Development and Validation of RP-HPLC Method for The Analysis of Cholecalciferol in Injectable Products

This study focuses on the development and validation of a robust Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) method for the quantification of cholecalciferol (vitamin D3) in commercially available pharmaceutical products. Vitamin D3 formulations, often produced in varying concentrations by pharmaceutical companies such as 1 mg/mL and 5 mg/mL, require precise dosage validation to ensure efficacy and safety. Accurate determination of cholecalciferol content is critical for quality control and compliance. In Pakistan, the most used method for estimating cholecalciferol (vitamin D3) is UV-visible spectroscopy, as recommended by the British Pharmacopoeia. This method involves the preparation of a diluent over 48 hours and the measurement of absorbance at 500 nm and 550 nm. However, it is prone to significant drawbacks, including time inefficiency and susceptibility to inaccuracies or losses during sample preparation. These limitations, coupled with potential interference from excipients overlapping with vitamin D3's absorption spectrum, make UV-visible spectroscopy an unreliable choice for routine analysis. To address these challenges, a more efficient and reliable Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) method was developed and validated in this study. The method employed a C18 column and a photodiode array (PDA) detector, with detection set at 265 nm. The mobile phase consisted of methanol and water in a ratio of 98:2 (v/v), ensuring optimal separation and detection. The method demonstrated excellent linearity, accuracy, precision, and specificity, making it a reliable tool for routine quality control testing. The RP-HPLC technique was found to be efficient, sensitive, and less time-consuming, making it highly suitable for determining cholecalciferol concentrations in injectable vitamin D3 formulations and ensuring the quality and efficacy of these products in the market.