic analysis of a sample of the extract and of a synthetic mixture made from authentic A7,Edimethyltryptamine and 5-methoxy'V',.V-dimethyltryptamine. The correspondence is evident. In additional studies with other columns identity was established for these components of the snuff and the authentic reference compounds (4). Relative retention times for a number of tryptamine-related indole bases and their derivatives are in Table I (4). Catecholamines. Methods for the separation, identification, and estimation of amines of the adrenalin family are of interest in several scientific areas. The chief problem in gas chromatography is that of finding suitable phases and derivatives which will provide the necessary separation. The present study was concerned with acyl derivatives. The reaction of adrenalin with acetic anhydride, in the absence of pyridine, leads only to partial acetylation; the addition of pyridine as a catalyst results in the formation of a fully acetylated product. This effect is shown in Figure 5 . A 3% DC-710 column was used for the separation, and the reaction mixture was injected directly. The methylene unit values for adrenalin, noradrenalin, nietanephrine, and normetanephrine, as the fully acetylated derivatives, were determined with an SE-30 phase, and it was found (Table 11) that the values were such that a complete separation would be difficult with this phase. The derivative$ from
Table It. Methylene Unit Values for Catecholamine Derivatives Determined with SE-30 Phase
b
Y
8 h
NORMETANEPHR I NE
K
MU values& Acetyl Propionyl
Xormetanephrine 22 1 lletanephrine 22 4 23 15 Noradrenalin 23 55 Ad r en a 1in a Determined with 1.257, 190" c.
Y K
24 55 24 75 26 55 26 7 SE-30 a t
8 0 YK
''L
\I I
5 MINUTES
I
10
Figure 7. Sep ration of acetyl derivatives of metanephrine and normetanephrine with SE-30 Conditions:
same as for Figure 6
adrenalin and noradrenalin, and from met,anephrine and normetanephrine, were run in pairs with an SE-30 phase with the results shown in Figures 6 and 7 . These results suggest that the separation should be carried out with a selective phase rather than a nonselective phase. The F-60-Z phase used by Vanden Heuvel, Gardiner, and Horning (8) and by Brooks and Horning (Part I) is useful for many separations carried out below about 220' C. When this phase is used in continuous service a t its upper limit t,he polyester component is gradually lost, and the characteristics of the phase change substantially. A search was therefore instituted for a stable phase or a mixture of phases which would have a relatively high selective retention effect for ester and amide groups, and which would be stable under isothermal or temperature programmed conditions up to about 250" C. A JXR-CHDMS phase was found to satisfy these conditions. Figure 8 shows the separation of the acetyl derivatives of adrenalin, noradrenalin, metanephrinr, and normetanephrine with a JXR-CHDMS phase under temperature programmed conditions (4' per minute temperature rise). A separation of all four catecholamine derivatives was achieved in a run of about 15 minutes. The separation illustrated in Figure 8 is acceptable for many purposes, but it was considered desirable to continue the investigation of separation patterns with respect to two considerations. .In increase in size of the functional group derivative frequently results in increased separation factors for steroid alcohols and steroid ketones, and the properties of the propionyl derivatives of the catecholamines were therefore
investigated. Methylene unit values for these derivatives were determined with a n SE-30 phase (Table 11). The separation of these derivatives with a nonselective phase was found to be less satisfactory than that observed for the acetyl derivatives, and a higher temperature or longer retention time was required for the separation. I t was considered undesirable to increase the temperature of the elution when a JXR-CHDMS column was employed, and for this reason it was considered that acetyl derivatives were more suitable than propionyl derivatives for separation or identification purposes. Levels of biological amines in human and animal tissues are usually low. Electron capture or electron affinity detection techniques permit the estimation of very small amounts of material, provided that the substances under study have strong electron capture properties. I t is known that perfluoro compounds with seven fluorine atoms in aliphatic systems show electron capture properties ( 2 ) ,and it is also known that trifluoroacetyl, pentafluoropropionyl, and heptafluorobutyryl derivatives of amines show approximately the same retention characteristics (8). This effect has also been observed for derivatives of sterols (3). The separation of adrenalin and noradrenalin as
! NORADRENALIN ADRENALIN
-
MIN. .C
15
IO
5
0
225.
205.
185'
165'
Figure 8. Separation of acetyl derivatives of adrenalin, noradrenalin, metanephrine, and normetanephrine with JXR-CHDMS Conditions: 0.6% JXR and 0.270 CHDMS on 80- to 1 00-mesh Gas Chrom P; temperature pro18 p i . nitrogen; hygrammed separation; drogen flame ionization detection system
I
MIN. 20 I
I
I
I
15
IO
5
I
I
I
170. 230. 210. 190. Figure 9. Separation of pentafluoropropionyl derivatives of adrenalin and noradrenalin with JXR-CHDMS .C
Conditions:
same as for Figure 8
pentafluoropropionyl derivatives with a JXR-CHDMS column was therefore investigated; the result is shown. in Figure 9. The separation is entirely satisfactory, and similar results were obtained for the separation of the corresponding derivatives of metanephrine and normetanephrine. The sensitivity of determination for perfluoroacyl derivatives of the catecholamines is currently under study with a view to determining the limits of sensitivity of detection for these compounds. The use of acet'yl derivatives in ordinary mass measurement procedures is also under investigation. LITERATURE CITED
(1) Brochmann-Hanssen, E., J. Pharm. Sci. 51, 1017 (1962). ( 2 ) Clark, S. J., Wotiz, H. H., Steroids 2, 535 (1963). ( 3 ) Fish, 11.1. S., Johnson, N. M., Homing, E. C., J . A m . Chem. SOC. 77, 5892 (1955). ( 4 ) Holmstedt, B., Vanden Heuvel, W. J. A,, Gardiner, W. L., Horning, E. C., Anal. Biochem., in press. ( 5 ) Homing, E. C., VandenHeuvel, W. J. A,, Ann. Rev. Biochem., 32, 709 (1963). ( 6 ) Horning, E. C., Vanden Heuvel, W.
J. A , , Creech, B. G., "Methods of Biochemical Analysis," Vol. XI, L). Glick, ed., Interscience, n'ew York, 1963. ( 7 ) Luukkainen, T., Vanden Heuvel, W. J. A,, Haahti, E. 0. A,, Homing, E. C., Biochem. Biophys. Acta 52, 599 (1961). (8) Vanden Heuvel, W. J. A., Gardiner, W. L., Homing, E. C., ANAL. CHEM. 36, 1550 (1964). ( 9 ) Vanden Heuvel, W. J . A,, Homing, E. C., "Biochemical ,,Applications of Gas Chromatography, H. Szymanski,
ed., Plenum Press, New York, in press. Received for review March 26, 1964. Accepted May 14, 1964. 2nd International Symposium on Advances in Gas Chromatography, University of Houston, Houston, Texas, March 23-26, 1964. This work was supported in part by Grant HE05435 of the National Institutes of Health. VOL. 36,
NO. 6 ,
JULY 1964
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