2-fluoro-1-methylpyridinium p-toluene sulfonate: a new LC-MS/MS derivatization reagent for vitamin D metabolites

Vitamin D analysis by mass spectrometry faces several analytical challenges, including inefficient ionization of vitamin D compounds, non-specific fragmentation patterns, interferences from epimers, isomers and isobars, as well as very low concentration levels for some of the metabolites in blood samples. Derivatization has been a solution to several of these problems. In this study, we used 2-fluoro-1-methylpyridinium p-toluene sulfonate (FMP-TS) for the first time for derivatization of vitamin D3 metabolites, with the aim of increasing detection sensitivity and allowing for full chromatographic separation of vitamin D isomers and epimers. UHPLC-MS/MS was used for the measurement of five major vitamin D3 metabolites in human serum. Compared to Amplifex and 4-phenyl-1,2,4-triazolin-3,5-dion (PTAD), the FMP-TS reaction required less time to be performed (only 15 min). Multiple experimental conditions of the method were optimized and validated to ensure the accuracy, precision and reliability of the method. In-house and commercially available quality control samples were used to assure the quality of the results for 25-hydroxyvitamin D3. The method showed very good linearity and intra- and inter-day accuracy and precision; coefficients of determination (r2) ranged between 0.9977-0.9992, relative recovery from 95-111% and coefficient of variation from 0.9-11.3. Stability tests showed that the extracted derivatized serum samples were stable for 24 h after storage at -20 oC. Additionally, 24,25(OH)2D3 and 1,25(OH)2D3-FMP derivatives were stable for 1 week when stored at -80 oC. The optimized method was successfully applied to human samples of healthy individuals for quantitative determination of vitamin D3, the two epimers of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3.