Simultaneous determination of methionine and total homocysteine in human plasma by gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric method for the simultaneous determination of methionine and total homocysteine in human plasma is described. dl-²H₄]Methionine and dl-²H₈]homocystine were used as internal standards. The method involved reduction of the disulfide bond with dithiothreitol, purification by cation-exchange chromatography using a BondElut SCX cartridge and derivatization with isobutyl chlorocarbonate in water–ethanol–pyridine. Quantitation was performed by selected-ion monitoring of the quasi-molecular ions of N(O,S)-isobutyloxycarbonyl ethyl ester (IBC-OEt) derivatives for methionine and ²H₄]methionine, respectively, and the fragment ions (M+H–COOisoBu–COOEt]⁺) for IBC-OEt derivatives for homocysteine and ²H₄]homocysteine, respectively. The sensitivity, specificity, accuracy and precision of the method were demonstrated to be satisfactory for measuring concentrations of methionine and total homocysteine in human plasma.