HPLC determination of Vitamin D3 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 D3 and 25-hydroxyvitamin D3 (25-OH-D3) in a sample of human plasma. The system uses a BioTrap precolumn for the on-line sample cleanup. A sample of 1 ml of human plasma was treated with 2 ml 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 250 μL of a solution of methanol 5 mmol l−1 phosphate buffer, pH 6.5 (4:1 (v/v)), and a 200 μl aliquot of this solution was injected onto the BioTrap precolumn. After washing during 5 min with a mobile phase constituted by a solution of 6% acetonitrile in 5 mmol l−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 5 mmol l−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 265 nm. The method was linear in the range 3.0–32.0 ng ml−1 with a limit of quantification of 3.0 ng ml−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 D3 and 25-OH-D3 in 16 min, with an adequate precision and sensitivity. However, the overlap of the sample cleanup step with the analysis increases the sampling frequency to five samples h−1. The method was successfully applied for the determination of Vitamin D3 and 25-OH-D3 in plasma from 46 female volunteers, ranging from 50 to 94 years old. Vitamin D3 and 25-OH-D3 concentrations in plasma were found from 4.30–40.70 ng ml−1 (19.74 ± 9.48 ng ml−1) and 3.1–36.52 ng ml−1 (7.13 ± 7.80 ng ml−1), respectively. These results were in good agreement with data published by other authors.