Tables I and II indicate that olefins of move the retention time of a trace (8) Halász, L, Horváth, C.
Horváth, C., Nature 197,
the cis configuration 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 outstanding for
its suitability in temperature-pro-
grammed gas chromatography.
Finally, the retention times of the
hydrocarbons can deliberately be shifted
by adding columns with strongly polar
phases such as aluminum oxide. In this
manner it should always be possible to
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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 of Biochemistry, Baylor University College of Medicine, Houston, Texas
Conditions are described for the
gas chromatographic separation of
the catecholamines and of 31 other
amines of the phenylalkylamine, imid-
azole, 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 repro-
ducibility of gas chromatographic work
was aided by the stability of the
acetyl derivatives. Regularities noted
in retention factors assignable to sub-
stituents are of potential value in
qualitative analysis. Other com-
pounds, notably methyl esters of N-
acetylamino acids and of aromatic
acids representing catecholamine me-
tabolites, 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 chromato-
graphic estimation indicated satisfac-
tory retrieval of (3-hydroxyphenyl-
ethylamine and tyramine, but sub-
stantial loss of dihydroxyamines.
of microanalyti-
development
The cal procedures applicable to organic
amines is warranted by the physiological
and pharmacological importance of
1540 · ANALYTICAL CHEMISTRY
compound outside the range of the main
component and thus to facilitate es-
sentially its quantitative determination.
LITERATURE CITED
(1) Breshchenko, E. M., Khim i. Teknol,
Topliva i Masel 32 (1954).
(2) Bruderreck, H., Schneider, W., Halász,
I., Anal. Chem. 36, 461 (1964).
(3) Desty, D. H., Haresnape, J. H.,
Why man, B. H. F., Ibid., 32, 302
(1959).
(4) Eggertson, F. T., Knight, H. S.,
Groennings, S., Ibid., 28, 303 (1956).
(5) Halász, I., Heine, E., Nature 194, 971
(1962).
(6) Halász, I., Horváth, C., Anal. Chem.
36, 1178(1964).
(7) Halász, I., Horváth, C., Ibid., 36,
499 (1964).
many of these compounds (19). Par-
ticular interest attaches to the cate-
cholamines, to their natural and syn-
thetic 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. Con-
siderable progress has been made in the
isolation of amines and their partial
separation by ion exchange chromatog-
raphy (9, 12). For the identification
and estimation of individual compounds,
gas chromatography is likely to be the
most useful of the presently available
techniques (1).
The first significant gas chromato-
graphic 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. Broch-
mann-Hanssen and Svendsen (2) showed
that the products (presumably Schiff’s
bases or oxazolidines) from reaction of
ephedrine and related amines with
acetone were satisfactory derivatives
for gas chromatography. They also
studied acetylated amines, reporting
poor results with triacetylepinephrine
but stating that the trimethylsilyl
ether of the triacetyl derivative gave a
single symmetrical peak. Limited data
have been presented by other inves-
tigators (11,20).
71 (1963).
(9) Halász, I., Kabaker, G., unpublished
results.
(10) Heine, E., Ph.D. thesis, Universitat
Frankfurt am Main, West Germany,
1963.
(11) Horváth, C., Ph.D. thesis, Univer-
sitat Frankfurt am Main, West Ger-
many, 1963.
(12) Lange, N. A., “Handbook of Chemis-
try,” Handbook Publishers, Inc., San-
dusky, Ohio, 1956.
(13) Simmons, M. C., Snyder, L. R.,
Anal. Chem. 30, 32 (1958).
Received for review March 26, 1964.
Accepted May 21, 1964. Presented at
2nd International Symposium on Ad-
vances in Gas Chromatography, Univer-
sity of Houston, Houston, Texas, March
23-26, 1964.
This paper describes conditions under
which the natural amines of the phenyl-
alkylamine, 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 reg-
ularities in retention behavior assist
characterization.
Since the completion of this work,
Sen and McGeer (IS) have described
the gas chromatography of trimethyl-
silyl derivatives of catecholamines;
these compounds showed excellent gas
chromatographic properties but unlike
the acetyl derivatives did not dis-
tinguish epinephrine from norepine-
phrine, or metanephrine from nor-
metanephrine.
EXPERIMENTAL
Gas Chromatography. All separa-
tions were conducted isothermally
with a Barber-Colman Model 10 or
an E.I.R. instrument; argon ioniza-
detectors
tion were employed.
Column packings were made with Gas
Chrom P (Applied Science Laboratories
Inc.) which had been sieved to 80- to
100- or 100-to 120-mesh, acid-washed
and treated with dichlorodimethylsilane
1
Present address, Department of Chem-
istry, The University, Glasgow, W.2.,
Scotland. On leave of absence, from the
Atheroma Research Unit of the Medical
Research Council, Glasgow, Scotland,
May-September 1963.
before the stationary phase was applied of other acids were prepared with di-
by a slurry filtration process. The azomethane in ether. Table I. Retention Data0 for Amine-
procedures were carried out as de- The majority of the amines and amino Acetone Condensation Products
scribed by Horning, Vanden Heuvel, and acids examined in this work were
Creech (8). Two types of packing were racemic forms or L-enantiomers; their 7% F-60/- 10%
used: 10% neopentyl glycol succinate absolute configuration had no bearing Stationary phase 1%Z NGS
polyester (NGS) and a two-component on the results and has accordingly not Relative retention
phase comprising 7% of silicone oil F-60 been specified in the tables. of derivative
(Dow Corning Corp.) with 1% of ethyl- Recovery of Amines from Aqueous Column temp., °C. 165°6 183°= 215°d
ene glycol succinate-phenylmethylsilox- Solution. A standard aqueous solu- Amine
ane copolymer EGSS-Z (Applied tion containing the hydrochlorides of ß-Phenylethylamine 0.11 0.12
Science Laboratories). Freshly pre- ß-hydroxyphenylethylamine, (20 Mg·, as ß-Hydroxyphenyl-
pared columns were preconditioned at free amine); tyramine (30 Mg·); octo- ethylamine 0.18 0.20 0.22
210° to 225° C. in a stream of argon, pamine (50 Mg·) and normetanephrine Tyramine 0.49 0.46 1.14
for 12 to 24 hours, or until a satisfactory (60 Mg·) was employed. Aliquots were Octopamine 1.00 0.88 3.3
baseline was obtained. Columns were taken (a) for titration with sodium hy- Homovanillylamine 0.73 0.68 0.86
N ormetanephrine 1.28 1.15 2.24
operated at 170°, 198° and 216° C. droxide, evaporation, and acetylation of 0.09 0.11 0.06
Flash heater and detector cell tempera- the dry residue with acetic anhydride Amphetamine
and pyridine; (b) for addition to 100 Tryptamine 1.8 3.4
tures were maintained 30° to 50° C.
...

above column temperature. Glass ti-
tube columns, 6 feet long X 4-mm. i.d.
were used. Samples were dissolved in
ethyl acetate (or other suitable solvent)
and injected with a Hamilton syringe.
The sample volumes were generally in
the range 0.1 to 2 µ\., and the amounts
of sample injected were of the order of
0.1 Mg. for rapidly eluted compounds
and 1 Mg- for those with long retention
times. These quantities gave peaks of
convenient magnitude when the instru-
ments were used at moderate sensitivity
(detector voltage 800, amplifier sensi-
tivity setting nominally 1 X 10~7 ma.
for full-scale recorder deflection). The
limit of mass detection was not de-
termined.
Retention data were determined rela-
tive to anthracene as a reference stand-
ard. Quantitative comparisons were
based on peak areas estimated by tri-
angulation, and in some cases on the
product of peak height and retention
time (4).
Reference Compounds and Reag-
ents. Amines were obtained, mostly
as salts, from commercial sources.
Where necessary, the free bases were
liberated by titration with sodium
hydroxide solution (less than the
required amount was used with the
catecholamines); on an analytical scale
(10 to 1000 Mg·) it was convenient to
evaporate the resulting solutions to dry-
ness in a vacuum desiccator and to
take up the residual amines in a suitable
solvent or reagent. Acetylation was
effected quantitatively by treatment of
the amine with redistilled acetic an-
hydride (10 to 20 m1·) and pyridine
(distilled over potassium hydroxide)
(10 to 20 m1·), followed by evaporation
of the excess reagents in a vacuum
desiccator. Acetylated amines could
be stored in solution in ethyl acetate
at 5° C. for several weeks without
appreciable change.
Amino acid methyl esters were pre-
pared by refluxing the acid in meth-
anolic hydrogen chloride (ca. 5% HC1;
prepared from acetyl chloride and meth-
anol) for 4 hours, evaporating to dry-
ness, and liberating the free base with
alkali. In the case of tryptophan the
methylation was effected by overnight
treatment at room temperature. N-
Acetylamino methyl esters were ob-
tained by treatment with acetic an-
hydride and pyridine, followed by evap-
oration of the reagents. Methyl esters
ml. of water; and (c) for addition to
100 ml. of urine wdiich had been thor-
oughly extracted with ethyl acetate at
pH 1 to remove acidic and neutral
material.
The solutions from (b) and (c) were
buffered by the addition of solid po-
tassium carbonate and treated with
acetic anhydride, added in portions of
10, 5, and 5 ml. over a period of 2 hours.
The pH was kept above 8 by the ad-
dition 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 m1·) and pyridine (20
m1.) 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 gaschromatography 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 ac-
cordingly made, following previous
work (7), of the chromatographic be-
havior of the products formed by dis-
solving primary amines in dry acetone.
In the case of ß-hydroxy amines, oxa-
zolidines (13) may be formed under
these conditions. It is clear from the
retention data in Table I that if the
ß-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) col-
umn, 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 ß-hydroxyl
group into tyramine and homovanil-
lylamine are 2.9 and 2.6, respectively.
5-Methoxytrypt-
amine ... 4.5
“
Anthracene =
1.00.
b
Anthracene, ca. 15 minutes.
c
ca. 6.5 minutes.
Anthracene,
d
Anthracene, ca. 9 minutes.
Similarly, with a less polar F-60-Z
column at 183° C., the retention factors
associated with the p-hydroxyl group
(4.2, 4.6) greatly exceed those found
for the introduction of a ß-hydroxyl
group (1.8, 1.9, 1.7). The chemical
basis of these correlations requires
further study, but the data strongly
suggest that the compounds either
survive chromatography unchanged or
undergo specific transformations so
that 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 bond-
ing, involving the phenolic 4-hydroxyl
group, greatly reduces its interaction
with an NGS phase; the retention
factors were found to be 0.76 and 0.67
for this column. With an F-60-Z
column there is much less selective
retention due to polar groups, and the
addition of the 3-methoxyl group
causes an increase in retention ap-
proximately commensurate with the
increased molecular weight.
Acetylated Amines. The catechol-
amines gave evidence of substantial
decomposition when their acetone
solutions were submitted to gas chro-
matography, suggesting that a gen-
eral procedure for biological amines
would require ether or ester forma-
tion for the functional groups. Tri-
methylsilvl derivatives of the simpler
amines were examined briefly; these
compounds showed satisfactory chro-
matographic properties. Prior atten-
tion, 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 na-

VOL. 36, NO. 8, JULY 1964 · 1541

 Table III. Retention Factors for ß-
Phenylethylamine Derivatives Derived
from Data in Table II for 7% F-60/1 %
Z at 198° C.
Formal
transformation Examples Factors
/3-H —*-
/3-AcO 1 — 2 2.77
3 — 4 2.68
5 —6 2.48
7 — 8 2.59
12 — 13 2.59
Figure 1. Separation of acetylated phenylethylamines at 17 — 18 2.28
198° C. with an F-60-Z phase 4-H — 4-AeO 1 — 3 5.64
2 — 4 5.47
3,4-diH —
3,4-diAcO 1 — 7 19.3
2 — 8 18.1
tural and synthetic amines were ac- also found to be fully separated at this 13— 16 14.6
cordingly examined as their fully temperature, but the retention times 4-H — 4-MeO 1 — 22 3.0
acetylated derivatives, obtained for (62 and 87 minutes, respectively) were 3,4-diH —
this purpose by treatment with acetic 3,4-diMeO — 9 6.0
long, and it was more
1
suitable to
3,4,5-triH —
anhydride and pyridine. The acet- operate at 216° C. and accept a less 3,4,5-triMeO 1 — 15 11.1
ylated di- and trihydroxy amines complete separation illustrated in Figure 3,4-diH — 3-MeO,
showed very long retention times with 2. 4-AcO 1 — 5 11.3
an NGS column, and most of the work Retention data collected in Table 2 — 6 10.2
—NHAc — —NMeAc 4—19 0.91
was therefore carried out with the II are means of several determinations. 6—10 0.89
two-component phase, 7% F-60 with The values were highly reproducible 8 — 11 0.89
1% EGSS-Z, which had been devised by (± to 12%) over periods of one or two —N HAc — —N Mea 3 — 21 0.16
Holmstedt, Vanden Heuvel, Gardiner, weeks, but they diminished gradually
and Horning (7). over longer periods of time. Retention
Phenylalkylamines. Acetylated factors attributable to the introduction tion value in the region of 0.37 X 5.5-
phenylalkylamines gave well defined of substituents, or to other transforma- i.e., about 2.0. This compound is
symmetrical peaks. Figure 1 depicts tions, may be calculated from these not yet available for trial.
typical results obtained at 198° C. data and some examples are given in Histamine and Tryptamine
with an amine mixture including ß- Table III. The values of the factors Derivatives. Acetyl derivatives of
hydroxyphenylethylamine and five rep- strongly suggest that the various sub- histamine and Ar-methylhistamine gave
resentative amines; the different acetyl stituents, with the possible exception of peaks with evidence of slight tailing.
derivatives are completely separated, the /3-acetoxvl group, which merits Acetylated tryptamines gave sym-
and the resolution of metanephrine further investigation, are preserved metrical peaks. Figure 3 shows a
from normetanephrine is notable. intact during chromatography. More- separation of tryptamine, melatonin
Epinephrine and norepinephrine were over, although these data are quite and serotonin, as acetyl derivatives.
limited, they show' that it is feasible to Figure 4 indicates that tryptamine,
apply group retention factors to predict histamine and Ar-methylhistamine are
Table II. Relative Retention Data" for approximate retention times. Thus, distinguishable in the presence of
the relative retention time for tetra- various phenylethylamine derivatives.
Acetylated Amines on 7% F-60/1 % Z acetylnorepinephrine at 198° C. could Relative retention data for acetylated
Relative retention have been predicted from the value for heterocyclic amines are given in Table
Column temp., °C. 198°6 216°c triacetyldopamine and the mean value IV. Certain regularities are discern-
No. Parent amine for the /3-acetoxyl group retention ible, and factors derived for some
factor, computed from five other ex- transformations are given in Table V.
1 /3-Phenylethylamine 0.39
2 /3-Hydroxyphenyl- amples, to be 7.53 X 2.56—i.e., The introduction of the 5-acetoxyl or
ethylamine 1.08 19.3. Similarly, one would expect the 5-methoxyl group in tryptamines causes
3 Tyramine 2.20 1.77 diacetyl derivative of p-hydroxy-a- the retention to change by a factor
4 Octopamine 5.91 4.39 similar to that found for the correspond-
5 Homovanillylamine 4.44 3.43 methylphenylethylamine (p-hydroxy-
6 Normetanephrine 11.0 7.43 amphetamine) to have a relative reten- ing 4-substituents in phenylethylamines.
7 Dopamine 7.53 5.44
8 Norepinephrine 19.5 12,05
9 Homoveratrylamine 2.35 2.00
10 Metanephrine 9.81 6.80
11 Epinephrine 17.3 11.2
12 Amphetamine 0.37
13 Norephedrine 0.96 0! 84
14 Vanillylamine 3.45 2,75
15 Mescaline 4.34
16 3,4-Dihydroxynor-
ephedrine 14.1 9.18
17 Deoxyephedrine 0.49
18 Ephedrine 1.12 1 '.05
19 Synephrine 5.35 4.17
20 Phenylephrine 4.71 3.36
21 Hordenine 0.36
22 4-Methoxv-0-phenyl-
ethylamine 1.20
“Anthracene = 1.00.
6
Anthracene, ca. 5 minutes.
Anthracene, ca. 2.8 minutes.
"

1542 · ANALYTICAL CHEMISTRY

 Figure 3. Separation of tryptamine, melatonin, and serotonin as acetyl derivatives at 216° C. with an F-60 Z phase

The agreement between retention fac- the preservation of the detection sys-
tors which serve to distinguish primary, tem during this time.
The relative response computed on a Table IV. Relative Retention Data”
secondary, and tertiary amines, of all
three nuclear types, is very satisfactory molar basis is given in the last column for Heterocyclic Amines and Their
and the factors are evidently applicable of Table VI. The three diaceto.xy- Acetylated Derivatives on 7% F-60/-
to the calculation of retention times. .V-acetyl derivatives gave approxi- 1%Z
Reproducibility of Retention Data. mately 80% of the response observed Relative
The principle features observed during for the monoacetoxy-.V-acetyl de- retention
the course of the work are illustrated rivatives. In separate experiments Column temp., °C. 198° 216°
by the results in Table VI, for columns these effects were found to be repro- No. Amine
operated at 198° C. The relative ducible, and comparative values for 23 A'-Methyltryptamine 2.34
retention values for column No. 1 derivatives of the following amines were 24 ', '-Dimethyltrypt-
were measured after this column had obtained: vanillylamine, 85%; phenyl- amine 1.00 0.98
25 5-Hydroxy-AyY-
been in use for several weeks and are ephrine, 82%, 3,4-dihydroxynorephe- dimethyltryptamine
closely matched by those recorded drine, 41%. (bufotenin) 3.71
at a similar stage for column No. 2. Recovery of Amines from Aqueous 26 5-Methoxytryptamine 2.84
A gradual decrease in retention values, Solution. These methods permit the 27 5-Methoxy-,Y,.Y-
noted over a period of three weeks, is qualitative and substantially quanti- dimethyltryptamine 2.60 2.22
28 .Y-Acetyltryptamine 8.73 6.35
attributable to the slow loss of EGSS- tative analysis of amines where mix- 29 .Y-Acetyl-A'-methyl-
Z from the stationary phase; deteriora- tures containing a few micrograms tryptamine 7.43 5.80
tion of the column in this respect was are available. There remains the 30 A'-Acetyl-S-acetoxy-
accelerated when it was used at 216° C. problem of isolating amines from a tryptamine (Diacetyl-
serotonin) 36.0
Table VI includes figures representing natural source such as urine, where
...

31 5-Acetoxy-AyY-
the quantitative response observed, their concentrations may be only of dimethyltryptamine 5.70 4.16
which show that for the injection of the order of 1 µg. per 100 ml. (9) and 32 A"-Acetyl-5-
acetylated amines in amounts ranging where the separation from gross methoxytryptamine 22.5 15.0
33 A"-Acetyl-.Y-methyl-
from 0.2 to 0.6 µg., reproducibility in amounts of other components is re- histamine 1.23 1.01
terms of an internal standard was quired. The task of isolating amines as 34 AyY-Diacetylhistamine 2.31 1.94
achieved even with different columns. a group is complicated by the marked
Runs carried out with a single column differences in properties between in- "Anthracene 1.00.
=

Retention time of anthracene at 198° C,

over periods not exceeding a few weeks dividual compounds and by their hy- ca. 5 minutes; at 216° C., ca. 2.8 minutes.
showed substantial replication of actual drophilic character. These features
peak heights; this was contingent upon have, however, been turned to advan-

Table V. Retention Factors for De-

rivatives of Tryptamine and Histamine,
derived from Data in Table IV for 7%
F-60/1% Z, at Two Column
Temperatures
Formal
transformation Examples Temp., °C.
198° 216°
Factors

5-H —* 5-MeO 28 ->· 32 2.58 2.36

24 — 27 2.60 2.27
5-H - 5-AcO 28 -* 30 5.67
24 — 31 5.70 4.16
5-OH — 5-AcO 25 31 1.12
—NHAc —
—NMeAc 28 -* 29 0.83 0.91
—NHAc
-NMe, 28 — 24 0.11 0.15
32 ->· 27 0.12 0.15
30 — 31 0.12
Figure 4. Separation of amines including histamine and N-dimethylhistamine at
198° C. with an F-60-Z phase

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

(7% F-60/1% Z) at 198° C.

Mole
ratios Mixture 1-column No. 1 Mixture 2-column No. 2
in Relative Peak Peak height, Relatived
mixtures retention6 height”, Relative Relative retention6 mm. Relative responsec response
Parent amine 1 and 2 (i) mm. response” (i) (iii) (iv) (ii) (íü) (iv) (ii) (iii) (iv) per mole
0-Hydroxyphenyl-
ethylamine 0.6 1.07 202 0.56 1.10
1.07 1.07 58 60 66 0.53 0.58 0.63 97 ±8
Tyramine 1.00 2.16 179 1.00 2.24
2.20 2.17 54 51 52 1.00 1.00 1.00 100 ...

Homovanillylamine 1.25 4.41 109 1.24 4.57

4.48 4.37 34 31 1.29 1.24 100 ±2
Octopamine 1.5 5.93 79 1.22 6.03
6.15 5.89 24 23 29 1.23 1.24 1.51 85 ±9
Metanephrine 1.75 9.60 53 1.32 9.72
9.90 9.45 17 16 16 1.39 1.32 1.34 76 ±2
N ormetanephrine 2.0 10.9 61 1.72 11.3
11.1 10.7 18 16 18 1.68 1.59 1.70 84 ±3
0
The quantities in the sample injected corresponded to 0.3 ¿ig. of tyramine. In experiment (i) (June 27) the detector was particularly
sensitive. Experiments (ii), (iii) and (iv) were run on Aug. 27, Aug. 30 and Sept. 18, respectively: the gradual change in retention
values is apparent.
6
Anthracene (ca. minutes) =
1.00.
Computed arbitrarily as the product of peak height and relative retention, and expressed as a proportion of the value found for tyr-
c

amine.
d
Tyramine is taken as standard =
100%, and the figures are based on the mean response in the runs (i) to (iv).

tage in recent ion exchange-chromato-

graphic procedures {12), which provide
an elegant technique for fractionating
urinary amines, and which may be
adapted to subsequent gas chromato-
graphic analysis.
The possibility of recovering amines
from aqueous media by acetylation at
pH 8 {6, 21), extraction, and reacetyla-
tion of the extracted compounds under
anhydrous conditions to ensure acetyla-
tion of the benzylic hydroxyl group
was evaluated. Samples of four amines
were treated in this way with the
results cited in Table VII. The re-
covery of octopamine and normetane-
phrine was only 30 to 40%, but it
was notable that no difference between
the recovery from water and from
urine was observed.
Analysis of Catecholamine Metabo-
lites. The principal acidic products
of the oxidative metabolic deamina-
tion of the catecholamines, notably

Figure 5. Separation of several aromatic acids as methyl esters before and after
acetylation
Table VII. Calculated Recovery of
Amines from Aqueous Solution by
Acetylation, Extraction, Reacetylation,
and Gas Chromatographic Estimation Table VIII. Relative Retention Data" for Methylated Catecholamine Metabolites
Amounts and Related Compounds on 7% F-60/1% Z
added to Relative retention
100 ml. Percentage
aqueous recovery" Acetylated methyl
phase, From From Methyl ester ester
Amine mixture Mg- water urine Column temp., °C. 198°6 170°” ©

/3-Hydroxyphenyl- Parent acid

ethylamine 20 95 c

30 95 100 Anthranilic 0.15 Not examined

Tyramine Vanillic 0.35 0.61
Octopamine 50 35 40
Homovanillic 0.48 0.48 0.98
Normethanephrine 60 30 35
0.95 1.12 3.02
4-Hydroxy-3-methoxymandelic
“
Based on peak heights relative to the Veratric 0.45 0.44 Lnaltered
directly acetylated mixture. Means of Hippuric 0.87 0.98 Enaltered
duplicate chromatograms, rounded off to Ferulic 1.36 Not examined
the nearest 5%. "
Anthracene = 1.00.
6
Previously extracted with ethyl acetate 6
5 minutes.
at pH 1. Anthracene, ca.
”
Anthracene, ca. 13 minutes.
c
Peak obscured by other components.

1544 e ANALYTICAL CHEMISTRY

4-hy droxy-3-methoxy mandelic acid,
homovanillic acid, and vanillic acid, Table IX. Relative Retention Data for Acetylated Amino Acid Methyl Esters on
have received much analytical atten- 7% F-60/1% Z at 198° C.
tion (14, 16)· Gas chromatographic Retention factor
separations have been devised by RCH2 RCHC02Me
Sweeley, Williams, and their col- Relative retention0 I I
Parent acid of derivatives NHAc NHAc
laborators (17, 22, 23). The problem
of interference of the peak from methyl Phenylalanine 0.78 2.0
Tyrosine 3.89 1.8
hippurate has been overcome by acid- Histidine 4.67 2.0
catalysed acetylation under conditions Tryptophan 16.7 1.9
which cleave the amide bond in methyl 0
Anthracene (ca. 5 minutes) =
1.00.
hippurate (24).
Another approach to this separation
is illustrated in Figure 5, which shows
dilute aqueous solution has been dem-
that simple acetylation of a mixture
onstrated. A few compounds were Table X. Relative Retention Data" for
containing methyl hippurate and methyl found to have rather similar retention Reference Standards on 7% F-60/1 %
4-hydroxy-3-methoxymandelate affords Z at 198° and 216°
the acetyl derivative of the latter ester, times, leading to partial fusion of the
with a long retention time on 7% peaks. The separation of any pair of Relative retention
these should present little difficulty if
F-60-Z which suffices to separate it Column temp., °C. 198°6 216°=
the selectivity of the phase is changed.
from methyl hippurate. Retention 1-Tetradecanol 0.48
With these elementary refinements,
data for these and related compounds 1-Hexadecanol 1.04
are collected in Table VIII.
gas chromatographic methods should 1-Octadecanol 2.24
be a powerful supplement to paper 1-Eicosanol 4.82
Chromatography of Amino Acid
Derivatives. Four amino acids, rep- chromatography (9, 12, 18) and thin 1-Docosanol 10.23
layer chromatography. 1-Tetracosanol 21.7
resenting biological precursors of typ- Methyl palmitate 1.24 1.11
ical amines, have been studied with Methyl stearate 2.68 2.25
ACKNOWLEDGMENT behenate 12.35
an F-60-Z column in the form of Methyl
their acetylated methyl esters. The Methyl lignocerate 18.3
We thank Bo Holmstedt and W. J. 23.2
Squalene
relative retention times were almost A. Vanden Heuvel for stimulating dis- °
Anthracene = 1 00.
exactly double those of the corre- cussions, and Wanda L. Gardiner for 6
Anthracene, ca. 5 minutes.
sponding acetylated amines; the rele- assistance in the preparation of gas '
Anthracene, ca. 2.8 minutes.
vant data are presented in Table IX. chromatography columns.
This observation suggests that an
F-60-Z column might serve for the LITERATURE CITED
separation of certain amino acids. A. H., Gowenlock, ed., p. 39, Elsevier,
Retention Data for Reference (1) Brochmann-Hanssen, E., J. Pharm. Amsterdam, 1963.
Sci. 51, 1017 (1962). (15) Sen, N. P., McGeer, P. L., Biochem.
Standards. Data for several ref- (2) Brochmann-Hanssen, E., Svendsen, Biophys. Res. Comm. 13, 390 (1963).
erence compounds are quoted in A. B., Ibid., 51, 393, 938 (1962). (16) Smith, P., Varley, H., “The Clinical
Table X, in order to provide a means (3) Callingham, B. A., Cass, R., Varley, Chemistry of Monoamines,” A. H.
of comparing or characterizing the H., “The Clinical Chemistry of Mono- Gowenlock, ed., p. 31, Elsevier, Am-
amines,” A. H. Gowenlock, ed., p. 19, sterdam, 1963.
phases. Retention data for some of Elsevier, Amsterdam, 1963. (17) Sweeley, C. C., Williams, C. M.,
these compounds on other phases are (4) Carroll, K. K., Nature 191, 377 Anal. Biochem. 2, 83 (1961).
cited in the ASTM Compilation of (1961); 192,965 (1961). (18) Takesada, M., Kakimoto, Y., Sano,
Gas Chromatographic Data (10). (5) Pales, . M., Pisano, J. J., Anal. L, Kaneko, Z., Nature 199, 203 (1963).
Biochem. 3, 337 (1962). (19) Varley, H., “The Clinical Chem-
(6) Hagopian, M., Dorfman, R. I., istry of Monoamines,” A. H. Gowen-
CONCLUSIONS Gut, M., Ibid., 2, 387 (1961). lock, ed., Elsevier, Amsterdam, 1963.
(7) Holmstedt, B., Vanden Heuvel, W. (20) Vessman, J., Schill, G., Svensk. Farm.
These procedures provide a general J. A., Gardiner, W. L., Horning, E. C., Tidskr. 66, 601 (1962).
method for the qualitative analysis of Ibid., in press. (21) Welsh, L. H., J. Am. Pharm. Assoc.
(8) Horning, E. C., Vanden Heuvel, W. J. 44, 507 (1955).
all the principal phenylalkylamines en- A., Creech, B. G., Methods Biochem. (22) Williams, C. M., Anal. Biochem. 4,
countered in normal and abnormal Analy. 11, 69 (1963). 423 (1962).
metabolism, together with histamine, (9) Kakimoto, Y., Armstrong, M. D., J. (23) Williams, C. M., Greer, M., Clínica
Biol. Chem. 237, 208 (1962). Chim. Acta 7, 880 (1962).
tryptamine, and their congeners. Sat- (10) Lewis, J. S., “Compilation of Gas (24) Williams, C. M., Leonard, R. H.,
isfactory quantitative relationships have Chromatographic Data,” American So- Anal. Biochem. 5, 362 (1963).
been achieved in experiments with ciety for Testing and Materials, Phila-
standard mixtures containing ca. 0.2 delphia, Pa., 1963. Received for review March 26, 1964.
to 2 ¿íg. of amines, including metane- (11) Parker, K. D., Fontan, C. R., Kirk, Accepted May 14, 1964. 2nd Inter-
P. L., Anal. Chem. 35, 356 (1963). national Symposium on Advances in Gas
phrine and normetanephrine; estimation (12) Perry, T. L., Schroeder, W. A., J. Chromatography, University of Houston,
of the catecholamines themselves has Chromatog. 12, 358 (1963). Houston, Texas, March 23-26, 1964.
not yet been adequately investigated. (13) Pfanz, H., Kirchner, G., Ann. 614, C. J. W. Brooks acknowledges financial
149 (1958). support provided through the courtesy
Virtually complete recovery of ß- (14) Ruthven, C. R. J., Varley, H., “The of John M. Knox (Department of Der-
phenylethylamine and of tyramine from Clinical Chemistry of Monoamines,” matology).

VOL. 36, NO. 8, JULY 1964 · 1545