Simultaneous determination of methionine sulfoxide and methionine in blood plasma using gas chromatography-mass spectrometry

Abstract

Methionine sulfoxide is an oxidation product of methionine with reactive oxygen species via 2-electron-dependent mechanism. Such oxidants can be generated from activated neutrophils; therefore, methionine sulfoxide can be regarded as a biomarker of oxidative stress in vivo. We describe here a method for the simultaneous determination of methionine sulfoxide and methionine in blood plasma using gas chromatography-mass spectrometry with isotopically labeled compounds as internal standards. This method comprises the inclusion of [ Me- 13C, Me- 2H 3]methionine sulfoxide and [ Me- 13C, Me- 2H 3]methionine into plasma, the removal of plasma proteins using acetonitrile, the purification of amino acids with cation-exchange chromatography, and the derivatization of methionine sulfoxide and methionine to their corresponding tert-butyldimethylsilyl derivatives using N-( tert-butyldimethylsilyl)- N-methyltrifluoroacetamide. Quantitation was performed by electron impact mode. The levels of methionine sulfoxide in healthy human blood plasma were 4.0±1.0 μM (means ± SD, n=8), indicating that approximately 10% of methionine is detected as the oxidized form in healthy human plasma. The ratio of methionine sulfoxide in total methionine increased with treatment of human blood with phorbol 12-myristate 13-acetate, while this ratio remained constant in plasma from alloxan-induced hyperglycemic rabbits. These results indicate that this method is applicable for plasma samples and methionine sulfoxide can represent oxidative stress caused by nonradical oxidation in vivo.