| Summary: | This work deals with up-to-date optimization of cholesterol content determination when saponification and extraction procedures as well as HPLC conditions were studied. As found, optimal conditions for saponification process were identified by 15 min heating in the presence of 0.015 L of methanolic KOH solution with a concentration 1 mol/L with subsequent 0.015 L n-hexane–chloroform binary mixture (1:1, <i>v/v</i>) double extraction. HPLC separation consisted of isocratic elution with flow rate of 0.5 mL/min mobile phase composed of acetonitrile/methanol 60:40 (<i>v/v</i>) and stationary phase Zorbax Eclipse Plus C18 column 2.1 × 100 mm, 3.5 μm particle size diameters with detector wavelength 205 nm. The method passed through in-house validation criteria and its suitability was verified by analysis of butter reference materials. In final, the average content of cholesterol content in butter was determined at 2271.0 mg/kg. Thus, the method is suitable for the determination of cholesterol content in butter and probably also in other dairy products.
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