Enhanced 3-epi-25-hydroxyvitamin D3 signal leads to overestimation of its concentration and amplifies interference in 25-hydroxyvitamin D LC-MS/MS assays

Background 3-epi-25-hydroxyvitamin D-3 (3-epi-25OHD(3)) interferes in most liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for 25-hydroxyvitamin D (25OHD). The clinical significance of this is unclear, with concentrations from undetectable to 230 nmol/L reported. Many studies have quantified 3-epi-25OHD(3) based on 25OHD(3) calibrators or other indirect methods, and we speculated that this contributes to the observed variability in reported 3-epi-25OHD(3) concentrations. Methods We compared continuous MS/MS infusions of 3-epi-25OHD(3) and 25OHD(3) solutions, spiked both analytes into the same serum matrix and analysed patient samples to assess the effect of three different quantitation methods on 3-epi-25OHD(3) concentration. Experiments were performed on an LC-MS/MS system using a phenyl column which does not resolve 3-epi-25OHD(3), and a modified method utilizing a Zorbax SB-CN column that chromatographically resolves 3-epi-25OHD(3) from 25OHD(3). Results A greater 3-epi-25OHD(3) signal, compared with 25OHD(3), was observed during equimolar post-column continuous infusion of analyte solutions, and following analysis of a serum pool spiked with both analytes. 3-epi-25OHD(3) signal enhancement was dependent on mobile phase composition. Compared with 3-epi-25OHD(3) calibrators, indirect quantitation methods resulted in up to 10 times as many samples having 3-epi-25OHD(3) concentrations >= 10 nmol/L, and an approximately fourfold increase in the maximum observed 3-epi-25OHD(3) concentration to 95 nmol/L. Conclusions Enhanced 3-epi-25OHD(3) signal leads to overestimation of its concentrations in the indirect quantitation methods used in many previous studies. The enhanced signal may contribute to greater interference in some 25OHD LC-MS/MS assays than others. We highlight that equimolar responses cannot be assumed in LC-MS/MS systems, even if two molecules are structurally similar.