An HPTLC method for quantification of cholesteryl esters from human plasma and rat liver microsomes

Abstract

ABSTRACTCholesteryl oleate present as a neutral lipid in low‐density lipoprotein has been speculated to be a biomarker for atherosclerosis. Methods which are at hand for the quantification of cholesteryl oleate are either costly or entail the use of radioactive compounds. Charring of TLC plates has been used to identify cholesteryl esters for a long time but has never been applied to quantification of cholesteryl esters in biological matrices. Here, we report a novel method based on planar chromatography for the analysis of the products of the acyl CoA–cholesterol acyltransferase (ACAT) assay, viz. cholesteryl esters. Using silica gel 60 F254 as stationary phase, compounds were spotted on the plate and run using a solvent system comprising n‐hexane–diethyl ether–glacial acetic acid (90:10:1, v/v/v). The plates were developed by dipping in anisaldehyde–sulfuric acid reagent and were scanned at 546 nm for quantification. The developed method shows good linear relationship in the concentration range of 100–500 ng/band with a correlation coefficient (r) value of 0.9996. The method was validated for accuracy, precision and robustness. Percentage recovery of the method was found to be in the range 96.88–103.01% with intra‐ and inter‐day precision analysis yielding <2% relative standard deviation at nominal concentrations for analysis. The limits of detection and quantification were found to be 6.45 and 19.54 ng, respectively. The method was validated for robustness by making deliberate changes in mobile phase composition, volume and temperature of analysis, and the standard deviations of peak areas for these intentional changes were found to be 1.07, 1.02 and 1.30 respectively. The method was applied to the estimation of cholesteryl esters in plasma samples from patients diagnosed with hypercholesterolemia. No interferences were found from the biological matrices used in the assay. The proposed method could be of immense potential for estimation of cholesteryl oleate as a marker of ACAT activity, for screening of ACAT inhibitors in drug discovery process and in the prognosis of atherosclerosis. Copyright © 2013 John Wiley & Sons, Ltd.