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

For quantification of 25-hydroxyvitamin D3 (25OH-D3), 25-hydroxyvitamin D2 (25OH-D2), 3-epi-25-hydroxyvitamin D3 (3-epi-25OH-D3) and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2-D3) in human serum a high performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) method was developed and validated. After protein precipitation further purification is achieved with on-line sample preparation using a reversed phase (RP) C-4 column. Chromatographic separation is realized by a RP-column with core shell material and pentafluorophenyl (PFP) selectivity. Atmospheric pressure chemical ionization in the positive ion mode with multi‐reaction monitoring is used for analyte detection. Baseline separation of the analytes is achieved below 10min. The method is linear over the range 4.0–265.3nmol/L for 25OH-D3, 3.9–183.6nmol/L for 25OH-D2, 2.0–133.8nmol/L for 3-epi-25OH-D3 and 2.8–129.9nmol/L for 24R,25(OH)2-D3 (r2>0.998). The limit of quantification is 4.0nmol/L for 25OH-D3, 3.9nmol/L for 25OH-D2, 2.0nmol/L for 3-epi-25OH-D3 and 2.8nmol/L for 24R,25(OH)2-D3. The CVs for the intra-day and inter-day precision are