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.