Determination of 24,25-dihydroxyvitamin D(3) in human plasma using liquid chromatography-mass spectrometry after...

A practical LC-MS method for determination of (24R)-24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)] in human plasma has been developed using derivatization with a Cookson-type reagent, 4-[4-(6-methoxy-2-benzoxazolyl)phenyl]-1,2,4-triazoline-3,5-dione (MBOTAD). The derivatization with MBOTAD significantly improved the ionization efficiency of the analyte with a detection limit of 18 fmol [equivalent to 7.5 pg of 24,25(OH)(2)D(3)] per injection. The method employed protein precipitation with acetonitrile, purification with OASIS HLB cartridge and silica gel column, derivatization with MBOTAD and atmospheric pressure chemical ionization MS detection. The mass spectrometer was operated in the positive-ion mode of mass chromatography and [26,26,26,27,27,27-(2)H(6)]-24,25(OH)(2)D(3) was used as an internal standard. The intra- and inter-assay coefficients of variation were below 3.4 and 2.5%, respectively, and the analytical recovery of 24,25(OH)(2)D(3) was quantitative. Assay linearity was obtained in the range of 0.05-1.2 ng per tube and the limit of quantitation was 0.23 ng/mL for a 0.3 mL plasma aliquot. The developed method was applied to plasma samples obtained from volunteers and gave satisfactory results.