Quantitation of branched-chain α-keto acids as theirN-methylquinoxalone derivatives: Comparison ofO- andN-alkylation versus -silylation

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Abstract
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 α‐keto acids. In the present study, N‐methyl‐quinoxalone derivative was developed and its use in quantification in human studies verified. O‐phenylenediamine and α‐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 α‐ketoisocaproate, α‐keto‐β‐methylvalerate and α‐ketoisovalerate, ‘single ion monitoring’ could be performed for the quantification of isotopic enrichment as well as plasma concentration of these three branched‐chain α‐keto acids. Isotopic enrichment from 0.25 to 7.5 at% excess could be measured easily, with an average coefficient of variation of −1 in a 200‐μl aliquot could be measured. Methyl migration was an interesting feature of the mass spectrometric fragmentation pattern of the α‐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.