Simultaneous quantification of four vitamin D metabolites in human serum using high performance liquid chromatography tandem mass spectrometry for vitamin D profiling

► HPLC-MS/MS method for vitamin D profiling in human serum samples ► Simultaneous quantification of 25OH-D3, 3-epi-25OH-D3, 25OH-D2 and 24R,25(OH)2-D3 ► Chromatographic separation of 25OH-D3 and 3-epi-25OH-D3 below 10 min ► Significant amounts of 3-epi-25OH-D3 and 24R,25(OH)2-D3 in serum samples detected. ► 24R,25(OH)2-D3 level increased disproportionally with increasing 25OH-D3 level.