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

Abstract

Our aim was to develop and validate a method for separate quantitation of 25‐hydroxyvitamin D2 (25OHD2) and 25‐hydroxyvitamin D3 (25OHD3) and its epimer (epi‐25OHD3), with complete chromatographic resolution of 25OHD3 and epi‐25OHD3. Serum (100 uL) was diluted with internal standards (d3‐25OHD2 and d6‐25OHD3) 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/H2O 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 25OHD2, 25OHD3, and epi‐25OHD3 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.