Determination of R- and S-3-methyl-2-oxopentanoate enantiomers in human plasma: suitable method for label enrichment analysis.

A sensitive method for the determination of S- and R-3-methyl-2-oxopentanoate enantiomers (KMV, alpha-keto-beta-methylvalerate) in physiological fluids suitable for isotope enrichment analysis is described: after extraction with acid, 2-oxo acids are separated from interfering amino acids by cation-exchange chromatography. Reductive amination of the branched-chain 2-oxo acids by use of L-leucine dehydrogenase yields the corresponding L-amino acids. L-Isoleucine and L-alloisoleucine which are formed from S- and R-3-methyl-2-oxopentanoate, respectively, are then quantified by amino acid analysis. The method was used for determination of the R-IS-3-methyl-2-oxopentanoate ratio in plasma of healthy subjects and patients with diabetes mellitus and maple syrup urine disease. Applicability for gas chromatographic-mass spectrometric analysis of 13C-label enrichment in plasma S-3-methyl-2-oxopentanoate is demonstrated.