Simultaneous Determination of 25-Hydroxyvitamin D_2 and 25-Hydroxyvitamin D_3 in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry Employing Derivatization with a Cookson-Type Reagent

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A rapid and highly sensitive LC-MS-MS method for simultaneous determination of 25-hydroxyvitamin D_2[25(OH)DH_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 20μl 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.
公益社団法人日本薬学会の論文
2001-07-01