Simultaneous Determination of Cholecalciferol and 25- Hydroxycholecalceferol in Lipid-based Self-nanoemulsifying formulations and Marketed Product Vi-de 3® by UHPLC-UV

Abstract

Background: The purpose of the current study was to develop a selective, precise, fast economical and advanced reverse phase ultra-high-performance liquid chromatography (UHPLC UV) method and validate it for the simultaneous estimation of cholecalciferol and its analogue 25- hydroxycholecalciferol in lipid-based self-nano emulsifying formulation (SNEDDS). Methods: The chromatographic separation was simply performed on a Dionex® UHPLC systems (Ultimate 3000, Thermo scientific) by using HSS C18 (2.1x50 mm, 1.8 µm) analytical column. The elution was carried out isocratically with the mobile phase consisting of acetonitrile and methanol in the ratio of 50:50 %v/v with a flow rate of 0.4 ml/min, followed by the UV detection at 265 nm. The injection volume was 1µl and the column temperature was maintained at 45°C. FDA regulatory guidelines were used to develop and validate the method. Results: The current developed UHPLC-UV method was found to be rapid (run time 2 min), and selective with the high resolution of cholecalciferol and 25-hydroxycholecalciferol (RT=0.530 min & 1.360 min) from different lipid matrices. The method was highly sensitive (Limit of Detection and Lower Limit of Quantification were 0.13 ppm & 0.51ppm, and 0.15 ppm & 0.54 ppm, respectively). The linearity, accuracy and precision were determined as suitable over the concentration range of 0.5-50.0 ppm for both the analytes. Conclusion: The proposed UHPLC-UV method can be used for the determination of cholecalciferol and 25-hydroxycholecalciferol in SNEDDS and marketed Vi-De 3® as pure forms (intact) with no interference of excipients or drug-related substances.