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 (R) UHPLC systems (Ultimate 3000, Thermo scientific) by using HSS C-18 (2.1x50 mm, 1.8 mu m) 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 1 1 and the column temperature was maintained at 45 degrees C. FDA regulatory guidelines were used to develop and validate the method.
Results: The current developed UHPLC-UV method was found to he 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 line at 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 (R) as pure forms (intact) With no interference of excipients or drug-related substances.