Jones oxidation and high performance liquid chromatographic analysis of cholesterol in biological samples

A simple pre-column derivatization procedure for HPLC analysis of cholesterol in biological samples was developed. Cholesterol was treated with chromic acid and sulfuric acid in acetone (the Jones oxidation) and cholest-4-en-3,6-dione was formed. The reaction was finished in 5 min at room temperature and the product showed a strong UV absorbance at 250 nm that enabled an HPLC detection limit of 0.2 pmol. With stigmasterol as an internal standard, the reaction was applied to the analysis of total and free cholesterol in serum and high-density lipoproteins and the analysis showed a within-run and total coefficient of variation of about 0.2% and 0.5%, respectively.