Improved accuracy of an tandem liquid chromatography–mass spectrometry method measuring 24R,25-dihydroxyvitamin D₃ and 25-hydroxyvitamin D metabolites in serum using unspiked controls and its application to determining cross-reactivity of a chemiluminescent microparticle immunoassay

Measurement of serum 25-hydroxyvitamin D [25(OH)D] is considered the best indicator of vitamin D status. Two minor vitamin D metabolites are common interferences encountered in 25(OH)D assays. The first is 3-epi-25-hydroxyvitamin D₃ [3-epi-25(OH)D₃], which if not chromatographically resolved from 25-hydroxyvitamin D₃ [25(OH)D₃], can overestimate 25(OH)D concentrations. The second is 24R,25-dihydroxyvitamin D₃ [24R,25(OH)₂D₃], which can cross-react with the antibodies in 25(OH)D immunoassays. Our aim was to develop an LC–MS/MS method capable of detecting both 3-epi-25(OH)D₃ and 24R,25(OH)₂D₃ in serum without the use of a derivatization agent. We report an isotope dilution LC–MS/MS method, with electrospray ionization in the positive mode, that can simultaneously detect 24R,25(OH)₂D₃, 25(OH)D₃, 3-epi-25(OH)D₃, and 25-hydroxyvitamin D₂. The method employs a cost-effective liquid–liquid extraction using only 150 μL of sera and a total run time of 10 min. Method performance was assessed by using quality controls made from pooled sera as an alternative to sera spiked with analytes. Biobanked samples, originally analyzed by chemiluminescent microparticle immunoassay (CMIA), were re-analyzed with this method to determine the contribution of 24R,25(OH)₂D₃ cross-reactivity to 25(OH)D measurement bias. The CMIA over-estimation of 25(OH)D measurements relative to LC–MS/MS was found to depend on both 25(OH)D and 24R,25(OH)₂D₃ concentrations.