Determination of Serum 25-hydroxy Cholecalciferol Using High-Performance Liquid Chromatography: A Reliable Tool for Assessment of Vitamin D Status

This study was undertaken to design and set up a rather simple, reliable, and less expensive high-performance liquid chromatography (HPLC)-based method to assay 25(OH)D as a diagnostic tool for vitamin D assessment. Serum proteins were precipitated using ethanol and, after 10 minutes incubation at room temperature, methanol:isopropanol. The extraction was performed using hexane followed by evaporation under nitrogen flow. The sediment was then reconstituted in methanol and passed through a polypropylene filter. To run the chromatographic analysis, 20 μL of the filtrate was injected to the column. Peaks of 25(OH)D2 and 25(OH)D3 were both detected using a UV detector set at 265 nm. With a flow rate of 1.2 mL/minute, peaks of D3 and D2 vitamers were detected around 9.5 and 10.7 minutes, respectively. The intra- and inter-assay variations were 8.1% and 12.6%, respectively, and the recovery percent was found to be 100 ± 5%. To compare the procedure with conventional methods, 90 serum samples from subjects (48 females and 42 males) aged 40.5 ± 13.9 yrs, were analyzed for 25(OH)D using HPLC, competitive protein-binding assay (CPBA), and radioimmunoassay (RIA). Generally, CPBA and RIA assays both showed over-estimation of serum 25(OH)D, compared to HPLC. Though all three methods correlated significantly with each other, with the strongest between HPLC and RIA (r = 0.87, p < 0.001), both RIA and CPBA were found unreliable in detection of some deficient samples.