Gas Chromatographic−Mass Spectrometric Analysis of Biomarkers Related to Folate and Cobalamin Status in Human Serum after Dimercaptopropanesulfonate Reduction and Heptafluorobutyl Chloroformate Derivatization

Abstract

Methylmalonic acid and total homocysteine belong to useful clinical indicators of cobalamin and folate status and are commonly measured separately. A sensitive and rapid method has been developed for simultaneous determination of both biomarkers and related metabolites in serum or plasma by isotope dilution gas chromatography-mass spectrometry (GC/MS). Thiols bound in disulfide bonds were released with 2,3-dimercapto-1-propanesulfonic acid (DMPS), and after deproteinization, they were phase-transfer derivatized with heptafluorobutyl chloroformate (HFBCF) in a single step. The reducing capability of the DMPS agent was comparable to that of the dithiothreitol, but exceeded the latter in much cleaner extracts obtained. The new method enabled GC/MS screening of amino acidic metabolites, including cystathionine and thiol-containing dipeptides, as their N(S,O)-heptafluorobutoxycarbonyl heptafluorobutyl ester derivatives in serum or plasma. Accurate quantitation of seven biomarkers was accomplished by using deuterated internal standards; the detection limits ranged from 7 to 20 nmol/L, the between-day precision from 1.5 to 8.8%, and the recoveries were between 83 and 103%. The results suggest that the new combined procedure of DMPS reduction and HFBCF derivatization make the method efficient for diagnostics of folate and cobalamin status as well as for screening of amino acidic metabolites in body fluids.