Rapid HPLC Method for Measurement of Vitamin D₃ and 25(OH)D₃ in Blood Plasma

The present work describes a novel, simplified high-performance liquid chromatography (HPLC) method for evaluation of vitamin D₃ and its 25-hydroxy metabolite in blood plasma. The retrieval of the analytes from the blood plasma matrix is based on a single-step extraction using acetonitrile. The method is specific, sensitive, and ensures good reproducibility. The recovery of the analytes, precision, and reproducibility obtained using the present approach gave results comparable to or better than more complex, laborious, and time-consuming procedures. This method is suitable for evaluation of the host’s vitamin D physiological status, as well as for rapid analysis of blood plasma samples in suspected cholecalciferol toxicity. With a significantly shortened time of analysis (10 minutes), the present method allows the possibility for processing of a large number of samples rapidly, efficiently, and at a low cost.