Design-of-Experiment Approach for HPLC Analysis of 25-Hydroxyvitamin D: A Comparative Assay with ELISA

Although high-performance liquid chromatography–mass spectrometry (HPLC–MS) is adopted as the method of choice for the determination of vitamin D and its metabolites in plasma, yet the unavailability of this expensive detection technique in many clinical laboratories makes ultraviolet (UV) detection the alternative of choice in many places worldwide. In this regard, determination of parameters affecting HPLC separation of vitamins D2, D3 and their hydroxyl metabolites in plasma in a systematic way would put an end to irrelevant trials for more optimization. A new robust HPLC–UV was developed, optimized using DryLab®2000 and validated for the determination of vitamins D2 and D3 and their 25-hydroxyl metabolites in plasma to achieve best resolution and least runtime where the metabolites elute in <10 min, where vitamin D2 is considered a feasible internal standard. Chromatographic parameters affecting resolution of the four peaks were specifically defined by a two-dimensional resolution map. Forty-six plasma samples were analyzed by the optimized method as well as by an ELISA kit to compare results and to judge validity of ELISA as a technique of clinical importance. Statistical analyses proved that the investigated assays were incomparable. Variation among subjects was detected by HPLC but not ELISA, concluding that HPLC–UV is the better tool in determining vitamin D status than ELISA.