Rapid HPLC determination of total homocysteine and other thiols in serum and plasma: Sex differences and correlation with cobalamin and folate concentrations in healthy subjects

High-performance liquid chromatography with fluorescence detection has been utilized for the rapid determination of total homocysteine, cysteine, and cysteinylglycine in human serum and plasma. Our earlier procedure (Anal Biochem 1989; 178:208), which used monobromobimane to specifically derivatize thiols, has been extensively modified to allow for rapid processing of samples. As a result, >80 samples a day can be assayed for total homocysteine, cysteine, and cysteinylglycine. The method is sensitive (lower limit of detection ≤4 pmol in the assay) and precise (intra- and interassay CV for homocysteine, 3.31% and 4.85%, respectively). Mean total homocysteine concentrations in plasma and serum were significantly different, both from healthy male donors (9.26 and 12.30 μmol/L, respectively; P <0.001) and healthy female donors (7.85 and 10.34 μmol/L, respectively; P <0.001). The differences in total homocysteine between sexes were also significant (P = 0.002 for both plasma and serum). Similar differences were found for cysteine and cysteinylglycine. We found a significant inverse correlation between serum cobalamin and total homocysteine in men (P = 0.0102) and women (P = 0.0174). Serum folate also inversely correlated with total homocysteine in both sexes.