Identification and Structural Elucidation of Vitamin D3 Metabolites in Human Urine Using LC-MS-MS

An analytical method was developed for the identification of primary vitamin D₃ metabolites in human urine using liquid chromatography tandem mass spectrometry in positive mode. Urine samples were purified using C₁₈ solid-phase extraction cartridges and analytical separations were performed by reversed phase liquid chromatography in gradient mode using ammonium acetate (0.01 mol L^?1) and acetonitrile as the mobile phases. Identification and structural elucidation of the metabolites were carried out by comparison with mass spectral fragmentation behavior of vitamin D₃ and retention characteristics. Three primary urinary vitamin D₃ metabolites were identified as 25-hydroxyvitamin D₃, 1α,25-dihydroxyvitamin D₃ and vitamin D₃ sulphate, respectively.