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Synthesis of Deuterium-Labeled Vitamin D Metabolites as Internal Standards for LC-MS Analysis.

ABSTRACT: Blood levels of the vitamin D₃ (D₃) metabolites 25-hydroxyvitamin D₃ (25(OH)D₃), 24R,25-dihydroxyvitamin D₃, and 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) are recognized indicators for the diagnosis of bone metabolism-related diseases, D₃ deficiency-related diseases, and hypercalcemia, and are generally measured by liquid-chromatography tandem mass spectrometry (LC-MS/MS) using an isotope dilution method. However, other D₃ metabolites, such as 20-hydroxyvitamin D₃ and lactone D₃, also show interesting biological activities and stable isotope-labeled derivatives are required for LC-MS/MS analysis of their concentrations in serum. Here, we describe a versatile synthesis of deuterium-labeled D₃ metabolites using A-ring synthons containing three deuterium atoms. Deuterium-labeled 25(OH)D₃ (2), 25(OH)D₃-23,26-lactone (6), and 1,25(OH)₂D₃-23,26-lactone (7) were synthesized, and successfully applied as internal standards for the measurement of these compounds in pooled human serum. This is the first quantification of 1,25(OH)₂D₃-23,26-lactone (7) in human serum.

SUBMITTER: Nagata A

PROVIDER: S-EPMC9025501 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Synthesis of Deuterium-Labeled Vitamin D Metabolites as Internal Standards for LC-MS Analysis.

Nagata Akiko A Iijima Kazuto K Sakamoto Ryota R Mizumoto Yuka Y Iwaki Miho M Takiwaki Masaki M Kikutani Yoshikuni Y Fukuzawa Seketsu S Odagi Minami M Tera Masayuki M Nagasawa Kazuo K

Molecules (Basel, Switzerland) 20220409 8

Blood levels of the vitamin D3 (D3) metabolites 25-hydroxyvitamin D3 (25(OH)D3), 24R,25-dihydroxyvitamin D3, and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) are recognized indicators for the diagnosis of bone metabolism-related diseases, D3 deficiency-related diseases, and hypercalcemia, and are generally measured by liquid-chromatography tandem mass spectrometry (LC-MS/MS) using an isotope dilu ...[more]

PMID: 35458625

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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

Synthesis of Deuterium-Labeled Vitamin D Metabolites as Internal Standards for LC-MS Analysis.

Publications

Synthesis of Deuterium-Labeled Vitamin D Metabolites as Internal Standards for LC-MS Analysis.

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