Quantitation of branched-chain alpha-keto acids as their N-methylquinoxalone derivatives: comparison of O- and N-alkylation versus -silylation.

Quantitative estimation of isotopic enrichment and concentrations of keto analogs of branched-chain amino acids in biological fluid has been used for the study of protein metabolism in animal and human studies. At present, O-trimethylsilyl-quinoxalinol derivative is used widely in the quantification of branched-chain alpha-keto acids. In the present study, N-methyl-quinoxalone derivative was developed and its use in quantification in human studies verified. O-phenylenediamine and alpha-keto acid react in acidic media to yield phenolic and amide tautomers. O-trimethylsilyl-quinoxalinol derivative of the phenolic tautomer is used at present for quantification by chemical ionization/selected ion monitoring. We have prepared N-methyl-quinoxalone derivative using N,N-dimethyl formamide dimethyl acetal. This derivative is characterized by a major amide form and a minor phenolic form. The mass spectrum has a characteristic fragment, which facilitates easy identification and quantitation by electron impact/selected ion monitoring. Because m/z 174 was observed as the base peak for alpha-ketoisocaproate, alpha-keto-beta-methylvalerate and alpha-ketoisovalerate, "single ion monitoring' could be performed for the quantification of isotopic enrichment as well as plasma concentration of these three branched-chain alpha-keto acids. Isotopic enrichment from 0.25 to 7.5 at% excess could be measured easily, with an average coefficient of variation of less than 8%. Plasma concentrations as low as 10 microM l-1 in a 200-microliters aliquot could be measured. Methyl migration was an interesting feature of the mass spectrometric fragmentation pattern of the alpha-keto acids. The mechanism of methyl migration is proposed and discussed. This paper also describes some of the studies involved in the formation of isomeric O- and N-alkyl, -quinoxaline and -quinoxalone using a number of N,N-dimethyl formamide dialkyl acetals.