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Development of a high-performance liquid chromatography method for quantitative determination of cholesterol/cholesteryl ester in cellular matrices

biochemistry
cell-biology
green-chemistry

Abstract

Background. Cholesterol levels play a crucial role in the development of cardiovascular diseases, diabetes, and steatohepatitis. However, existing methodologies for quantifying cellular cholesterol levels are plagued by limitations such as poor reproducibility and cumbersome sample processing procedures. This study will explore the process of quantitative analysis of cholesterol and cholesterol esters in cells. The aim is to establish a high-performance liquid chromatography (HPLC) method for the quantitative analysis of intracellular cholesterol and cholesterol esters.

Methods. The optimized procedure involved enzymatic hydrolysis of cellular lysates through 2 h incubation at 37°C, followed by triglyceride elimination using potassium hydroxide treatment. A novel dual-phase extraction system employing n-hexane/isopropanol (3:2, v/v) demonstrated superior recovery of lipid analytes. After phase separation by centrifugation, the organic layer was concentrated and reconstituted in methanol for chromatographic analysis. Critical chromatographic parameters were optimized as follows: isocratic elution using acetonitrile/isopropanol (53:47, v/v) at a 1.0 mL/min flow rate, column temperature at 30°C, and UV detection at 240 nm with 10 μL injection volume. The method's validity was confirmed through parallel oil red O staining validation.

Results. Key analytical performance metrics revealed exceptional linearity (R² > 0.996) across a broad concentration range (2.5-160 μg/mL), with recovery rates exceeding 91.35%. The protocol exhibited remarkable reproducibility and precision, establishing it as a reliable analytical platform for cellular lipid quantification. This integrated approach addresses critical limitations in conventional lipid analysis methods by combining enzymatic specificity with optimized solvent extraction, while maintaining compatibility with standard cell biology laboratory workflows.

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