Abstract

Background: Accurate cholesterol measurements are essential for the prevention and management of cardiovascular diseases. Quality assurance of cholesterol measurements requires reference methods.

Methods: An isotope dilution liquid chromatography mass spectrometric (ID/LC/MS) method was developed. Serum samples were sampled volumetrically using automated dilutors, treated with potassium hydroxide and equilibrated with 3,4-¹³C₂ cholesterol. The natural cholesterol and the internal standard were extracted with hexane and oxidized to cholest-4-en-3,6-diones with chromic acid. The oxidation products were separated on reversed phase LC and detected by tandem MS. The method was calibrated using aqueous cholesterol calibrators and the calibration function was established with a polynomial regression.

Results: The correlation coefficients of the calibration curves were always >0.9999. The coefficients of variation (CV) of the volumetric sampling and the LC/MS analysis averaged 0.22% and 0.50%, respectively, and the total measurement CV was 0.60%. Other sources of measurement uncertainty were minor. Results on certified reference materials agreed within 1% of the certified values.

Conclusions: An ID/LC/MS method for serum cholesterol has been developed. The method is simple and accurate and may be used as a candidate reference method.