Simultaneous quantification of 25-hydroxyvitamin D 3 -3-sulfate and 25-hydroxyvitamin D 3 -3-glucuronide in human serum and plasma using liquid chromatography-tandem mass spectrometry coupled with DAPTAD-derivatization.

25-hydroxyvitamin D3 -3-sulfate (25-OHD3 -S) and 25-hydroxyvitamin D3 -3-glucuronide (25-OHD3 -G) are major conjugative metabolites of vitamin D3 found in the systemic circulation and potentially important reservoirs for 25-hydroxyvitamin D3 . Simultaneous and accurate quantification of these metabolites could advance assessment of the impact of vitamin D3 on health and disease. In this study, a highly sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous quantification of 25-OHD3 -S and 25-OHD3 -G in human serum or plasma. Following protein precipitation, the analytes of interest were partially purified by solid-phase extraction and subjected to derivatization with 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD). Quantification of the analytes was based on multiple reaction monitoring (MRM) operated in the positive ion mode, and deuterated internal standards were used for each conjugative metabolite. Applying this method to the analysis of 25-OHD3 -S and 25-OHD3 -G concentrations in human serum or plasma samples achieved satisfactory reproducibility, accuracy and sensitivity. We subsequently used this method to simultaneously determine serum concentrations of the two metabolites in archived samples from a rifampin treatment study. Drug treatment had no effect on metabolite concentrations, but significantly increased the 25-OHD3 -S/25-OHD3 concentration ratio (p=0.01). The availability of this new method should improve sample throughput and our ability to quantify and monitor circulating 25-OHD3 -S and 25-OHD3 -G concentrations.