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

Objectives: For quantification of 25-hydroxyvitamin D-3 (25OH-D-3), 25-hydroxyvitamin D-2 (25OH-D-2). 3-epi-25-hydroxyvitamin D-3 (3-epi-25OH-D-3) and 24R,25-dihydroxyvitamin D-3 (24R,25(OH)(2)-D-3) in human serum a high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed and validated. Design and methods: 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. Results: Baseline separation of the analytes is achieved below 10 min. The method is linear over the range 4.0-2653 nmol/L for 25OH-D-3, 3.9-183.6 nmol/L for 25OH-D-2, 2.0-133.8 nmol/L for 3-epi-25OH-D-3 and 2.8-129.9 nmol/L for 24R,25(OH)(2)-D-3 (r(2)>0.998). The limit of quantification is 4.0 nmol/L for 25OH-D-3, 3.9 nmol/L for 25OH-D-2, 2.0 nmol/L for 3-epi-25OH-D-3 and 2.8 nmol/L for 24R,25(OH)(2)-D-3. The CVs for the intra-day and inter-day precision are <5% and <4%, respectively. Metabolite levels for a set of 50 human serum samples have been determined and resulted in the detection of considerable amounts of 3-epi-25OH-D-3 and 24R,25(OH)(2)-D-3. Conclusions: This highly specific HPLC-MS/MS method is suitable for vitamin D profiling. There is a correlation between 25OH-D-3 and 24R,25(OH)(2)-D-3. Serum concentration of 24R,25(OH)(2)-D-3 increases disproportionally with increasing concentration of 25OH-D-3. (C) 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.