The comparison of HPLC and spectrophotometric method for cholesterol determination

Lukáš Kolarič,Peter Šimko

doi:10.5219/1302

Abstract

The present study was carried out to compare two different analytical methods (HPLC and spectrophotometric) for determination of cholesterol content in milk while cholesterol in food is important not only for the nutritional value setting of foods but also due to the validation of a fast, reliable and economical method for studying the possible mechanism of its reduction. Spectrophotometric determination of cholesterol content was based on the Liebermann-Burchard (LB) reaction among cholesterol, ethyl acetate, acetic anhydride, plus concentrated H2SO4 and measuring absorbance of formed color at 625 nm. HPLC method was performed by column chromatography on reverse phase C18 with DAD detection at 205 nm. The methods were applied to the milk sample. The achieved LOD and LOQ for HPLC were 2.13 mg.kg-1 and 6.45 mg.kg-1, respectively, while for spectrophotometric method were 12.55 and 38.04 mg.kg-1. The difference between cholesterol content determined by both methods was statistically insignificant at p <0.05. Therefore, it can be concluded that both methods are suitable for determination of cholesterol content in milk, however, HPLC method exhibited higher sensitivity and lower limits of detection or quantification, respectively.

Highlights

Cholesterol is a key compound in most biological systems
High-performance liquid chromatography (HPLC) method was performed by column chromatography on reverse phase C18 with DAD detection at 205 nm
It can be concluded that both methods are suitable for determination of cholesterol content in milk, HPLC method exhibited higher sensitivity and lower limits of detection or quantification, respectively

Summary

Introduction

Cholesterol is a key compound in most biological systems. It is an essential compound in cellular membrane functions of animals and the precursor of important endogenous substances. Cholesterol is obtained from two sources: endogenous synthesis and exogenous ingestion from food (Ramalho, Casal and Oliveira, 2011). From a nutritional point of view, cholesterol is not found in significant amounts in plant sources, is mostly present in foods of animal origin, namely cheese, egg, beef, pork, poultry, fish, and shrimp. High levels of low-density lipoprotein cholesterol are a major cardiovascular risk factor. Once dietary cholesterol intake is increasing, the plasma cholesterol levels rise and increases the risk of cardiovascular diseases and atherosclerosis (Albuquerque et al, 2016)

Methods

Results

Conclusion