B-147 Enhancing the Detection of Vitamin D Metabolites by LC-MS/MS Using a Novel Derivatization Kit

Abstract Background Vitamin D is crucial for various biological functions and exists in various tissues, including bones, intestines, and kidneys. 25-hydroxyvitamin D serves as a key marker for assessing vitamin D status in the body. Currently, LC-MS/MS is the gold standard for measuring vitamin D levels. The JeoQuant Kit for LC/MS Analysis of Vitamin D Metabolites (JEOL, Tokyo, Japan), which is used for the determination of vitamin D metabolites in serum based on the DAP-PA (Seki et al, 2019), enhances the ionization efficiency in the LC-MS/MS system for four vitamin D metabolites (25(OH)D3, 25(OH)D2, 24,25(OH)2D3 and 3-epi-25(OH)D3), enabling accurate fractionation and quantification. DAP-PA was developed as a caged Cookson-type reagent, demonstrating significantly enhanced stability compared to previous non-caged Cookson-type reagents. Our research aimed to assess whether the kit works practically. Methods The JeoQuant Kit contains internal standards of stable isotope-labelled compounds at appropriate concentrations, derivatization reagents, quality control materials derived from human serum and calibration materials. An Acquity H-Class UPLC coupled with an Acquity TQD mass spectrometer (Waters, MA, US) was used for analysis. CAPCELL CORE C18 2.1mm*100mm (Osaka-soda, Tokyo, Japan) was used as the analytical column. For the pretreatment, an Isolute SLE+ column (Biotage, Tokyo, Japan) was used. Eluent of LC-MS/MS was used 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile, with binary gradient. Results Using attached control L/H, reproducibility was shown by intra- and inter-assay CVs which were below 10% and 15%, respectively. Storage tests of derivatization agents at -20°C, 4°C, and room temperature for one week showed no significant difference on peak areas (one-way ANOVA). Through this derivatization, it was possible to achieve more than a 20-fold increase in sensitivity compared to measurements without derivatization. Notably, low concentrations of 3-epi-25(OH)D3 were distinctly separated and detected. Conclusions This study showed that the JeoQuant Kit effectively increases the sensitivity of vitamin D metabolites through derivatization. It also emphasized the reagent's notable stability. Furthermore, it enabled the detection of 3-epi-25(OH)D3 with less sensitive LC-MS/MS models, offering clear separation from 25(OH)D3. The kit, which is complete with calibrants, internal standards, and quality control samples, simplifies analytical