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ABSTRACT

A simple and rapid ultra performance liquid chromatography (UPLC) method for simultaneous determination of vitamin D-2 (VD-2), vitamin D-3 (VD-3), 25-hydroxyvitamin D-2 (25(OH)VD-2), and 25-hydroxyvitamin D-3 (25(OH)VD-3) in human plasma was developed and validated. Plasma samples were deproteinized with acetonitrile and then extracted with hexane. After evaporation, the residue was dissolved in the mobile phase, centrifuged, and the clear solution was injected into the system. Using dodecanophenone as an internal standard (IS), analytes separation was achieved by UPLC BEH C-18 column and a mobile phase that consisted of methanol, acetonitrile, and water (pH 3.0) and used in a gradient elution mode with a total run time of 14 minutes. The eluents were monitored by photodiode array detector (wavelength 265 nm). The relationship between plasma concentration of each of the four analytes and its peak area ratio (analyte to IS) was linear over the range of 2.5-100 ng/ml. Inter-day coefficient of variation and bias were ≤ 13.4% and ≤ 18%, respectively, for all the four analytes. Mean extraction recovery ranged from 77%-93% for the four analytes and was 85% for the IS. 

KEYWORDS

25-Hydroxyvitamin D-2, 25-Hydroxyvitamin D-3, Vitamin D-2, Vitamin D-3, Dodecanophenone, UPLC.

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