A simple and precise LC-MS/MS method for the simultaneous determination of serum 25-hydroxyvitamin D3 and D2 without interference from the C3 epimer

Abstract

Serum 25-hydroxy metabolites of vitamin D₃ and D₂ (25-OH D₃ and 25-OH D₂) are significant biomarkers for vitamin D deficiency, which has drawn widespread attention as a notable public health problem; therefore, monitoring serum 25-OH D levels plays an increasingly important role in clinical tests and preventive medicine. Some current analytical methods used to measure human serum 25-OH D₃ and 25-OH D₂ suffer from complex operations, inaccuracies, or interference from the C₃ epimer of the metabolites. In this study, a simple, precise, and accurate LC-MS/MS method has been developed to determine serum 25-OH D₃ and 25-OH D₂ levels, markedly eliminating interference from the C₃ epimer through successful chromatographic separation performed on a pentafluorophenyl column. Detection of the analytes was carried out in multiple-reaction monitoring mode, and quantitative analysis was supported by the use of isotope-labeled 25-OH D₃ and D₂ as internal standards. The relative standard deviations of intra-run and total assays were less than 2.9% and 5.5%. The detection limits were as low as 0.27 and 0.31 ng ml⁻¹ for 25-OH D₃ and 25-OH D₂, with average recoveries of 99.65% and 99.93%, respectively. The accuracy of the protocol was also validated by measuring the National Institute of Standards and Technology standard serum reference materials, and the results were consistent with the certified values. In addition, the utility of the method was evaluated in a vitamin D status study to assess reference intervals and associations between vitamin D and risk factors for cardiovascular diseases in healthy Chinese volunteers. The method is simple, precise, and accurate, and can be utilized to measure 25-OH D₃ and 25-OH D₂ levels in human serum in clinical settings.