Development and validation of an isotope dilution ultra‐high pressure liquid chromatography‐tandem mass spectrometry method for quantitation of serum 25‐hydroxyvitamin D3, D2 and 3‐epi‐D3

Our aim was to develop and validate a method for separate quantitation of 25‐hydroxyvitamin D 2 (25OHD 2 ) and 25‐hydroxyvitamin D 3 (25OHD 3 ) and its epimer (epi‐25OHD 3 ), with complete chromatographic resolution of 25OHD 3 and epi‐25OHD 3 . Serum (100 uL) was diluted with internal standards (d 3 ‐25OHD 2 and d 6 ‐25OHD 3 ) and extracted with hexanes. The organic layer was evaporated, reconstituted and injected onto a Kinetex PFP column (2.1 × 100 mm × 1.7 um) using an Accela UHPLC system coupled with a triple quadrupole Vantage mass spectrometer. All analytes were isocratically eluted with 72% MeOH/H 2 O flowing at 0.4 mL/min within 14 min. The mass spectrometer was set in positive APCI mode and two SRM transitions (quantitation and confirmation ions) were monitored for each analyte. Calibration was performed in 4% albumin‐PBS and verified with NIST standard reference materials. Inter‐assay precision was < 10% when concentrations were > 12.5 nmol/L. Method bias was < 5%. Limits of detection were 2 to 5 nmol/L. The method showed excellent performance in proficiency testing programs. Convenience samples from blood donors (n=36) and pregnant women (n=35) showed median values (nmol/L) for 25OHD 2, 25OHD 3, and epi‐25OHD 3 of 0.3, 43, 0.7 and 1.8, 88, 4, respectively. This method is accurate, precise, robust and suitable for the assessment of vitamin D status in populations.