Usefulness of Derivatization in High-Performance Liquid Chromatography/Tandem Mass Spectrometry of Conjugated Vitamin D Metabolites

The usefulness of derivatization in high-performance liquid chromatography/ion trap tandem mass spectrometry (electro-spray ionization) of conjugated vitamin D metabolites was examined. 24,25-Dihydroxyvitamin D3 [24,25(OH)2D3]-3-and -24-glucuronides (G) were converted to the adducts with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), the methyl esters and the acetates. The combination of the derivatization with PTAD and methylation improved the detection limit in both the positive- and negative-ion modes. The PTAD adducts of 24,25(OH)2D3-3G and -24G also gave a characteristic product ion indicating the conjugation position by the positive-MS/MS/MS, which was not observed in the intact forms. 25-Hydroxyvitamin D3 3-sulfate gave the deprotonated ion in the negative-ion mode, but no characteristic ion was observed in the positive-ion mode. On the contrary, its PTAD adduct gave a molecular-related ion in the positive-ion mode, the MS/MS/MS of which provided structural information, that is, the conjugation position and the existence of a sulfuric acid group.