Analysis of [(2)H7]methionine, [(2)H4]methionine, methionine, [(2)H4]homocysteine and homocysteine in plasma by gas chromatography-mass spectrometry to follow the fate of administered [(2)H7]methionine

Homocysteine plays a key role in several pathophysiological conditions. To assess the methionine-homocysteine kinetics by stable isotope methodology, we developed a simultaneous quantification method of [(2)H(7)]methionine, [(2)H(4)]methionine, methionine, [(2)H(4)]homocysteine and homocysteine in rat plasma by gas chromatography-mass spectrometry (GC-MS). [(13)C]Methionine and [(13)C]homocysteine were used as analytical internal standards to account for losses associated with the extraction, derivatization and chromatography. For labeled and non-labeled homocysteine measurements, disulfide bonds between homocysteine and other thiols or proteins were reduced by dithiothreitol. The reduced homocysteine and methionine species were purified by cation-exchange chromatography and derivatized with isobutyl chlorocarbonate in water-ethanol-pyridine. Quantification was carried out by selected ion monitoring of the molecular-related ions of N(O,S)-isobutyloxycarbonyl ethyl ester derivatives on the chemical ionization mode. The intra- and inter-day precision of the assay was less than 6% for all labeled and non-labeled methionine and homocysteine species. The method is sensitive enough to determine pharmacokinetics of labeled methionine and homocysteine.