Application Note: Method Optimization for LC-MS Analysis of Vitamin D Metabolite Critical Pairs in Serum (Sigma-Aldrich)
In this study, chromatographic screening studies for the analysis of vitamin D metabolites were expanded to include dihydroxy metabolites along with 25-epi-homologs. Screening consisted of evaluating various column stationary phases, organic modifiers and buffers for the resolution of critical pair isobaric compounds. The goal was to develop chromatographic conditions for the quantitation of hydroxy and dihydroxy vitamin D2 and D3 metabolites, including the isobaric epimers, from human serum samples. In addition to chromatographic method optimization, sample preparation techniques were evaluated to determine the impact of biological matrix on the sensitivity and accuracy of the LC-MS method.