Separation and Quantitation of Total Plasma Homocysteine and Methylmalonic Acid by LC-MS/MS Analysis

Homocysteine and methylmalonic acid are important biomarkers used in the diagnosis of a number of cobalamin disorders. Some of these disorders include hyperhomocysteinemia, homocystinuria, methylmalonic acidemia, and megaloblastic anemia. For proper diagnosis of cobalamin disorders, it is important to quantitate the levels of these analytes. When using homocysteine for quantitation, it is best to use total plasma homocysteine. This includes all of the oxidized and bound forms of the homocystine dimer. In order to calculate total plasma homocysteine, often derivatization is used. In the sample preparation used for this analysis, a sulfhydryl reagent called “dithiothreitol,” or DTT, is used. This eliminates the need for lengthy derivatization in the sample preparation and works effectively in converting all of the dimer forms of homocystine to the monomer form, homocysteine. When analyzing methylmalonic acid, it is important to keep in mind that methylmalonic acid has a naturally occurring isomer, succinic acid. In order to quantitate methylmalonic acid accurately, there needs to be chromatographic separation of these two isomers. This work includes a quick sample preparation using protein precipitation and the use of DTT. The rapid, 4-minute LC-MS/MS method allows for resolution of all analytes, including the two isomers. This method showed reproducible retention times and selectivity over the course of testing as well as good precision and accuracy for all calibrators, quality controls, and standard addition plasma samples.