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Excretion products of algae
ANALYTICAL
78, 365-373 (1977)
BIOCHEMISTRY
Excretion
Products of Algae
Identification of Biogenic Amines by Gas-Liquid Chromatography and Mass Spectrometry of Their Trifluoroacetamides V. HERRMANN Institut
fiir Chemische Corrensstrasse
AND F. J~~TTNER
Pjlanzenphysiologie 41, 7400 Tiibingen.
der Universitiit, Germany
Received May 5, 1976: accepted November 5. 1976 The trifluoroacetyl derivatives of 12 biogenic amines were separated by gas-liquid chromatography and identified by mass spectrometry. Vacuum sublimation was used to separate nanomole quantities of amines from algal media and natural waters that contained algae. Trimethylamine, I-aminopropane, 2-aminopropane, and ethanolamine were found in the culture medium of Ochromonas. Chlamydomonas-enriched backwater contained butanolamine. I-Aminopropane was found in the media of Nitzschia, Ankistrodesmus. and Microcystis. Concentrations were approximately 300 pg/liter for Ankistrodesmus and 240 pg/liter for Microcystis media.
Many different methods have been described for the separation of biogenic amines: Some of the more typical are briefly mentioned. Stein von Kamienski (24) separated the amines by paper chromatography, and Ilert and Hartmann (6) used their dinitrophenyl derivatives for separation by thin-layer chromatography. A tic system for dansylated amines was worked out by Seiler and Wiechmann (20). Kanazawaet al. (lo), Hatano et al. (5) and Wang (27) used ion-exchange columns to separate free mono-, di-, and polyamines. For gas-liquid chromatographic (glc) separations of selected amines, Edwards and Blau (3) employed the dinitrophenyl derivatives, Knights (12), the silyl derivatives, and Pailer and Hiibsch (16) and Irvine and Saxby (7), the acyl derivatives. Of the latter derivation methods, the trifluoroacetylation is very advantageous because of its reliability and rapid quantitative reaction. Since the products which were formed exhibited high volatility and good glc separation properties, we employed this method for the separation of biogenic amines excreted by algae. A further advantage of these derivatives is their informative mass fragmentation pattern which was to be expected according to the observations of Karoum et al. (11) with phenolic amines and Crathorne and Saxby (2) with some alkylamines. Combining gas-liquid chromatography with mass spectrometry facilitates the identification of unknown biogenic amines even if they are only available in the nanomole range. 365 Copyright All
riehts
0 of
1977 revroduction
by
Academic in any
Press.
Inc.
form
reserved.
ISSN
ooO3-2697
366
HERRMANN
AND JijTTNER
EXPERIMENTAL Growth of algae. The algae,Ankistrodesmus braunii 202-7c, Microcystis aeruginosa LB 1450-1, and Ochromonas danica L 933-7 were obtained
from the Sammlung von Algenkulturen des Pllanzenphysiologischen Instituts der Universitst Giittingen, Germany. The strain of Nitzschia actinastroides used was an isolate from the Lake of Konstance (South-West Germany). Chlamydomonas-enriched water was collected during a bloom of this organism in the backwaters of the Ruhr River near Geisecke.’ Precultivation of the algae was performed in 300-ml culturing tubes (13) and 30-liter tower-type cultivation plants (9) were used for the subsequent mass cultivation. The details of the cultivation parameters used were similar to those as reported for Ochromonas by Jiittner and Friz (8)) and for Ankistrodesmus by Severge et al. (21) for Microcystis (Aphanizomenon flos-aquae strain Hindak 1963/143 Laborator Experimentalni Algologie, Tieboii, &SR) by Jiittner (9), and for Nitzschia by Miiller (15). The algae were harvested during the declining logarithmic growth. Separation of the culture media from the algae was performed with a continuous-flow centrifuge. Isolation of amines by vacuum sublimation. For isolation of the amines, the algal medium (25-30 liter) was acidified (pH 1) with HCl and taken to dryness in a rotary flash evaporator. Early in this investigation, the amines were isolated by extraction with butanol-1. After the addition of 50 ml of butanol- 1,2 N NaOH was added, and the butanol layer was separated and acidified. The amines were concentrated as their hydrochlorides and, as such, were prepared for head-space analysis. Later, the separation process was improved by application of vacuum sublimation. For this, 1 ml of the amine hydrochloride solution was transferred into a 50-ml round-bottom flask with a ground-glass joint; it was frozen, overlayered with 1 ml of distilled water, refrozen, and then overlayered with 1 ml of 2 N NaOH. The flask was immediately fitted to an all stainless steel vacuum sublimation apparatus. To start the reaction, the contents of the flask were thawed out to ensure adequate mixing. Before sublimation (2.5 hr, 0.06 Torr) was started, the sample was frozen again to prevent bumping. The trap was cooled with liquid nitrogen. After sublimation was completed, 25 ml of 1 N HCl was poured into the trap immediately after opening it. The amine hydrochlorides were concentrated, transferred into l-ml conical reaction vials, and taken to dryness under vacuum, after which 50-~1 each of ethyl acetate and trifluoroacetic anhydride were added. The reaction was 1 We are greatly indebted to Dr. H. Miiller, Limnologisches Institut der Universitat Freiburg, 775 Konstanz-Egg, for providing a culture of Nirzschiu actinasrroides and to Dipl. Biol. G. Klein, Institut fiir Wasserforschung, 5841 Geisecke, for collecting Chlumydomonasenriched water.
BIOGENIC
AMINES
367
1' 1
10
100
1000
pg 1 -aminopropanc FIG. 1. Detector response as a function of sample size of I-aminopropane vacuum sublimation.
separated by
completed within 10 min at 80°C. From each sample, a 2-4 vol was taken and injected into the gas chromatograph. Gas-chromatographic separation of the biogenic amines. The gasliquid chromatographic separation of the trifluoroacetamides and lowboiling free amines was performed on a 1.8m x 3.2mm (i.d.) column packed with 3% OV-17 on high-performance Chromosorb W AW DMCS (80- 100 mesh). After a 3-min postinjection interval at 50°C the temperature was increased at a rate of lO”C/min until it reached 250°C. Both the flame ionization detector and injection port temperatures were maintained at 28O”C, and nitrogen was used as the carrier gas (30 ml/min). The oncolumn decomposition of the amine hydrochlorides (1 ~1) was performed at 30°C according to Umbreit et al. (26). Tertiary, highly volatile amines TABLE RECOVERY
Compoundb MA IBA 3-MBA 1,5-PA
1
OF BIOGENIC
AMINE@
Percentage of recovery (%)
Compound
Percentage of recovery (%)
99 8.5 74 83
I-AP 2-AP EOA PEA
82 83 58 83
LIBiogenic amines (500-pg samples) were recovered after sublimation for 2.5 hr at 0.06 Torr. b Abbreviations used: MA, methylamine; TMA, trimethylamine; IBA, isobutylamine = I-amino-2-methyl-propane; 3-MBA, 3-methylbutylamine; I-AP, I-aminopropane; 2-AP, 2-aminopropane; EOA, ethanolamine; PEA, P-phenylethylamine; 1,4-BA, 1,4-diaminobutane; 1J-PA, 1,5-diaminopentane; BA, butanolamine.
368
HERRMANN
AND JijTTNER
6o- k-+--l ' .c f y fL
60-
LO-
20 -
0
I 30
FIG. 2. Recovery of I-aminopropane
1 60
u 90
I 120
1 150
1 160
min
as a function of the sublimation time (4-mg samples
2-AP
I-AP h
16 min FIG. 3. Head space analysis of biogenic amines of Ochromonas culture medium (3% OV-17 on Chromosorb W AW DMCS, 30°C. 30 ml of N,/min). TMA, trimethylamine: 2-AP, 2-aminopropane; I-AP. I-aminopropane.
BIOGENIC
369
AMINES
-
1
z
I
I
I
4
8
12
I
16 min
FIG. 4. Gas chromatogmm of an authentic mixture of 12 trifluoroacetylated biogenic amines (3% OV-17 on Chromosorb W AW DMCS, postinjection interval of 3 mitt, temperature progression: 50-200°C rate: 6”C/min, carrier gas: 30 ml of N,/min). (1) Ethylamine, (2) 2-aminopropane, (3) I-aminopropane, (4) isobutylamine, (5) 2-methylbutylamine, (6) (8) cysteamine, (9) butanolamine, (10) 3-methylbutylamine, (7) ethanolamine, P-phenylethylamine, (11) I .4-diaminobutane, (12) 1.5diaminopentane
were examined by head-space analysis in 5-ml vials sealed with siliconerubber septa. After the addition of 1 ml of 2 N NaOH to the amine hydrochlorides, the sample was heated to 5O”C, and 1 ml of gas was injected into the gas chromatograph. Absolute values of amines were calculated from peak areas, in comparison to authentic samples. RESULTS AND DISCUSSION Preparation of Arnines
Selective separation of nanomole amounts of biogenic amines from algal culture media and water from natural sources is complicated by the
370
HERRMANN
AND JijTTNER
TABLE MASS
SPECTRA
OF
13 TRIFLUOROACETAMIDES TEN
Compound MA
EA
141
I-AP
MOST
INTENSE
MOlfSUlElI weight 127
155
2 IN FRAGMENT
Fragment
THE
SEQUENCE
OF THEIR
IONS
ion (m/e)
58 ( IW”
69 (38.4)
78 (7.6)
45 (6.7)
50 (4.8)
59 (3.8)
51
126 (loo)
141 (65.4)
69 (63.1)
72 (53.5)
44 (34.5)
78 (16.6)
45 (11.9)
126 (loo)
69 (32.2)
43 (25.0)
140 (19.6)
41 (19.6)
114 (17.9)
42 (16.1)
78 (14.3)
86 (13.4)
49
0.8)
97 (2.8)
33 (2.8)
51 (7. I)
97
(6.5)
56 (1.9) 140 (6.5)
(9.8)
2-AP
155
140 (100)
69 (35.7)
43 (33.6)
41 (22.1)
45 (20.0)
42 (17.8)
70 (13.6)
155 (12.6)
47 (10.5)
86 (9.4)
IBA
169
127 (100)
56 (77.8)
126 (62.1)
41 (53.5)
43 (50.7)
58 (39.2)
69 (37.8)
57 (2 I .4)
114 (15.0)
loo (12.8)
2-MBA
183
127 (loo)
41 (61.4)
57 (46.0)
70 (37.4)
69 (30.0)
126 (34.0)
55 (22.7)
58 (21.4)
114 (18.0)
71 (12.7)
3.MBA
183
55 (100)
126 (90.9)
70 WO)
127 (81.8)
41 (65.9)
57 (63.6)
43 (54.5)
69 (47.7)
114 (3 1.8)
42 (27.2)
EOA
253
126 (100)
69 (82.9)
70 (35.4)
139 (30.3)
140 (15.8)
78 (13.9)
42 (10.1)
97 (6.9)
184 (5.7)
47 (5.7)
BA
281
126 mo)
55 (36.3)
69 (34.6)
139 (20.8)
54 (16.4)
98 (11.5)
41 (10.2)
78 (7.8)
167 (6.4)
140 (6.0)
CA
269
126 (1cQ)
69 (76.8)
156 (54.6)
172 (28.7)
78 (17.5)
140 (13.8)
59 13.8
87 (12.9)
157 (10.1)
139 (10.1)
PEA
217
104 (100)
91 (63.7)
105 (12.5)
65 (12.5)
69 (10.0)
126 (7.5)
92 (7.5)
78 (6.2)
77 (6.2)
51 (6.2)
I .4-BA
280
126 (loo)
167 (53.4)
69 (41.5)
127 (34. I)
55 (28.2)
166 (17.8)
151 (16.3)
54 (15.5)
41 (15.5)
78 (12.6)
I.5-PA
294
126 (100)
68 (63.5)
69 (56.1)
55 (41.5)
127 (3 I .8)
41 (29.3)
168 (26.8)
loo (22.0)
180 (14.6)
140 (14.6)
a Number within parentheses is the percentage of relative intensity.
large amount of salts which appear after concentration. Although alkaline steam distillation is a very useful method for the separation of amines from nonvolatile compounds, this procedure separates diamines and alkanolamines only to a limited extent (24), and some of them may be produced by uncontrolled degradation of other compounds (25). Lowering the temperature, however, reduces the reactivity and hence, the formation of degradation products. Therefore, we employed vacuum sublimation which was selective and gentle. Amounts of amines up to 1.5 pg can be separated from a large excess of other compounds by this method (Fig. 1). The yield for simple amines varies between 58 and 9%) with an average, for the eight compounds tested, of 81%, higher than that for amines obtained by extraction methods (Table 1). Amine loss is due to
BIOGENIC TABLE OCCURRENCE
Organisms
a Presence
+ (+);
absence
3
OF BIOGENIC AMINES IN CULTURE NATURAL WATERS OF SOME ALGAE
MA
Ochrornonas Nitzschia Ankistrodesmus Microcystis Chlamydomonas
371
AMINES
MEDIA
AND
TMA
l-AP
2-AP
EOA
BA
+ -
+ + + + -
+ -
+ -
+
(-)
uncontrolled adsorption on the surface of the sublimation apparatus and to the formation of aerosols. The yield is dependent on the sublimation time: Maximum yields were obtained, for example, for 1-aminopropane after 2.5 hr if a j-ml sample was used (Fig. 2). Good results were obtained for volatile amines, including diaminopentane. Tryptamine, tyramine, cysteamine, and histamine were not sublimable in significant amounts using a vacuum of 0.06 Torr. The sublimate is a very pure mixture of amines that is contaminated by only minor amounts of other substances. Although this method is very advantageous for analysis by gas-liquid chromatography, very high-boiling amines cannot be separated this way. An alternative procedure for the latter compounds is the extraction of amines with solvents, one of which, butanol- 1, produced the best results in comparison with diethyl ether and chloroform. Gas-liquid chromatographic analysis of amine fractions obtained by both extraction and sublimation methods revealed that the former contained a larger amount of contaminating substances than the latter. Gas-liquid Biogenic
Chromatographic Amines
and Mass
Spectrometric
Analysis
of
A very convenient method for analyzing low-boiling amines by gas-liquid chromatography is direct head-space analysis after decomposition from their salts with alkali (Fig. 3). Although this method simplifies the mass spectrometric analysis, quantification of gas analyses is rather difficult. To substantially reduce handling intricacies, the conversion of the hydrochloride salts to the free amines on base-loaded columns was suggested for the lower aliphatic amines (26). However, this system is not favorable to the separation of free-form bifunctional amines. Pailer and Htibsch (16) have used the trifluoroacetyl derivatives for the separation of primary and secondary amines, and much better results were obtained
372
HERRMANN
AND
JijTTNER
when this technique was applied to the diamines. The addition of pyridine, as stated by the authors, had no effect on the reactivity of the amine hydrochlorides under the conditions used and was, therefore, omitted. A test chromatogram of 12 biogenic trifluoroacetamides is shown in Fig. 4. The mass spectrometric identification of these biogenic amines by the relative intensity of typical fragment ions was possible in all cases (Table 2). The molecular ions of the amines studied were either of low intensity or nondetectable. With the exception of P-phenylethylamine, cysteamine, ethanolamine, and methylamine, the formation of the fragment ion m/e 114 (CF,CONH,) was very characteristic. Primary aliphatic amines exhibited the corresponding homologous series m/e 126, 140 . . . (CF,CONHCH, ). The fragmentation pattern of trifluoroacetylated amines was intensively discussed by Saxby (18,19). Biogenic
Amines in Algal Culture Media
and in Natural
Waters
Of the previously mentioned biogenic amines, methylamine, trimethylamine, I-aminopropane, 2-aminopropane, ethanolamine, and butanolamine were detected both in algal culture media and in Ruhr River water which contained a Chlamydomonas bloom. The presence of these amines in natural waters and in culture media of various algae is indicated in Table 3. One of the excretion products, 1-aminopropane, was found to have a wide distribution. It was detected in the media of four different classes of algae, whereas trimethylamine and methylamine, which previously had been observed frequently in algae (17,23), were only detected once each in two different species. Other biogenic amines, e.g., 2-aminopropane and butanolamine, had not been found previously in algae. The algae investigated not only excrete a wide spectrum of various amines, but those amines which they liberate exhibit a characteristic composition. The amine concentration in the medium at the beginning of the stationary phase of algal growth was between 300 &liter of I-aminopropane with Ankistrodesmus and 240 PgAiter with Microcystis. With Nitzschia , the medium contained 50 pg/liter of methylamine in addition to 66 pglliter of 1-propylamine. These concentrations are within the range of physiological activity and, thus, may be of ecological importance. The formation of amines by algae has long been postulated, since the odor of fish was often noticed in water reservoirs along with the mass production of algae (22,14); however, analyses for amines were only rarely performed. Rolle et al. (17) reported finding methylamine and dimethylamine in the exhaust air of a mass culture of green algae, and Hartmann and Aufermann (4) detected 3-methylbutylamine in the medium after feeding a marine red alga, Polysiphonia urceolata, with labeled leucine.
BIOGENIC
AMINES
373
The appearance of biogenic amines in water can, accordingly, be caused directly by algae, and must not necessarily be the result of the bacterial decomposition of harmless algal excretion products which have been detected in large numbers (1,8). The occurrence of amines in the medium of an axenic culture of Ochrumonas is proof that they are formed by the algae themselves. ACKNOWLEDGMENT We are greatly indebted to the Deutsche Forschungsgemeinschaft
for financial support.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
20. 21. 22. 23. 24. 25. 26. 27.
Chang, W. -H., and Tolbert, N. E. (1970) Plant Physiol. 46, 377-385. Crathorne, B., and Saxby, M. J. (1973) J. Chromatogr. 82, 373-376. Edwards, D. J., and Blau, K. (1972) Anal. Biochem. 45, 387-402. Hartmann, T., and Aufermann, B. (1973) Mar. Biol. 21, 70-74. Hatano, H., Sumizu, K., Rokushika, S., and Murakami, F. (1970) Anal. Biochem. 35, 377-383. Bert. H. J., and Hartmann. T. (1972) J. Chromatogr. 71, 119- 125. Irvine, W. J., and Saxby, M. J. (1969) Phytochemistty 8,473-476. Jiittner. F.. and Friz, R. (1974) Arch. Microbial. 96, 223-232. Jtittner, F. (1976) Z. Natur Forsch. 31c, 491-495. Kanazawa, T., Yanagisawa, T., and Tamiya. H. (1966) Z. PJlanzenphysiol. 54, 57-62. Karoum, F., Cattabeni, F., Costa, E., Ruthven, C. R. J., and Sandler, M. (1972) Anal. Biochem. 47, 550-561. Knights, B. A. (1%7) J. Gas Chromatogr. 5, 273. Kuhl, A., and Lorenzen, H. (1964) in Methods in Cell Physiology (Prescott, D. M., ed.). Vol. 1, p. 159, Academic Press, New York. Liebmann, H. (1960) Handbuch der Frischwasser- und Abwasser-Biologie. Vol. 2, R. Oldenburg, Munchen. Miiller, H. (1972) Arch. Hydrobiol. Suppl. 38, 399-484. Pailer, M., and Hiibsch, W. J. (1%6) Monatsh. Chem !l7, 1541-1553. Rolle, J., Payer, R., and Soeder, C. J. (1971) Arch. Mikrobiol. 77, 185-195. Saxby, M. J. (1969) Org. Muss Specfrom. 2, 33-36. Saxby, M. J. (1969) Org. Mass Spectrom. 2, 835-842. Seiler, N., and Wiechmann, M. (1965) Experienria 21, 203-204. Severge. A., Jiittner, F., Breitmaier, E., and Jung, G. (1976) Biochim. Biophys. Acta. 437, 289-300. Seydel, E. (1914) Mitt. Fisch. Ver. Prov. Brandenburg 5, 87-91. Smith, T. A. (1975) Phytochemistry 14, 865-890. Stein von Kamienski, E. (1957) PIantu 50, 291-314. Steiner, M., and Hartmann, T. (1968) Planta 79, 113- 121. Umbreit, G. R., Nygren, R. E., and Testa, A. I. (1969) J. Chromatogr. 43, 25-32. Wang, L. C. (1972) Plant Physiol. 50, 152- 156.
78, 365-373 (1977)
BIOCHEMISTRY
Excretion
Products of Algae
Identification of Biogenic Amines by Gas-Liquid Chromatography and Mass Spectrometry of Their Trifluoroacetamides V. HERRMANN Institut
fiir Chemische Corrensstrasse
AND F. J~~TTNER
Pjlanzenphysiologie 41, 7400 Tiibingen.
der Universitiit, Germany
Received May 5, 1976: accepted November 5. 1976 The trifluoroacetyl derivatives of 12 biogenic amines were separated by gas-liquid chromatography and identified by mass spectrometry. Vacuum sublimation was used to separate nanomole quantities of amines from algal media and natural waters that contained algae. Trimethylamine, I-aminopropane, 2-aminopropane, and ethanolamine were found in the culture medium of Ochromonas. Chlamydomonas-enriched backwater contained butanolamine. I-Aminopropane was found in the media of Nitzschia, Ankistrodesmus. and Microcystis. Concentrations were approximately 300 pg/liter for Ankistrodesmus and 240 pg/liter for Microcystis media.
Many different methods have been described for the separation of biogenic amines: Some of the more typical are briefly mentioned. Stein von Kamienski (24) separated the amines by paper chromatography, and Ilert and Hartmann (6) used their dinitrophenyl derivatives for separation by thin-layer chromatography. A tic system for dansylated amines was worked out by Seiler and Wiechmann (20). Kanazawaet al. (lo), Hatano et al. (5) and Wang (27) used ion-exchange columns to separate free mono-, di-, and polyamines. For gas-liquid chromatographic (glc) separations of selected amines, Edwards and Blau (3) employed the dinitrophenyl derivatives, Knights (12), the silyl derivatives, and Pailer and Hiibsch (16) and Irvine and Saxby (7), the acyl derivatives. Of the latter derivation methods, the trifluoroacetylation is very advantageous because of its reliability and rapid quantitative reaction. Since the products which were formed exhibited high volatility and good glc separation properties, we employed this method for the separation of biogenic amines excreted by algae. A further advantage of these derivatives is their informative mass fragmentation pattern which was to be expected according to the observations of Karoum et al. (11) with phenolic amines and Crathorne and Saxby (2) with some alkylamines. Combining gas-liquid chromatography with mass spectrometry facilitates the identification of unknown biogenic amines even if they are only available in the nanomole range. 365 Copyright All
riehts
0 of
1977 revroduction
by
Academic in any
Press.
Inc.
form
reserved.
ISSN
ooO3-2697
366
HERRMANN
AND JijTTNER
EXPERIMENTAL Growth of algae. The algae,Ankistrodesmus braunii 202-7c, Microcystis aeruginosa LB 1450-1, and Ochromonas danica L 933-7 were obtained
from the Sammlung von Algenkulturen des Pllanzenphysiologischen Instituts der Universitst Giittingen, Germany. The strain of Nitzschia actinastroides used was an isolate from the Lake of Konstance (South-West Germany). Chlamydomonas-enriched water was collected during a bloom of this organism in the backwaters of the Ruhr River near Geisecke.’ Precultivation of the algae was performed in 300-ml culturing tubes (13) and 30-liter tower-type cultivation plants (9) were used for the subsequent mass cultivation. The details of the cultivation parameters used were similar to those as reported for Ochromonas by Jiittner and Friz (8)) and for Ankistrodesmus by Severge et al. (21) for Microcystis (Aphanizomenon flos-aquae strain Hindak 1963/143 Laborator Experimentalni Algologie, Tieboii, &SR) by Jiittner (9), and for Nitzschia by Miiller (15). The algae were harvested during the declining logarithmic growth. Separation of the culture media from the algae was performed with a continuous-flow centrifuge. Isolation of amines by vacuum sublimation. For isolation of the amines, the algal medium (25-30 liter) was acidified (pH 1) with HCl and taken to dryness in a rotary flash evaporator. Early in this investigation, the amines were isolated by extraction with butanol-1. After the addition of 50 ml of butanol- 1,2 N NaOH was added, and the butanol layer was separated and acidified. The amines were concentrated as their hydrochlorides and, as such, were prepared for head-space analysis. Later, the separation process was improved by application of vacuum sublimation. For this, 1 ml of the amine hydrochloride solution was transferred into a 50-ml round-bottom flask with a ground-glass joint; it was frozen, overlayered with 1 ml of distilled water, refrozen, and then overlayered with 1 ml of 2 N NaOH. The flask was immediately fitted to an all stainless steel vacuum sublimation apparatus. To start the reaction, the contents of the flask were thawed out to ensure adequate mixing. Before sublimation (2.5 hr, 0.06 Torr) was started, the sample was frozen again to prevent bumping. The trap was cooled with liquid nitrogen. After sublimation was completed, 25 ml of 1 N HCl was poured into the trap immediately after opening it. The amine hydrochlorides were concentrated, transferred into l-ml conical reaction vials, and taken to dryness under vacuum, after which 50-~1 each of ethyl acetate and trifluoroacetic anhydride were added. The reaction was 1 We are greatly indebted to Dr. H. Miiller, Limnologisches Institut der Universitat Freiburg, 775 Konstanz-Egg, for providing a culture of Nirzschiu actinasrroides and to Dipl. Biol. G. Klein, Institut fiir Wasserforschung, 5841 Geisecke, for collecting Chlumydomonasenriched water.
BIOGENIC
AMINES
367
1' 1
10
100
1000
pg 1 -aminopropanc FIG. 1. Detector response as a function of sample size of I-aminopropane vacuum sublimation.
separated by
completed within 10 min at 80°C. From each sample, a 2-4 vol was taken and injected into the gas chromatograph. Gas-chromatographic separation of the biogenic amines. The gasliquid chromatographic separation of the trifluoroacetamides and lowboiling free amines was performed on a 1.8m x 3.2mm (i.d.) column packed with 3% OV-17 on high-performance Chromosorb W AW DMCS (80- 100 mesh). After a 3-min postinjection interval at 50°C the temperature was increased at a rate of lO”C/min until it reached 250°C. Both the flame ionization detector and injection port temperatures were maintained at 28O”C, and nitrogen was used as the carrier gas (30 ml/min). The oncolumn decomposition of the amine hydrochlorides (1 ~1) was performed at 30°C according to Umbreit et al. (26). Tertiary, highly volatile amines TABLE RECOVERY
Compoundb MA IBA 3-MBA 1,5-PA
1
OF BIOGENIC
AMINE@
Percentage of recovery (%)
Compound
Percentage of recovery (%)
99 8.5 74 83
I-AP 2-AP EOA PEA
82 83 58 83
LIBiogenic amines (500-pg samples) were recovered after sublimation for 2.5 hr at 0.06 Torr. b Abbreviations used: MA, methylamine; TMA, trimethylamine; IBA, isobutylamine = I-amino-2-methyl-propane; 3-MBA, 3-methylbutylamine; I-AP, I-aminopropane; 2-AP, 2-aminopropane; EOA, ethanolamine; PEA, P-phenylethylamine; 1,4-BA, 1,4-diaminobutane; 1J-PA, 1,5-diaminopentane; BA, butanolamine.
368
HERRMANN
AND JijTTNER
6o- k-+--l ' .c f y fL
60-
LO-
20 -
0
I 30
FIG. 2. Recovery of I-aminopropane
1 60
u 90
I 120
1 150
1 160
min
as a function of the sublimation time (4-mg samples
2-AP
I-AP h
16 min FIG. 3. Head space analysis of biogenic amines of Ochromonas culture medium (3% OV-17 on Chromosorb W AW DMCS, 30°C. 30 ml of N,/min). TMA, trimethylamine: 2-AP, 2-aminopropane; I-AP. I-aminopropane.
BIOGENIC
369
AMINES
-
1
z
I
I
I
4
8
12
I
16 min
FIG. 4. Gas chromatogmm of an authentic mixture of 12 trifluoroacetylated biogenic amines (3% OV-17 on Chromosorb W AW DMCS, postinjection interval of 3 mitt, temperature progression: 50-200°C rate: 6”C/min, carrier gas: 30 ml of N,/min). (1) Ethylamine, (2) 2-aminopropane, (3) I-aminopropane, (4) isobutylamine, (5) 2-methylbutylamine, (6) (8) cysteamine, (9) butanolamine, (10) 3-methylbutylamine, (7) ethanolamine, P-phenylethylamine, (11) I .4-diaminobutane, (12) 1.5diaminopentane
were examined by head-space analysis in 5-ml vials sealed with siliconerubber septa. After the addition of 1 ml of 2 N NaOH to the amine hydrochlorides, the sample was heated to 5O”C, and 1 ml of gas was injected into the gas chromatograph. Absolute values of amines were calculated from peak areas, in comparison to authentic samples. RESULTS AND DISCUSSION Preparation of Arnines
Selective separation of nanomole amounts of biogenic amines from algal culture media and water from natural sources is complicated by the
370
HERRMANN
AND JijTTNER
TABLE MASS
SPECTRA
OF
13 TRIFLUOROACETAMIDES TEN
Compound MA
EA
141
I-AP
MOST
INTENSE
MOlfSUlElI weight 127
155
2 IN FRAGMENT
Fragment
THE
SEQUENCE
OF THEIR
IONS
ion (m/e)
58 ( IW”
69 (38.4)
78 (7.6)
45 (6.7)
50 (4.8)
59 (3.8)
51
126 (loo)
141 (65.4)
69 (63.1)
72 (53.5)
44 (34.5)
78 (16.6)
45 (11.9)
126 (loo)
69 (32.2)
43 (25.0)
140 (19.6)
41 (19.6)
114 (17.9)
42 (16.1)
78 (14.3)
86 (13.4)
49
0.8)
97 (2.8)
33 (2.8)
51 (7. I)
97
(6.5)
56 (1.9) 140 (6.5)
(9.8)
2-AP
155
140 (100)
69 (35.7)
43 (33.6)
41 (22.1)
45 (20.0)
42 (17.8)
70 (13.6)
155 (12.6)
47 (10.5)
86 (9.4)
IBA
169
127 (100)
56 (77.8)
126 (62.1)
41 (53.5)
43 (50.7)
58 (39.2)
69 (37.8)
57 (2 I .4)
114 (15.0)
loo (12.8)
2-MBA
183
127 (loo)
41 (61.4)
57 (46.0)
70 (37.4)
69 (30.0)
126 (34.0)
55 (22.7)
58 (21.4)
114 (18.0)
71 (12.7)
3.MBA
183
55 (100)
126 (90.9)
70 WO)
127 (81.8)
41 (65.9)
57 (63.6)
43 (54.5)
69 (47.7)
114 (3 1.8)
42 (27.2)
EOA
253
126 (100)
69 (82.9)
70 (35.4)
139 (30.3)
140 (15.8)
78 (13.9)
42 (10.1)
97 (6.9)
184 (5.7)
47 (5.7)
BA
281
126 mo)
55 (36.3)
69 (34.6)
139 (20.8)
54 (16.4)
98 (11.5)
41 (10.2)
78 (7.8)
167 (6.4)
140 (6.0)
CA
269
126 (1cQ)
69 (76.8)
156 (54.6)
172 (28.7)
78 (17.5)
140 (13.8)
59 13.8
87 (12.9)
157 (10.1)
139 (10.1)
PEA
217
104 (100)
91 (63.7)
105 (12.5)
65 (12.5)
69 (10.0)
126 (7.5)
92 (7.5)
78 (6.2)
77 (6.2)
51 (6.2)
I .4-BA
280
126 (loo)
167 (53.4)
69 (41.5)
127 (34. I)
55 (28.2)
166 (17.8)
151 (16.3)
54 (15.5)
41 (15.5)
78 (12.6)
I.5-PA
294
126 (100)
68 (63.5)
69 (56.1)
55 (41.5)
127 (3 I .8)
41 (29.3)
168 (26.8)
loo (22.0)
180 (14.6)
140 (14.6)
a Number within parentheses is the percentage of relative intensity.
large amount of salts which appear after concentration. Although alkaline steam distillation is a very useful method for the separation of amines from nonvolatile compounds, this procedure separates diamines and alkanolamines only to a limited extent (24), and some of them may be produced by uncontrolled degradation of other compounds (25). Lowering the temperature, however, reduces the reactivity and hence, the formation of degradation products. Therefore, we employed vacuum sublimation which was selective and gentle. Amounts of amines up to 1.5 pg can be separated from a large excess of other compounds by this method (Fig. 1). The yield for simple amines varies between 58 and 9%) with an average, for the eight compounds tested, of 81%, higher than that for amines obtained by extraction methods (Table 1). Amine loss is due to
BIOGENIC TABLE OCCURRENCE
Organisms
a Presence
+ (+);
absence
3
OF BIOGENIC AMINES IN CULTURE NATURAL WATERS OF SOME ALGAE
MA
Ochrornonas Nitzschia Ankistrodesmus Microcystis Chlamydomonas
371
AMINES
MEDIA
AND
TMA
l-AP
2-AP
EOA
BA
+ -
+ + + + -
+ -
+ -
+
(-)
uncontrolled adsorption on the surface of the sublimation apparatus and to the formation of aerosols. The yield is dependent on the sublimation time: Maximum yields were obtained, for example, for 1-aminopropane after 2.5 hr if a j-ml sample was used (Fig. 2). Good results were obtained for volatile amines, including diaminopentane. Tryptamine, tyramine, cysteamine, and histamine were not sublimable in significant amounts using a vacuum of 0.06 Torr. The sublimate is a very pure mixture of amines that is contaminated by only minor amounts of other substances. Although this method is very advantageous for analysis by gas-liquid chromatography, very high-boiling amines cannot be separated this way. An alternative procedure for the latter compounds is the extraction of amines with solvents, one of which, butanol- 1, produced the best results in comparison with diethyl ether and chloroform. Gas-liquid chromatographic analysis of amine fractions obtained by both extraction and sublimation methods revealed that the former contained a larger amount of contaminating substances than the latter. Gas-liquid Biogenic
Chromatographic Amines
and Mass
Spectrometric
Analysis
of
A very convenient method for analyzing low-boiling amines by gas-liquid chromatography is direct head-space analysis after decomposition from their salts with alkali (Fig. 3). Although this method simplifies the mass spectrometric analysis, quantification of gas analyses is rather difficult. To substantially reduce handling intricacies, the conversion of the hydrochloride salts to the free amines on base-loaded columns was suggested for the lower aliphatic amines (26). However, this system is not favorable to the separation of free-form bifunctional amines. Pailer and Htibsch (16) have used the trifluoroacetyl derivatives for the separation of primary and secondary amines, and much better results were obtained
372
HERRMANN
AND
JijTTNER
when this technique was applied to the diamines. The addition of pyridine, as stated by the authors, had no effect on the reactivity of the amine hydrochlorides under the conditions used and was, therefore, omitted. A test chromatogram of 12 biogenic trifluoroacetamides is shown in Fig. 4. The mass spectrometric identification of these biogenic amines by the relative intensity of typical fragment ions was possible in all cases (Table 2). The molecular ions of the amines studied were either of low intensity or nondetectable. With the exception of P-phenylethylamine, cysteamine, ethanolamine, and methylamine, the formation of the fragment ion m/e 114 (CF,CONH,) was very characteristic. Primary aliphatic amines exhibited the corresponding homologous series m/e 126, 140 . . . (CF,CONHCH, ). The fragmentation pattern of trifluoroacetylated amines was intensively discussed by Saxby (18,19). Biogenic
Amines in Algal Culture Media
and in Natural
Waters
Of the previously mentioned biogenic amines, methylamine, trimethylamine, I-aminopropane, 2-aminopropane, ethanolamine, and butanolamine were detected both in algal culture media and in Ruhr River water which contained a Chlamydomonas bloom. The presence of these amines in natural waters and in culture media of various algae is indicated in Table 3. One of the excretion products, 1-aminopropane, was found to have a wide distribution. It was detected in the media of four different classes of algae, whereas trimethylamine and methylamine, which previously had been observed frequently in algae (17,23), were only detected once each in two different species. Other biogenic amines, e.g., 2-aminopropane and butanolamine, had not been found previously in algae. The algae investigated not only excrete a wide spectrum of various amines, but those amines which they liberate exhibit a characteristic composition. The amine concentration in the medium at the beginning of the stationary phase of algal growth was between 300 &liter of I-aminopropane with Ankistrodesmus and 240 PgAiter with Microcystis. With Nitzschia , the medium contained 50 pg/liter of methylamine in addition to 66 pglliter of 1-propylamine. These concentrations are within the range of physiological activity and, thus, may be of ecological importance. The formation of amines by algae has long been postulated, since the odor of fish was often noticed in water reservoirs along with the mass production of algae (22,14); however, analyses for amines were only rarely performed. Rolle et al. (17) reported finding methylamine and dimethylamine in the exhaust air of a mass culture of green algae, and Hartmann and Aufermann (4) detected 3-methylbutylamine in the medium after feeding a marine red alga, Polysiphonia urceolata, with labeled leucine.
BIOGENIC
AMINES
373
The appearance of biogenic amines in water can, accordingly, be caused directly by algae, and must not necessarily be the result of the bacterial decomposition of harmless algal excretion products which have been detected in large numbers (1,8). The occurrence of amines in the medium of an axenic culture of Ochrumonas is proof that they are formed by the algae themselves. ACKNOWLEDGMENT We are greatly indebted to the Deutsche Forschungsgemeinschaft
for financial support.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
20. 21. 22. 23. 24. 25. 26. 27.
Chang, W. -H., and Tolbert, N. E. (1970) Plant Physiol. 46, 377-385. Crathorne, B., and Saxby, M. J. (1973) J. Chromatogr. 82, 373-376. Edwards, D. J., and Blau, K. (1972) Anal. Biochem. 45, 387-402. Hartmann, T., and Aufermann, B. (1973) Mar. Biol. 21, 70-74. Hatano, H., Sumizu, K., Rokushika, S., and Murakami, F. (1970) Anal. Biochem. 35, 377-383. Bert. H. J., and Hartmann. T. (1972) J. Chromatogr. 71, 119- 125. Irvine, W. J., and Saxby, M. J. (1969) Phytochemistty 8,473-476. Jiittner. F.. and Friz, R. (1974) Arch. Microbial. 96, 223-232. Jtittner, F. (1976) Z. Natur Forsch. 31c, 491-495. Kanazawa, T., Yanagisawa, T., and Tamiya. H. (1966) Z. PJlanzenphysiol. 54, 57-62. Karoum, F., Cattabeni, F., Costa, E., Ruthven, C. R. J., and Sandler, M. (1972) Anal. Biochem. 47, 550-561. Knights, B. A. (1%7) J. Gas Chromatogr. 5, 273. Kuhl, A., and Lorenzen, H. (1964) in Methods in Cell Physiology (Prescott, D. M., ed.). Vol. 1, p. 159, Academic Press, New York. Liebmann, H. (1960) Handbuch der Frischwasser- und Abwasser-Biologie. Vol. 2, R. Oldenburg, Munchen. Miiller, H. (1972) Arch. Hydrobiol. Suppl. 38, 399-484. Pailer, M., and Hiibsch, W. J. (1%6) Monatsh. Chem !l7, 1541-1553. Rolle, J., Payer, R., and Soeder, C. J. (1971) Arch. Mikrobiol. 77, 185-195. Saxby, M. J. (1969) Org. Muss Specfrom. 2, 33-36. Saxby, M. J. (1969) Org. Mass Spectrom. 2, 835-842. Seiler, N., and Wiechmann, M. (1965) Experienria 21, 203-204. Severge. A., Jiittner, F., Breitmaier, E., and Jung, G. (1976) Biochim. Biophys. Acta. 437, 289-300. Seydel, E. (1914) Mitt. Fisch. Ver. Prov. Brandenburg 5, 87-91. Smith, T. A. (1975) Phytochemistry 14, 865-890. Stein von Kamienski, E. (1957) PIantu 50, 291-314. Steiner, M., and Hartmann, T. (1968) Planta 79, 113- 121. Umbreit, G. R., Nygren, R. E., and Testa, A. I. (1969) J. Chromatogr. 43, 25-32. Wang, L. C. (1972) Plant Physiol. 50, 152- 156.