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.