Problems in Commercial Kits of 25-Hydroxy Vitamin D and the Development of Simple, Robust and Faster HPLC Method

Vitamin D is a lipid-soluble compound that plays a key role in bone mineral metabolism. The commercial current kits and several published assay methods (High-performance liquid chromatography (HPLC) and Immunoassay) are complicated due to the use of multiple reagents, larger sample volume, high backpressure, longer extraction time, evaporation under nitrogen after extraction, significant interference and antibody cross-reactivity. Here we report a new HPLC method for the determination of 25-hydroxyvitamin D2 (25-OHD2) and 25-hydroxyvitamin D3 (25-OHD3) that is simple (no evaporation), rapid (10-minute run time) and robust. Serum sample (300 μl) is mixed with 300 μl acetonitrile containing lauraphenone as internal standard. After vortexing and centrifugation, the supernatant was loaded into C18 extraction cartridges, washed with 70% methanol and then eluted with 200 μl of a mixture of 70% ethanol and 30% isopropyl alcohol (IPA). The eluent was mixed with 50 μl of water and injected into the HPLC-UV system for analysis. The method proved to be linear in the range of 10-750 nmol/L of 25-OHD2 and 25-OHD3. The intra- and inter-assay precision was less than 10 for both compounds at four different concentrations. The method was compared with (LC-MS/MS) and the correlation coefficients (R2) were 0.9454 and 0.9673 for 25-OHD2 and 25-OHD3 respectively. The proposed HPLC method is simple, rapid, robust and free from the most common problems encountered with commercial kits. It can be used in a high-volume laboratory that uses the HPLC method for the simultaneous determination of 25-OHD2 and 25-OHD3 in serum samples.