This rapid, 4-minute LC-MS\/MS method allows simultaneous quantitative analysis of total homocysteine and methylmalonic acid without the need for derivatization or additional amino acid testing. Full chromatographic separation of homocysteine, methionine, cysteine, cystathionine, methylmalonic acid, and succinic acid in plasma was also achieved.<\/p>\n\n\n\n
Cobalamin, also known as vitamin B12, plays an essential role in many metabolic processes, including DNA synthesis, amino acid metabolism, and energy production. Cobalamin disorders can occur if these processes are disrupted by either genetic mutations or a deficiency in B vitamins, such as cobalamin (B12); folic acid (B9); or pyridoxine (B6). Clinical diagnosis of certain cobalamin disorders is based on critical metabolites in the methionine pathway as well as on methylmalonic acid. In particular, assays of total homocysteine, methionine, and methylmalonic acid in plasma are used for differential diagnosis. Immunoassays and LC-MS\/MS methods are used for the analysis of methionine pathway metabolites and methylmalonic acid, but both have limitations. Immunoassay is not as sensitive as LC-MS\/MS analysis, but with LC-MS\/MS, a lengthy and time-consuming derivatization is often needed during sample preparation.<\/p>\n\n\n\n
Homocysteine is an endogenous, sulfur-containing amino acid that is formed during the metabolism of methionine to cysteine. It is irreversibly catabolized by transsulfuration to cysteine or remethylated to methionine with the aid of B vitamins [1]. It is an important biomarker for homocystinuria, which is caused by a defect in methionine pathway enzymes and results in the accumulation of both methionine and homocysteine in blood and urine. It is also used to diagnose hyperhomocysteinemia, which is caused by a deficiency in vitamin B12 or B9 and characterized by elevated levels of homocysteine in plasma [2,3].<\/p>\n\n\n\n
In plasma, homocysteine occurs primarily in oxidized disulfide forms. Less than 1% occurs as the free reduced homocysteine monomer, whereas 80\u201390% occurs as a disulfide bound to plasma proteins, and the remaining 10-20% is found as either homocysteine-cysteine mixed disulfide or as homocystine (homocysteine dimer). The sum of these four forms is referred to as total plasma homocysteine, and it is the parameter used for diagnosis [4]. To convert all the oxidized disulfide forms of homocysteine to the free monomer form, dithiothreitol (DTT) can potentially be used as a sulfhydryl reducing agent (Figure 1).<\/p>\n\n\n