Metabolites in the methionine pathway, as well as methylmalonic acid (MMA), are important biomarkers in the diagnosis of certain cobalamin disorders. Raised plasma levels of methylmalonic acid, methionine, or total plasma homocysteine can be indicative of these disorders, and, therefore, the chromatographic separation of isomers and quantitation of these analytes is needed in diagnosing these different disorders.
A rapid four-minute LC-MS/MS method was developed for the simultaneous analysis of methionine pathway metabolites, including homocysteine, methionine, cysteine, and cystathionine, as well as methylmalonic acid and succinic acid in plasma. Complete chromatographic separation of all compounds was achieved using a Raptor Polar X column under HILIC conditions. The method employed a simple sample reduction procedure using DTT, replacing time-consuming derivatization procedures. Reproducible retention times and selectivity were demonstrated throughout testing. Acceptable precision and accuracy results for the quantitative analysis of total plasma homocysteine and methylmalonic acid, as well as accuracy of the surrogate matrix by the used of standard addition testing, were also achieved.
