ANALYTICAL
BIOCHEMISTRY
90, 359-364 (1978)
Identification of Keto Acids in Arctic Bramble, Rubus arcticus L. as Methyl Esters of their 2,4-Dinitrophenylhydrazones HEIKKI
KALLIO,
* REINO R. LINKO, * TAPANI AND IRMELI PUNTARI*
PYYSALO, t
*Depariment of Chemistry and BiochemistrylLuboratory of Food Chrmistty, Unibvrsiiy Turku. SF-20500 Turku 50. Finland, and TTechnical Research Centre of Finland, Laboratory for Food Research and Technology, SF-02150 Espoo 15, Finland
of
Received May 9. 1978 The major keto acids in arctic bramble. Rubus art’tic,us L. were investigated. The acids were isolated with anionic and cationic ion-exchange resins, converted to 2,4-dinitrophenylhydrazones, and purified with an Al,O:, column. The derivatives were separated on a silica gel G thin-layer plate and esterified with methanal-HCI and the methyl esters of the keto acid 2,4-dinitrophenylhydrazones formed were analyzed on an OV-l-glass capillary gas-liquid chromatography column and with mass spectrometry. 2-Oxoglutaric, pyruvic. oxaloacetic, and glyoxylic acids were identified. The mass spectra of the derivatives are presented.
Only a few studies concerning the contents of keto acids in fruits and berries have been carried out. Wyman and Palmer (1) detected eight keto acids in banana fruit and identified pyruvic, 2-oxoglutaric. oxaloacetic, and glyoxylic acids. The same acids also have been identified in apple (2) and grape (3). Heatherbell (4) identified 2-oxoglutaric and oxaloacetic acids in grapefruit, mountain pawpaw, tangelo, and apricot; Johnson and Carroll (5) determined pyruvate in grape; and Chan et al. (6) identified 2-oxoglutaric acid in papaya. Most methods of keto acid analysis in fruits and berries are based on paper chromatography and thin-layer chromatography (tic) (l-3). The analysis of 2,4-DNPHs’ of keto acids as developed by Isherwood and Niavis (7) and Meister and Abendschein (8) for paper chromatography has been further developed for tic and spectrophotometric analysis by Stan and Schormtiller (9). The derivatives commonly used in gas-liquid chromatographic (glc) analysis are methyl esters (10,l l), methyl oximes of methyl or trimethylsilyl esters (12,13), trimethylsilyl oximes of trimethylsilyl esters (14,1.5), or trimethylsilyl derivatives of quinoxalones ’ Abbreviation
used: 2.4-DNPH. 2,4-dinitrophenylhydrazone. 359
OOO3-2697/78/0901-0359$02OO/O Copyright Q 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
360
KALLIO
ET AL.
(16,17). Nowadays, however, the glc methods developed specially for clinical and microbiological purposes are not in common use for the analysis of keto acids in fruits and berries. In a previous paper by Kallio and Linko (18) it was shown that the methyl esters of 2,4-DNPHs of keto acids are very applicable to glc analyses. The aim of the present study was to improve the method further by analyzing these keto acid derivatives with a combined capillary column gas-liquid chromatography-mass spectrometry (glc-ms), applying it to studies of the keto acids of arctic bramble. MATERIALS
AND METHODS
The berries (arctic bramble Rubus arcticus ssp. arcticus L.) were cultivated in central Finland (Agricultural Research Centre South Savo Experimental Station, Mikkeli, Finland). They were frozen immediately after harvesting and stored at -20°C. The keto acids were extracted from the berries by blending with (99.5%) ethanol in a macerator. The homogenate was centrifuged and the acids were further purified by applying the supernate to a Dowex 5OW x 8 (Cl- form) (50- 100 mesh) cation exchanger which removed the anthocyanins and amino acids. The acids were eluted with distilled water and applied to a Dowex 1 x 8 (formate form) (loo200 mesh) anionic exchange column which was washed with distilled water until free of sugars. The keto acids were eluted with 6 N formic acid and the solvent was removed in a vacuum evaporator. The 2,4-DNPHs of the keto acids were prepared and purified in an Al,O, column and separated by tic on silica gel G according to the method of Stan and Schormiiller (9). For glc analyses the 2,4-DNPHs of the keto acids dissolved in ethyl acetate (either after the A1203 purification or after the tic separation) were esterified in 1.75 N methanol-HCl at 55°C for 0.5 h. To remove the water from the reaction mixture dichloromethane, which formed an azeotrope with the water, was added. The solvent was removed with nitrogen gas. The esterification procedure was repeated. The methyl esters of the keto acid 2,4-DNPHs were dissolved in ethyl acetate. The glc analyses of the methyl esters of the keto acid 2,4-DNPHs were carried out on a Varian Aerograph Model 2100-20 gas chromatograph with a flame ionization detector (18). An OV-I glass capillary column, 32 m long and of 0.75 mm i.d., was used. The conditions of the analyses were as follows: injector, 265°C; detector, 255°C; temperature program 200 to 25o”C, 2”/min. The flow rate of nitrogen, the carrier gas, was 30 mUmin. The glc-ms analyses of the derivatives were carried out on a Jeol JMSDlOO double-focusing mass spectrometer by using a packed 1% OV-1 column, 1 m long and of 3 mm i.d., and a temperature programming from 180 to 25o”C, 2”/min. The double-stage jet separator of the mass spectrometer was maintained at 250°C and the ion source at 280°C. The
IDENTIFICATION
361
OF KETO ACIDS
ionization energy was 70 eV and the electron current 300 PA. A scanning speed of 5 s from mle 1 to mle 400 was used. RESULTS AND DISCUSSION
Thin-layer chromatography fractionation and glc/ms analyses proved the existence of 2-oxoglutaric, pyruvic, oxaloacetic, and glyoxylic acids in arctic bramble. Figure 1 shows the tic chromatogram of keto acid 2,4DNPHs from ripe berries (B) and that of the reference mixture composed of synthetic compounds (A). The main components derived from arctic bramble appear clearly separated and the most abundant compound is 2oxoglutaric acid. Also pyruvic, oxaloacetic, and glyoxylic acids are clearly visible. The existence of 2-oxobutyric acid is possible. In glc analyses the methyl esters of keto acid 2,4-DNPHs were also well separated in an OV-1 capillary column. In addition, these keto acid derivatives were more stable than. e.g., the trimethylsilyl esters of keto acids (15), which are instable in the presence of water. The monocarboxylic keto acid derivatives caused double peaks because of isomerism.
cm -- 15
--10
B FIG. 1. Thin-layer chromatograms on silica gel G of the 2,4-DNPHs of keto acids derived from a mixture of reference compounds (A) and from ripe arctic bramble (B). Reference compounds: oxaloacetic acid (l), 2-oxoglutaric acid (2). glyoxylic acid (3). pyruvic acid (4). and 2-oxobutyric acid (5). Solvent: ethyl formate-petroleum ether (bp 60-8o”C)acetic acid (50:50:7. v/v/v).
362
KALLIO
ET AL.
IDENTIFICATION
OF KETO
ACIDS
8
364
KALLIO
ET AL.
With columns longer than 25 m we had problems in analyzing the dicarboxylic keto acids. In some chromatograms 2-oxobutyric acid was also tentatively identified. In Fig. 2 the mass spectra a, b, c, and d of the methyl esters of keto acid 2,4-DNPHs are shown representing glyoxylic, pyruvic, oxaloacetic, and 2-oxoglutaric acids, respectively. Spectra a through c were obtained from compounds isolated from the berries but spectrum d, representing 2oxoglutaric acid, was obtained using the reference compound. In the case of the derivatives of 2-oxoglutaric acid isolated from the berries a disturbing background was observed in the spectrum. Spectra a through c were identical with those of the authentic compounds used for reference. However, the 2,4-DNPHs are easily recognized by the lower part of the spectra. Some of the fragments are analogous with the spectra of 2,4DNPHs of both carbonyl compounds (aldehydes, ketones) (19) and methyl esters of mono- and dicarboxylic keto acids. These are m/e 51,63, 75, 77, 79, 91, 105, 122, 152, 180, and 181. Some of these are typical signs of aromatic compounds as m/e 51, (C,H,), 63 (C,H,), and 77 (C,H,). The fragments m/e 122, 135, 152, and 180 to 182 are especially dominant in the spectra of the methyl esters of the keto acid 2,4-DNPHs and can be used as specific signals in mass fragmentography. We found the method described above useful in the exact qualitative analysis of keto acids in biological material especially because of the ms analysis. The suitability of this method for quantitative analysis of keto acids has to be studied further. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Wyman, H., and Palmer. J. K. (1964) Plant Physiol. 39, 630. Kollas, D. A. (1964) Nature (London) 204, 758. Kliewer, W. M. (1966) P/ant Physiol. 41, 923. Heatherbell, D. A. (1974) J. Sci. Food Agr. 25, 1095. Johnson, L. A., and Carroll, D. E. (1973) J. Food Sci. 38, 21. Chan, H. T. Jr., Chang, T. S. K., Stafford, A. E., and Brekke, J. E. (1971)J. Agr. Food Chem. 19, 263. Isherwood, F. A., and Niavis, C. A. (1956) Biochem. J. 64, 549. Meister. A., and Abendschein, P. A. 1956) Anal Chem. 28, 171. Stan, H.-J., and Schormiiller, J. (1969) J. Chromatogr. 43, 103. Estes, F. L., and Bachmann, R. C. (1966) Anal. Chem. 38, 1178. Makita, M., and Wells, W. W. (1963) Anal. Biochem. 5, 523. Ishitoya, Y., Itoh, C.. Osawa, N., Hashimoto, L., Iwanaga, T., and Nambara, T. (1970) Clin. Chim. Acta 21, 233. Homing, M. Cl., Boucher, E. A., Moss. A. M., and Homing, E. C. (1968) Anaf. Left. 1, 713. Horii, Z., Makita, M., and Tamura, Y. (1965) Chem. Ind. 34, 1494. Rosenqvist. H., Kallio, H., and Nurmikko. V. (1972) Anal. Biochem. 46, 224. Hoffman, N. E., and Killinger, T. A. (1969) Anal. Chem. 41, 162. Langenbeck, U., Mohring. H.-U., and Dieckmann, K.-P. (1975)J. Chromatogr. 115,65. Kaiho, H., and Linko, R. (1973) J. Chromatogr. 76, 229. Stanley, J. B.. Brown, D. F.. Senn, V. J., and Dollear, F. G. (1975)J. FoodSci. 40, 1134.
BIOCHEMISTRY
90, 359-364 (1978)
Identification of Keto Acids in Arctic Bramble, Rubus arcticus L. as Methyl Esters of their 2,4-Dinitrophenylhydrazones HEIKKI
KALLIO,
* REINO R. LINKO, * TAPANI AND IRMELI PUNTARI*
PYYSALO, t
*Depariment of Chemistry and BiochemistrylLuboratory of Food Chrmistty, Unibvrsiiy Turku. SF-20500 Turku 50. Finland, and TTechnical Research Centre of Finland, Laboratory for Food Research and Technology, SF-02150 Espoo 15, Finland
of
Received May 9. 1978 The major keto acids in arctic bramble. Rubus art’tic,us L. were investigated. The acids were isolated with anionic and cationic ion-exchange resins, converted to 2,4-dinitrophenylhydrazones, and purified with an Al,O:, column. The derivatives were separated on a silica gel G thin-layer plate and esterified with methanal-HCI and the methyl esters of the keto acid 2,4-dinitrophenylhydrazones formed were analyzed on an OV-l-glass capillary gas-liquid chromatography column and with mass spectrometry. 2-Oxoglutaric, pyruvic. oxaloacetic, and glyoxylic acids were identified. The mass spectra of the derivatives are presented.
Only a few studies concerning the contents of keto acids in fruits and berries have been carried out. Wyman and Palmer (1) detected eight keto acids in banana fruit and identified pyruvic, 2-oxoglutaric. oxaloacetic, and glyoxylic acids. The same acids also have been identified in apple (2) and grape (3). Heatherbell (4) identified 2-oxoglutaric and oxaloacetic acids in grapefruit, mountain pawpaw, tangelo, and apricot; Johnson and Carroll (5) determined pyruvate in grape; and Chan et al. (6) identified 2-oxoglutaric acid in papaya. Most methods of keto acid analysis in fruits and berries are based on paper chromatography and thin-layer chromatography (tic) (l-3). The analysis of 2,4-DNPHs’ of keto acids as developed by Isherwood and Niavis (7) and Meister and Abendschein (8) for paper chromatography has been further developed for tic and spectrophotometric analysis by Stan and Schormtiller (9). The derivatives commonly used in gas-liquid chromatographic (glc) analysis are methyl esters (10,l l), methyl oximes of methyl or trimethylsilyl esters (12,13), trimethylsilyl oximes of trimethylsilyl esters (14,1.5), or trimethylsilyl derivatives of quinoxalones ’ Abbreviation
used: 2.4-DNPH. 2,4-dinitrophenylhydrazone. 359
OOO3-2697/78/0901-0359$02OO/O Copyright Q 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
360
KALLIO
ET AL.
(16,17). Nowadays, however, the glc methods developed specially for clinical and microbiological purposes are not in common use for the analysis of keto acids in fruits and berries. In a previous paper by Kallio and Linko (18) it was shown that the methyl esters of 2,4-DNPHs of keto acids are very applicable to glc analyses. The aim of the present study was to improve the method further by analyzing these keto acid derivatives with a combined capillary column gas-liquid chromatography-mass spectrometry (glc-ms), applying it to studies of the keto acids of arctic bramble. MATERIALS
AND METHODS
The berries (arctic bramble Rubus arcticus ssp. arcticus L.) were cultivated in central Finland (Agricultural Research Centre South Savo Experimental Station, Mikkeli, Finland). They were frozen immediately after harvesting and stored at -20°C. The keto acids were extracted from the berries by blending with (99.5%) ethanol in a macerator. The homogenate was centrifuged and the acids were further purified by applying the supernate to a Dowex 5OW x 8 (Cl- form) (50- 100 mesh) cation exchanger which removed the anthocyanins and amino acids. The acids were eluted with distilled water and applied to a Dowex 1 x 8 (formate form) (loo200 mesh) anionic exchange column which was washed with distilled water until free of sugars. The keto acids were eluted with 6 N formic acid and the solvent was removed in a vacuum evaporator. The 2,4-DNPHs of the keto acids were prepared and purified in an Al,O, column and separated by tic on silica gel G according to the method of Stan and Schormiiller (9). For glc analyses the 2,4-DNPHs of the keto acids dissolved in ethyl acetate (either after the A1203 purification or after the tic separation) were esterified in 1.75 N methanol-HCl at 55°C for 0.5 h. To remove the water from the reaction mixture dichloromethane, which formed an azeotrope with the water, was added. The solvent was removed with nitrogen gas. The esterification procedure was repeated. The methyl esters of the keto acid 2,4-DNPHs were dissolved in ethyl acetate. The glc analyses of the methyl esters of the keto acid 2,4-DNPHs were carried out on a Varian Aerograph Model 2100-20 gas chromatograph with a flame ionization detector (18). An OV-I glass capillary column, 32 m long and of 0.75 mm i.d., was used. The conditions of the analyses were as follows: injector, 265°C; detector, 255°C; temperature program 200 to 25o”C, 2”/min. The flow rate of nitrogen, the carrier gas, was 30 mUmin. The glc-ms analyses of the derivatives were carried out on a Jeol JMSDlOO double-focusing mass spectrometer by using a packed 1% OV-1 column, 1 m long and of 3 mm i.d., and a temperature programming from 180 to 25o”C, 2”/min. The double-stage jet separator of the mass spectrometer was maintained at 250°C and the ion source at 280°C. The
IDENTIFICATION
361
OF KETO ACIDS
ionization energy was 70 eV and the electron current 300 PA. A scanning speed of 5 s from mle 1 to mle 400 was used. RESULTS AND DISCUSSION
Thin-layer chromatography fractionation and glc/ms analyses proved the existence of 2-oxoglutaric, pyruvic, oxaloacetic, and glyoxylic acids in arctic bramble. Figure 1 shows the tic chromatogram of keto acid 2,4DNPHs from ripe berries (B) and that of the reference mixture composed of synthetic compounds (A). The main components derived from arctic bramble appear clearly separated and the most abundant compound is 2oxoglutaric acid. Also pyruvic, oxaloacetic, and glyoxylic acids are clearly visible. The existence of 2-oxobutyric acid is possible. In glc analyses the methyl esters of keto acid 2,4-DNPHs were also well separated in an OV-1 capillary column. In addition, these keto acid derivatives were more stable than. e.g., the trimethylsilyl esters of keto acids (15), which are instable in the presence of water. The monocarboxylic keto acid derivatives caused double peaks because of isomerism.
cm -- 15
--10
B FIG. 1. Thin-layer chromatograms on silica gel G of the 2,4-DNPHs of keto acids derived from a mixture of reference compounds (A) and from ripe arctic bramble (B). Reference compounds: oxaloacetic acid (l), 2-oxoglutaric acid (2). glyoxylic acid (3). pyruvic acid (4). and 2-oxobutyric acid (5). Solvent: ethyl formate-petroleum ether (bp 60-8o”C)acetic acid (50:50:7. v/v/v).
362
KALLIO
ET AL.
IDENTIFICATION
OF KETO
ACIDS
8
364
KALLIO
ET AL.
With columns longer than 25 m we had problems in analyzing the dicarboxylic keto acids. In some chromatograms 2-oxobutyric acid was also tentatively identified. In Fig. 2 the mass spectra a, b, c, and d of the methyl esters of keto acid 2,4-DNPHs are shown representing glyoxylic, pyruvic, oxaloacetic, and 2-oxoglutaric acids, respectively. Spectra a through c were obtained from compounds isolated from the berries but spectrum d, representing 2oxoglutaric acid, was obtained using the reference compound. In the case of the derivatives of 2-oxoglutaric acid isolated from the berries a disturbing background was observed in the spectrum. Spectra a through c were identical with those of the authentic compounds used for reference. However, the 2,4-DNPHs are easily recognized by the lower part of the spectra. Some of the fragments are analogous with the spectra of 2,4DNPHs of both carbonyl compounds (aldehydes, ketones) (19) and methyl esters of mono- and dicarboxylic keto acids. These are m/e 51,63, 75, 77, 79, 91, 105, 122, 152, 180, and 181. Some of these are typical signs of aromatic compounds as m/e 51, (C,H,), 63 (C,H,), and 77 (C,H,). The fragments m/e 122, 135, 152, and 180 to 182 are especially dominant in the spectra of the methyl esters of the keto acid 2,4-DNPHs and can be used as specific signals in mass fragmentography. We found the method described above useful in the exact qualitative analysis of keto acids in biological material especially because of the ms analysis. The suitability of this method for quantitative analysis of keto acids has to be studied further. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Wyman, H., and Palmer. J. K. (1964) Plant Physiol. 39, 630. Kollas, D. A. (1964) Nature (London) 204, 758. Kliewer, W. M. (1966) P/ant Physiol. 41, 923. Heatherbell, D. A. (1974) J. Sci. Food Agr. 25, 1095. Johnson, L. A., and Carroll, D. E. (1973) J. Food Sci. 38, 21. Chan, H. T. Jr., Chang, T. S. K., Stafford, A. E., and Brekke, J. E. (1971)J. Agr. Food Chem. 19, 263. Isherwood, F. A., and Niavis, C. A. (1956) Biochem. J. 64, 549. Meister. A., and Abendschein, P. A. 1956) Anal Chem. 28, 171. Stan, H.-J., and Schormiiller, J. (1969) J. Chromatogr. 43, 103. Estes, F. L., and Bachmann, R. C. (1966) Anal. Chem. 38, 1178. Makita, M., and Wells, W. W. (1963) Anal. Biochem. 5, 523. Ishitoya, Y., Itoh, C.. Osawa, N., Hashimoto, L., Iwanaga, T., and Nambara, T. (1970) Clin. Chim. Acta 21, 233. Homing, M. Cl., Boucher, E. A., Moss. A. M., and Homing, E. C. (1968) Anaf. Left. 1, 713. Horii, Z., Makita, M., and Tamura, Y. (1965) Chem. Ind. 34, 1494. Rosenqvist. H., Kallio, H., and Nurmikko. V. (1972) Anal. Biochem. 46, 224. Hoffman, N. E., and Killinger, T. A. (1969) Anal. Chem. 41, 162. Langenbeck, U., Mohring. H.-U., and Dieckmann, K.-P. (1975)J. Chromatogr. 115,65. Kaiho, H., and Linko, R. (1973) J. Chromatogr. 76, 229. Stanley, J. B.. Brown, D. F.. Senn, V. J., and Dollear, F. G. (1975)J. FoodSci. 40, 1134.
