Journal
BIOLOGICAL & PHARMACEUTICAL BULLETIN
Volume 24, Issue 7, Pages 738-743Publisher
PHARMACEUTICAL SOC JAPAN
DOI: 10.1248/bpb.24.738
Keywords
25-hydroxyvitamin D; LC-MS-MS; Cookson-type reagent; derivatization; human plasma
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A rapid and highly sensitive LC-MS-MS method for simultaneous determination of 25-hydroxyvitamin D-2 [25(OH)D-2] and 25-hydroxyvitamin D-3 [25(OH)D-3] in human plasma has been developed using derivatization with a Cookson-type reagent, 4-[2-(6, 7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQTAD). The derivatization with DMEQTAD significantly improved the ionization efficiencies of 25(OH)D-2 and 25(OH)D-3 with detection Limits of 20 and 12.5 fmol (8 and 5 pg) per injection, respectively. The method employed two steps of solvent extraction but did not require chromatographic purifications for sample pretreatment. The determination was carried out by mass chromatography of the protonated molecular ions formed by atmospheric pressure chemical ionization operating in the positive-ion mode after the derivatization, and 25-hydroxyvitamin D-4 was used as an internal standard. The intra-assay coefficients of variation were below 4.02 and 3.24% for 25(OH)D-2 and 25(OH)D-3, respectively, and the analytical recoveries of both compounds were quantitative. Assay linearity was obtained in the range of 0.05-1 ng per tube and the determination limit was 3 ng/ml for a 20 mul plasma aliquot, for each compound. The developed method was applied to plasma samples obtained from volunteers, two of whom had received vitamin D-2 supplementation, and gave satisfactory results.
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