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. 2023 Aug;64(8):100409.
doi: 10.1016/j.jlr.2023.100409. Epub 2023 Jul 3.

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

Anastasia Alexandridou  1 , Dietrich A Volmer  2
Affiliations

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

Anastasia Alexandridou et al. J Lipid Res. 2023 Aug.

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.

Keywords: 25-hydroxyvitamin D(3); FMP-TS; LC-MS/MS; chemical derivatization; vitamin D(3) metabolites.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

None
Graphical abstract
Fig. 1
Fig. 1
A: Chromatographic separation of the investigated FMP-derivatized vitamin D3 metabolites and their respective IS using a pentafluorophenyl stationary phase and (A) H2O + 0.1% formic acid, (B) ACN + 0.1% formic acid as mobile phase. B: Chromatographic separation of the investigated FMP-vitamin D3 metabolites in human serum.
Fig. 2
Fig. 2
Optimization of FMP-TS concentrations in dry ACN (+TEA, 1% v/v) at 40°C for 15 min (standard deviation is shown in the bar chart).
Fig. 3
Fig. 3
Optimization of incubation time (following optimization of all other experimental conditions; the highest individual yields were each normalized to 100%; standard deviation is shown in the bar chart).
See this image and copyright information in PMC

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