Determination of Isotope Enrichment in13C-or2H-Labelled Branched-Chain l-Amino Acids From Physiological Fluids by Gas Chromatography-Mass Spectrometry: Use of l-Leucine Dehydrogenase for Specific Preparation of the Quinoxalinol Derivatives

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Abstract
A procedure is described which allows the specific and sensitive estimation of 13C- or 2H-label enrichment in branched-chain l-amino acids from physiological fluids: Amino acids are isolated from deproteinized samples by cation exchange chromatography. Specific conversion of branched-chain l-amino acids to their respective 2-oxo acids is achieved by treatment with l-leucine dehydrogenase from Bacillus sp. Reaction of the 2-oxo acids with o-phenylenediamin yields the branched-chain quinoxalinol derivatives which are purified by solid phase extraction. Isotopic label enrichment is then determined by gas chromatographic-mass spectrometric analysis of the O-trimethylsilyl quinoxalinol derivatives using an ammonia-chemical ionisation mode and selected ion monitoring of the quasi-molecular ions [MH]+ and [MH + 1]+. Applicability of the method is demonstrated by serum analysis in an oral loading test with l-[1-13C]leucine.