We developed and evaluated a candidate serum reference material to help improve clinical routine measurement, and to provide traceability of the measurement results. D8-Homocystine, dithiothreitol, and acetonitrile were used as an internal standard, the reducing agent, and the protein precipitating agent, respectively. A triple quadrupole mass spectrometer with an electrospray ionization source was used for monitoring the transitions (m/z 140.0 → 94.0, 136.0 → 90.0) in multiple-reaction-monitoring mode. We used a calibration model relying on bracketing and gravimetric measurements to give SI-traceability and higher accuracy to serum value assignments. The method was evaluated for accuracy using NIST Standard Reference Material SRM1955. The results of the three concentrations (1, 2, and 3) of total homocysteine in human serum samples were determined by an isotope-dilution liquid chromatography-tandem mass spectrometry method; tHcy 1 is 28.8 ± 1.1 μmol/L, tHcy 2 is 17.93 ± 0.57 μmol/L, and tHcy 3 is 14.38 ± 0.46 μmol/L.
