Development and Validation of an Analytical Method for the Estimation of Standard Cholesterol and Egg Cholesterol by High Performance Thin Layer Chromatography (HPTLC) Rao N. Mallikarjuna1,*, Bagyalakshmi J.2, Ravi T.K.2 1Research Scholar, JNTUK, Department of Pharmaceutical Sciences, Kakinada 2Department of Pharmaceutical Analysis, College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, New Siddapudur, Coimbatore, Tamilnadu, India *Corresponding Author E-mail: mallimpharmmba@gmail.com
Online published on 3 April, 2013. Abstract A simple, fast, specific and precise High Performance Thin Layer Chromatographic method (HPTLC) has been developed for estimation of cholesterol standard and egg cholesterol. The chromatographic separation was carried out on precoated silica gel 60F254 aluminum plates using a mixture of toluene: acetone: glacial acetic acid (6:1.3:0.1v/v/v) as mobile phase and densitometric evaluation of spots was carried out at 215nm using Camag TLC scanner III with CATS 1.3.4 version software. The experimental parameters like band size of the spot application, chamber saturation time, solvent front migration, slit width etc. were critically studied and optimum conditions were evolved. The drug was satisfactorily resolved with Rf value 0.55±0.03. The accuracy and repeatability of the proposed method was ascertained by evaluating various validation parameters like linearity (5 to 25 μg/spot), precision (intra-day RSD 0.57 to 0.86%, inter-day RSD 0.58 to 0.90%), accuracy (96.01±0.20), and specificity according to ICH guidelines. The limits of detection and quantification were 1μg/spot and 3μg/spot respectively. HPTLC method provides a faster and cost effective method for the estimation of cholesterol. Top Keywords Cholesterol, HPTLC, Densitometric estimation, Method development and Validation. Top |