The comparison of HPLC and spectrophotometric method for cholesterol determination
Lukáš Kolarič | Peter Šimko
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.
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