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

ABSTRACT: Vitamin D analysis by MS faces several analytical challenges, including inefficient ionization, nonspecific fragmentation, interferences from epimers, isomers, and isobars, as well as very low concentration levels. In this study, we used 2-fluoro-1-methylpyridinium (FMP) p-toluene sulfonate for derivatization of vitamin D3 metabolites to increase detection sensitivity and allow for full chromatographic separation of vitamin D isomers and epimers. UHPLC-MS/MS was used for measurement of five vitamin D3 metabolites in human serum. Compared with Amplifex and 4-phenyl-1,2,4-triazolin-3,5-dion, the FMP p-toluene sulfonate reaction required less time to be performed. The method was optimized and validated to ensure accuracy, precision, and reliability. In-house and commercial quality control samples were used to assure the quality of the results for 25-hydroxyvitamin D3. The method showed very good linearity and intraday and interday accuracy and precision; coefficients of determination (r²) ranged between 0.9977 and 0.9992, relative recovery from 95 to 111%, and coefficient of variation from 0.9 to 11.3. Stability tests showed that the extracted derivatized serum samples were stable for 24 h after storage at -20°C; 24,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D3-FMP derivatives were stable for 1 week at -80°C. The method was applied to samples of healthy individuals for quantitative determination of vitamin D3, the two epimers of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3.

SUBMITTER: Alexandridou A

PROVIDER: S-EPMC10410174 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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

Alexandridou Anastasia A Volmer Dietrich A DA

Journal of lipid research 20230703 8

Vitamin D analysis by MS faces several analytical challenges, including inefficient ionization, nonspecific fragmentation, interferences from epimers, isomers, and isobars, as well as very low concentration levels. In this study, we used 2-fluoro-1-methylpyridinium (FMP) p-toluene sulfonate for derivatization of vitamin D3 metabolites to increase detection sensitivity and allow for full chromatographic separation of vitamin D isomers and epimers. UHPLC-MS/MS was used for measurement of five vita ...[more]

PMID: 37406930

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Project description:The present study systematically compares the sensitivity and selectivity of the analysis of multiple vitamin D metabolites after chemical derivatization using different reagents for liquid chromatography-tandem mass spectrometry (LC-MS/MS). Generally, chemical derivatization is applied to vitamin D metabolites to increase the ionization efficiency, which is particularly important for very low abundant metabolites. Derivatization can also improve the selectivity of the LC separation. A wide variety of derivatization reagents has been reported in recent years, but information on their relative performance and applicability to different vitamin D metabolites is, unfortunately, not available in the literature. To fill this gap, we investigated vitamin D3, 3β-25-hydroxyvitamin D3 (3β-25(OH)D3), 3α-25-hydroxyvitamin D3 (3α-25(OH)D3), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and compared response factors and selectivity after derivatizing with several important reagents, including four dienophile reagents (4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQ-TAD), Amplifex, 2-nitrosopyridine (PyrNO)) as well as two reagents targeting hydroxyl groups: isonicotinoyl chloride (INC) and 2-fluoro-1-methylpyridinium-p-toluenesulfonate (FMP-TS). In addition, a combination of dienophiles and hydroxyl group reagents was examined. For LC separations, reversed-phase C-18 and mixed-mode pentafluorophenyl HPLC columns using different compositions of the mobile phase were compared. With respect to detection sensitivity, the optimum derivatization reagent for the profiling of multiple metabolites was Amplifex. Nevertheless, FMP-TS, INC, PTAD, or PTAD combined with an acetylation reaction showed very good performance for selected metabolites. These reagent combinations provided signal enhancements on the order of 3- to 295-fold depending on the compound. Chromatographic separation of the dihydroxylated vitamin D3 species was readily achieved using any of the derivatization reactions, while for 25(OH)D3 epimers, only PyrNO, FMP, INC, and PTAD combined with acetylation enabled complete separation. In conclusion, we believe this study can serve as a useful reference for vitamin D laboratories, to help analytical and clinical scientists decide which derivatization reagent to choose for their application.

Dataset Information

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

Publications

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

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