Jounml of Chromatography, 118 (1976) 236-239 0 Efsevier scientific Pubiig Company, Amsterdam CHROM.
-
ES&d
in The Netherian&
8787
Mote Gas ehromatographic
separation of Krebs-cycle
metabdites
A. PZNELLI and A. COLQMBQ Department
of Pharmacology,
UnpJersity of Milan,
Via Vanvirelli 32, Miian
(Italy)
(Received September 24th, 1975)
Gas-liquid chromato,oraphic (GLC) techniques have been used for the evaluation of citric acid-cycle metabolites by sever-a! authors1-15. The simultaneous determination of Krebs-cycle acids by GLC is preferable to conventional enzymic methods because of the reduction in analysis time; these acids are usually analysed as methyl’-’ or silyP-I2 derivatives. Methylation has been performed with methanol and hydrogen chloride’**, methanol and boron trifIuoride3*J or diazomethane1J~54, but, despite the different esterification conditions used, no single method had been reported in which all the compounds are methylated simultaneously. Simple esterification of acids containing double bonds or 0x0 groups is unsuitable because it gives rise to multiple peaksqd_ Silylation associated with oximation appears to be non-destructive9-“; nevertheless, only a few papers report the GLC anaiysis of Krebs-cycle acids as oximes and silyl derivatives’4 methoximes and silyl derivatives” or quinoxalone and sibyl derivatives’*. A few communications report the levels of some citric acid-cycle acids extracted from tissues and measured as methyi esters (not as silyi derivatives); recoveries were variable and 10w’~-‘~. Thus, there is a need of a method for extracting citric acid-cycle acids and evaiuating them as methoxime and silyl derivatives by GLC; this paper is a preliminary report of such a method.
Standard acids (succinic, fumaric, malic, oxalacetic, citric, isocitric, cisaconitic and 2-oxoglutaric) were obtained from BDH (Poole, Great Britain) and Biochemia (Milan, Italy). The solvents were purchased from Carlo Erba (Milan, Italy) and distihed before use. The silylating reagents [trimethylchlorosiiane, hexamethyldisilazme and bis(trimethylsilyl)tuoroacetamide~ were obtained from Applied Science Lab. (State College, Pa., U.S.A.). For GLC, a Hewlett-Packard Model 5750 gas chromatograph, with a hydrogen Aame detector, was used; the coiled column (6 ft. x 5 mm I.D.) was packed with Gas-Chrom P coated with 5 ok of SE-30. Purified air, nitrogen and hydrogen for GLC were obtained from SIAD (Bergamo, Italy).
NQTEi3
237
METHDS
Preparation of derivative9 0xizmtion. The standard 0x0 acid (0.02-0.1 mg) was dissolved in 0.2 ml of pyridine and allowed to react with 0.3 mg of methoxylammonium chloride for 1 h in z F’FFE-fined screw-capped tube at room temperature. After removal of the reagents, the product was silylated. Silylation. The standard carboxylic acid (0.024.1 mg) was dissolved in a mixture of pyridine (200 pi), trimethylchiorosiiane (50 ~1) and hexamethyldisilazane (100 pi), or bis(trimethylsilyl)trifluoroacztamide (100 ,uI) and pyridine (200 PI), and allowed to react for I h at room temperature. An internal standard (3,3-dimethylglutaric acid or tricarballylic acid) was previously added to the reaction mixture.
Gas chromatography Because some of the derivatives were sensitive to water, the final reaction mixt-ure was injected directly on to the column without removal of reagents (excess of reagent is necessary to ensure quantitative reaction of molecules having multiple functions). The operating conditions of the gas chromatograph were generally: column temperature, 130” for diwrboxylic acids and 180” for tricarboxylic acids; detector temperature, 240”; inlet temperature, 260”; gas flow-rate, about 20 mI/min.
A
I
7
B
1 j I i
i
I
0
*.a
B
a.
TsEamIuTEs
\ 0
*
16
mu e IPUTES
citric acidcycle metaboiiteszs silyl or methoxime-trisxcthyIsiIy1 derivativeson an SE-30 cdumn. l%e acids are locked in the following order: succinic(I), ftunah@J 024aCdi~. maiic@Vj, 2-oxo&tzric(V), cis-azonitic(VI) 2& citri$VKI}. Tiie ~01~ texnperatk was 13W for the dicarboxrlic acids (-4) and HO0 for the tri~xylic acids (B).
Fig_ I. Separation
of microgramamounts of
3i
.-
238 RESULTS
AND
NOTES
DlscuSSION
The di- and tricarboxylic acids of the Krebs cycle are well sepa#ed
by iso-
thermal GLC at 130” and 180”, respectively (see Fig. 1); their relative retention times are shown in Tables I and If. The partial overlapping of peaks for citric and isocitric acids has been reported by other authors’l. The relative retention time of the conipounds studied were highly reproducible over a period of more than 12 months. TABLE 1
F9XENTION TIMES OF DICAREOXYLIC ACIDS, AS SILYL OR MET%IOXIME-§ILYL DERIVATIVES, RELATIVE TO 3,3_DIMETHYLGLUTARIC ACID ON SE-30 COLUMN The retention time of dimethyl@taric acid was 13.76 min at 130°. Relative retention time
Compound
Succinic acid Fumaric acid 3,3-Dimethylglutaricacid Oxalaeeticacid (methoxime) MaIic acid 2-Oxoglutaric acid (methoxime)
0.51
0.63
1.00 1.11 1.43 2.08
TABLE II RETENTION TIMES OF TRICARBOXYLIC ACIDS, AS SLLYL DERIVATIVES, TO TRICAREGALLYLIC ACID ON SE-30 COLUMN
RELATIVE
The retention time of triearballylicacid was 9.36 min at 180”. Compound
Relctive retention the
Triearballylicacid c&Aconitic acid Citric acid Isocitric acid
1.00 1.07 1.57 1.45
Fip 2. Relationshipbetweenpeak area and concentration of Werent amounts of (A) succini~(*), fumaric (e). malic (A) and 3,3_dimethylglutaric(0) acids; and (l3) ~xakeetic (@), 2-oxoglutaqic(O), cis-aconitic (*), citric (A) and isoeitic (0) acids.
NOTES
239
Under the experimental conditions described, conversion of the parent compounds into the derivatives was complete, at& no extraneous peaks were observed. D&&or response to increasing quantities of samples was rectilinear (see Fig. 21, and submicrogram amounts could easily be detected. The use of 3,3-dimethyl,olutaric and tricarbaliylic acid as internal standards allows peak identification and permits recovery calculations when the chemicals are added to samples at the beginning of the extraction procedttre. For these reasons, the GLC procedure described here is suggested as suitable for the evahtation of Krebs-cycie acids extracted from biological materials. REFERENCES 1 2 3 4 5 6 7 8 9 10 11
12 13
H. H. Luke, T. E. Freman and L. B. Kier, Anal. C/rem., 35 (1963) 1916. G. G. McKeown and S. I. Read, Ann!. Cheer., 37 (1965) 1780. A. Kuskis and P. Vishwarkarma, Can. .l. Biocizem. Physioi., 41 (1963) 2353. N. W. Alcock, Anaim Biodrem., 11 (1965) 335. C Kowala, Z. H. _Kranz and I(. E. Murray, Aust. J. Ct’rm., 54 (1962) 832. D. 1. Canvin, Can. J. Biuchem., 43 (1965) 1281. C. E. Dalgleisb, E. C. Hornins, M. G. Homing, K_ L Knox and K. Yarger, Biochem. J., 101 (1966) 792. F. L. Testes and R. C. Bachman, Anal. Che~n., 38 (1966) 1178. Y. Ishitoya, C. Itoh, N. Gsawa, I. Hashimoto and T_ Iwanaga, Clin. Chim. Acru, 27 (1970) 233. z. Horii. M. Mzkita and Y. Tamura, Chem. Ind_ (Lonubn). 34 (1965) 1494. U. Langenbeck and I. E. Seegmiller, J. Chromatogr., 78 (1973) 420. N. E. Hoi&an and T. A. Kiliinger, AI&. Chem., 41 (1969) 162. A. Kuskis 2nd P. Prioreschi, Anal. Biochenl.,19 (1967) 468. F. Sauer, J. D. Ernie and M. R. Binns, Eur. J. Biockm., 17 (1970) 350.
14 15 E. Mensen Da Silva, Arm!_ Chem., 43 (1971) 1031. 16 M. G. Homing,
E. A_ Bowclter, A. M. Moss and E. C. Homing,
Ano!. Letf., 1 (1968) 713.
-
ES&d
in The Netherian&
8787
Mote Gas ehromatographic
separation of Krebs-cycle
metabdites
A. PZNELLI and A. COLQMBQ Department
of Pharmacology,
UnpJersity of Milan,
Via Vanvirelli 32, Miian
(Italy)
(Received September 24th, 1975)
Gas-liquid chromato,oraphic (GLC) techniques have been used for the evaluation of citric acid-cycle metabolites by sever-a! authors1-15. The simultaneous determination of Krebs-cycle acids by GLC is preferable to conventional enzymic methods because of the reduction in analysis time; these acids are usually analysed as methyl’-’ or silyP-I2 derivatives. Methylation has been performed with methanol and hydrogen chloride’**, methanol and boron trifIuoride3*J or diazomethane1J~54, but, despite the different esterification conditions used, no single method had been reported in which all the compounds are methylated simultaneously. Simple esterification of acids containing double bonds or 0x0 groups is unsuitable because it gives rise to multiple peaksqd_ Silylation associated with oximation appears to be non-destructive9-“; nevertheless, only a few papers report the GLC anaiysis of Krebs-cycle acids as oximes and silyl derivatives’4 methoximes and silyl derivatives” or quinoxalone and sibyl derivatives’*. A few communications report the levels of some citric acid-cycle acids extracted from tissues and measured as methyi esters (not as silyi derivatives); recoveries were variable and 10w’~-‘~. Thus, there is a need of a method for extracting citric acid-cycle acids and evaiuating them as methoxime and silyl derivatives by GLC; this paper is a preliminary report of such a method.
Standard acids (succinic, fumaric, malic, oxalacetic, citric, isocitric, cisaconitic and 2-oxoglutaric) were obtained from BDH (Poole, Great Britain) and Biochemia (Milan, Italy). The solvents were purchased from Carlo Erba (Milan, Italy) and distihed before use. The silylating reagents [trimethylchlorosiiane, hexamethyldisilazme and bis(trimethylsilyl)tuoroacetamide~ were obtained from Applied Science Lab. (State College, Pa., U.S.A.). For GLC, a Hewlett-Packard Model 5750 gas chromatograph, with a hydrogen Aame detector, was used; the coiled column (6 ft. x 5 mm I.D.) was packed with Gas-Chrom P coated with 5 ok of SE-30. Purified air, nitrogen and hydrogen for GLC were obtained from SIAD (Bergamo, Italy).
NQTEi3
237
METHDS
Preparation of derivative9 0xizmtion. The standard 0x0 acid (0.02-0.1 mg) was dissolved in 0.2 ml of pyridine and allowed to react with 0.3 mg of methoxylammonium chloride for 1 h in z F’FFE-fined screw-capped tube at room temperature. After removal of the reagents, the product was silylated. Silylation. The standard carboxylic acid (0.024.1 mg) was dissolved in a mixture of pyridine (200 pi), trimethylchiorosiiane (50 ~1) and hexamethyldisilazane (100 pi), or bis(trimethylsilyl)trifluoroacztamide (100 ,uI) and pyridine (200 PI), and allowed to react for I h at room temperature. An internal standard (3,3-dimethylglutaric acid or tricarballylic acid) was previously added to the reaction mixture.
Gas chromatography Because some of the derivatives were sensitive to water, the final reaction mixt-ure was injected directly on to the column without removal of reagents (excess of reagent is necessary to ensure quantitative reaction of molecules having multiple functions). The operating conditions of the gas chromatograph were generally: column temperature, 130” for diwrboxylic acids and 180” for tricarboxylic acids; detector temperature, 240”; inlet temperature, 260”; gas flow-rate, about 20 mI/min.
A
I
7
B
1 j I i
i
I
0
*.a
B
a.
TsEamIuTEs
\ 0
*
16
mu e IPUTES
citric acidcycle metaboiiteszs silyl or methoxime-trisxcthyIsiIy1 derivativeson an SE-30 cdumn. l%e acids are locked in the following order: succinic(I), ftunah@J 024aCdi~. maiic@Vj, 2-oxo&tzric(V), cis-azonitic(VI) 2& citri$VKI}. Tiie ~01~ texnperatk was 13W for the dicarboxrlic acids (-4) and HO0 for the tri~xylic acids (B).
Fig_ I. Separation
of microgramamounts of
3i
.-
238 RESULTS
AND
NOTES
DlscuSSION
The di- and tricarboxylic acids of the Krebs cycle are well sepa#ed
by iso-
thermal GLC at 130” and 180”, respectively (see Fig. 1); their relative retention times are shown in Tables I and If. The partial overlapping of peaks for citric and isocitric acids has been reported by other authors’l. The relative retention time of the conipounds studied were highly reproducible over a period of more than 12 months. TABLE 1
F9XENTION TIMES OF DICAREOXYLIC ACIDS, AS SILYL OR MET%IOXIME-§ILYL DERIVATIVES, RELATIVE TO 3,3_DIMETHYLGLUTARIC ACID ON SE-30 COLUMN The retention time of dimethyl@taric acid was 13.76 min at 130°. Relative retention time
Compound
Succinic acid Fumaric acid 3,3-Dimethylglutaricacid Oxalaeeticacid (methoxime) MaIic acid 2-Oxoglutaric acid (methoxime)
0.51
0.63
1.00 1.11 1.43 2.08
TABLE II RETENTION TIMES OF TRICARBOXYLIC ACIDS, AS SLLYL DERIVATIVES, TO TRICAREGALLYLIC ACID ON SE-30 COLUMN
RELATIVE
The retention time of triearballylicacid was 9.36 min at 180”. Compound
Relctive retention the
Triearballylicacid c&Aconitic acid Citric acid Isocitric acid
1.00 1.07 1.57 1.45
Fip 2. Relationshipbetweenpeak area and concentration of Werent amounts of (A) succini~(*), fumaric (e). malic (A) and 3,3_dimethylglutaric(0) acids; and (l3) ~xakeetic (@), 2-oxoglutaqic(O), cis-aconitic (*), citric (A) and isoeitic (0) acids.
NOTES
239
Under the experimental conditions described, conversion of the parent compounds into the derivatives was complete, at& no extraneous peaks were observed. D&&or response to increasing quantities of samples was rectilinear (see Fig. 21, and submicrogram amounts could easily be detected. The use of 3,3-dimethyl,olutaric and tricarbaliylic acid as internal standards allows peak identification and permits recovery calculations when the chemicals are added to samples at the beginning of the extraction procedttre. For these reasons, the GLC procedure described here is suggested as suitable for the evahtation of Krebs-cycie acids extracted from biological materials. REFERENCES 1 2 3 4 5 6 7 8 9 10 11
12 13
H. H. Luke, T. E. Freman and L. B. Kier, Anal. C/rem., 35 (1963) 1916. G. G. McKeown and S. I. Read, Ann!. Cheer., 37 (1965) 1780. A. Kuskis and P. Vishwarkarma, Can. .l. Biocizem. Physioi., 41 (1963) 2353. N. W. Alcock, Anaim Biodrem., 11 (1965) 335. C Kowala, Z. H. _Kranz and I(. E. Murray, Aust. J. Ct’rm., 54 (1962) 832. D. 1. Canvin, Can. J. Biuchem., 43 (1965) 1281. C. E. Dalgleisb, E. C. Hornins, M. G. Homing, K_ L Knox and K. Yarger, Biochem. J., 101 (1966) 792. F. L. Testes and R. C. Bachman, Anal. Che~n., 38 (1966) 1178. Y. Ishitoya, C. Itoh, N. Gsawa, I. Hashimoto and T_ Iwanaga, Clin. Chim. Acru, 27 (1970) 233. z. Horii. M. Mzkita and Y. Tamura, Chem. Ind_ (Lonubn). 34 (1965) 1494. U. Langenbeck and I. E. Seegmiller, J. Chromatogr., 78 (1973) 420. N. E. Hoi&an and T. A. Kiliinger, AI&. Chem., 41 (1969) 162. A. Kuskis 2nd P. Prioreschi, Anal. Biochenl.,19 (1967) 468. F. Sauer, J. D. Ernie and M. R. Binns, Eur. J. Biockm., 17 (1970) 350.
14 15 E. Mensen Da Silva, Arm!_ Chem., 43 (1971) 1031. 16 M. G. Homing,
E. A_ Bowclter, A. M. Moss and E. C. Homing,
Ano!. Letf., 1 (1968) 713.
