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An Improved and Sensitive Method for Vitamin D3 Estimation by RPHPLC

Subodh Kumar, Diwesh Chawla and Ashok Kumar Tripathi

Despite plenty of sunlight, vitamin D deficiency (VDD) in India is an epidemic. 50-90% populations among all age groups are associated with VDD. Among the common methods (RIA, immunoassay etc.) available for vitamin D estimation, the analytical method like HPLC is considering as a gold standard. In the proposed study, we have developed a RP-HPLC method for the estimation of vitamin D3 with greater precision and accuracy. Separation was achieved on C18 column in isocratic mode using two different mobile phases i.e. acetonitrile: methanol (method I) and methanol: water with 0.1% formic acid (method II). The column was maintained at 40 °C and the mobile phase was pumped at flow rate of 0.4 mL min−1. The detection of eluent was carried out at λmax 265 nm. Retention time of vitamin D3 for method I and II was found to be 7.14 and 7.01 minutes, respectively, with R2>0.99. The standard curves were linear over the concentration range of 0.5-5 ng mL−1. The LOD and LOQ values for vitamin D3 for method I and II were found to be 1.64, 5.02 and 1.10, 3.60 ng mL−1, respectively. The percentage recovery was found to be 69-79% and 75-87% for method I and II, respectively. The % RSD of intra and inter-day precision of method I was found <2 and <7%, whereas, for method II, <2 and <4% respectively. In conclusion, method II showed greater precision and accuracy and also cost effective, therefore, it can be used for vitamin D3 estimation at laboratory scale.