Open Access Research Article Kumar, Pharm Anal Acta 2015, 6:8 DOI: 10.4172/2153-2435.1000410 Volume 6 • Issue 8 • 1000410 Pharm Anal Acta ISSN: 2153-2435 PAA, an open access journal Abstract Despite plenty of sunlight, vitamin D defciency (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 fow rate of 0.4 mL min −1 . The detection of eluent was carried out at λ max 265 nm. Retention time of vitamin D3 for method I and II was found to be 7.14 and 7.01 minutes, respectively, with R 2 >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 D 3 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 D 3 estimation at laboratory scale. An Improved and Sensitive Method for Vitamin D3 Estimation by RP- HPLC Subodh Kumar 1 , Diwesh Chawla 1 and Ashok Kumar Tripathi 2* 1 Central Research Laboratory, Multi-disciplinary Research Unit, University College of Medical Sciences, Delhi-110095, India 2 Biochemistry and Immunology Laboratory, Department of Biochemistry, University College of Medical Sciences, Delhi-110095, India Keywords: Vitamin D 3 ; RP-HPLC; Development and validation; DAD Introduction India is well known for its traditional, cultural and lingual diversity. It is a vast tropical country extending from 8.4º N latitude to 37.6° N latitude. Majority of its population live in areas receiving abundant sunlight throughout the year and hence it was assumed that Vitamin D (Vit D) defciency is uncommon in India [1] and globally [2-5]. However from various studies and data available in the published literature, Vit D defciency is very common in India in all the age groups and both sexes across the country [6-8]. Hence, Vitamin D status screening is essential as it allows for monitoring a patient’s response to Vitamin D therapy and also evaluation of treatment efect therefore, samples providing immediate and reliable results are highly desirable. Vitamin D is very important fat soluble vitamin in human and animal diet. It exists in two forms “viz”, Vitamin D 2 and D 3 . *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 December 10, 2014; Accepted August 13, 2015; Published 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:10.4172/21532435.1000410 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. Vitamin D 3 (cholecalciferol) is synthesized endogenously from 7-dehydrocholesterol afer ultraviolet irradiation or is absorbed from the diet [9,10]; plant/yeast derived ergocalciferol (Vitamin D 2 ) is formed exogenously by irradiation of ergosterol. Vitamin D plays an important role in the maintenance of normal levels of calcium and phosphorus in the blood stream and is essential for the proper development and maintenance of bone [11]. Scientifc evidence revealed that, it is not only associated with skeletal disorder but also plays animportant role in cancer, cardiovascular disease, autoimmune disease, hypertension, diabetes mellitus etc. [12-14]. Vitamin D is not a single compound but is a family of compounds that exhibit Vit D activity. Its measurement is important as a clinical indicator of nutritional vitamin D defciency, which is one of the causes of osteoporosis. Clinical laboratory scientists have a diverse selection of Vit D testing methods from which to choose. Te routine methods for measurement of vitamin D 3 concentration in human plasma were based on competitive protein binding and used vitamin D-binding protein and a tritium-labelled tracer. Tese methods were replaced by a simpler, rapid RIA, and a radio iodinated tracer was incorporated into the RIA in 1993 [15]. Quantitative HPLC assays have been developed based on P h a r m a c e u t i c a A n a l y t i c a A c t a ISSN: 2153-2435 Pharmaceutica Analytica Acta