Overestimation of 25-hydroxyvitamin D3 by increased ionisation efficiency of 3-epi-25-hydroxyvitamin D3 in LC–MS/MS methods not separating both metabolites as determined by an LC–MS/MS method for separate quantification of 25-hydroxyvitamin D3, 3-epi-25-h

• We report an LC–MS/MS method for measurement of (3-epi-)25(OH)D3, and 25(OH)D2.
• Separate calibrators and SIL-IS for each compound ensure accurate measurement.
• Structurally-related 3-epi-25(OH)D3 and 25(OH)D3 differ in ionisation efficiency.
• This leads to overestimation of 25(OH)D3 concentration in C18-based LC–MS/MS methods.

BackgroundAn LC–MS/MS method was developed for simultaneous quantification of 25-hydroxyvitamin D3 (25(OH)D3), 3-epi-25(OH)D3, and 25(OH)D2 in human serum. Methods: Sample preparation consisted of protein precipitation followed by off-line SPE. Calibration curves for each vitamin D metabolite were constructed in phosphate-buffered saline with 60 g/L albumin including its corresponding stable isotope labelled (SIL) internal standard. A pentafluorophenyl (PFP) analytical column was used to resolve 25(OH)D3 from 25(OH)D2 and 3-epi-25(OH)D3, followed by SRM registration using positive ESI-MS/MS. Accuracy was assessed from measurement of samples with NIST reference method procedure (RMP) assigned values. The PFP LC–MS/MS method was compared to an in-house C18 column LC–MS/MS method, not resolving 25(OH)D3 from 3-epi-25(OH)D3, using adult and newborn samples. Results: Intra-assay and inter-assay coefficients of variation were less than 4% and 7.5%, respectively for all three vitamin D metabolites; lower limits of quantification were 1, 1 and 2 nmol/L and linearity of methods were 1–500, 1–200 and 2–500 nmol/L for 25(OH)D3, 3-epi-25(OH)D3 and 25(OH)D2, respectively. The PFP LC–MS/MS method showed minimal bias to the NIST RMP. Method comparison revealed that in the C18 LC–MS/MS method, the 3-epi-25(OH)D3 concentration is overestimated inadvertently not only from co-elution of both analytes, but also by an additional 30–40% higher ionisation efficiency of 3-epi-25(OH)D3 when compared to 25(OH)D3. Conclusion: This accurate LC–MS/MS method allows the simultaneous measurement of 25(OH)D3, 3-epi-25(OH)D3, and 25(OH)D2 in human serum. Due to increased ionisation efficiency, the contribution of the 3-epi-25(OH)D3 metabolite to the total 25(OH)D3 concentration is significantly overestimated in MS methods that do not resolve 3-epi-25(OH)D3 from 25(OH)D3 and may compromise its use in infant samples known to have significant amounts of 3-epi-25(OH)D3.