An Improved and Sensitive Method for Vitamin D3 Estimation by RPHPLC
- *Corresponding Author:
- AK Tripathi
Biochemistry and Immunology Laboratory
Department of Biochemistry
University College of Medical Sciences (University of Delhi) and G.T.B. Hospital
Dilshad Garden, Delhi-110095, India
Tel: +91-11-22582972-74 Extn 5210, +91- 9811259019
Fax: +91-11- 22590495
E-mail: aktripathiucms @gmail.com
Received Date: December 10, 2014; Accepted Date: August 13, 2015; Published Date: August 17, 2015
Citation: Kumar S, Chawla D,Tripathi AK (2015) An Improved and Sensitive Method for Vitamin D3 Estimation by RP-HPLC. Pharm Anal Acta 6:410. doi: 10.4172/2153-2435.1000410
Copyright: © 2015 Kumar S. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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.