HPLC determination of Vitamin D(3) and its metabolite in human plasma with on-line sample cleanup.

A HPLC method with automated column switching and UV-diode array detection is described for the simultaneous determination of Vitamin D(3) and 25-hydroxyvitamin D(3) (25-OH-D(3)) in a sample of human plasma. The system uses a BioTrap precolumn for the on-line sample cleanup. A sample of 1ml of human plasma was treated with 2ml of a mixture of ethanol-acetonitrile (2:1 (v/v)). Following centrifugation, the supernatant was evaporated to dryness under a stream of dry and pure nitrogen. The residue was reconstituted in 250muL of a solution of methanol 5mmoll(-1) phosphate buffer, pH 6.5 (4:1 (v/v)), and a 200mul aliquot of this solution was injected onto the BioTrap precolumn. After washing during 5min with a mobile phase constituted by a solution of 6% acetonitrile in 5mmoll(-1) phosphate buffer, pH 6.5 (extraction mobile phase), the retained analytes were then transferred to the analytical column in the backflush mode. The analytical separation was then performed by reverse-phase chromatography in the gradient elution mode with the solvents A and B (Solvent A: acetonitrile-phosphate buffer 5mmoll(-1), pH 6.5; 20:80 (v/v); solvent B: methanol-acetonitrile-tetrahydrofuran, 65:20:15 (v/v)). The compounds of interest were detected at 265nm. The method was linear in the range 3.0-32.0ngml(-1) with a limit of quantification of 3.0ngml(-1). Quantitative recoveries from spiked plasma samples were between 91.0 and 98.0%. In all cases, the coefficient of variation (CV) of the intra-day and inter-day-assay precision was </=2.80%. The proposed method permitted the simultaneous determination of Vitamin D(3) and 25-OH-D(3) in 16min, with an adequate precision and sensitivity. However, the overlap of the sample cleanup step with the analysis increases the sampling frequency to five samplesh(-1). The method was successfully applied for the determination of Vitamin D(3) and 25-OH-D(3) in plasma from 46 female volunteers, ranging from 50 to 94 years old. Vitamin D(3) and 25-OH-D(3) concentrations in plasma were found from 4.30-40.70ngml(-1) (19.74 +/- 9.48ngml(-1)) and 3.1-36.52ngml(-1) (7.13 +/- 7.80ngml(-1)), respectively. These results were in good agreement with data published by other authors.