Serum cholesterol determined by liquid chromatography with 6-chlorostigmasterol as internal standard

We describe an accurate and precise method for determining serum cholesterol by high-performance liquid chromatography (HPLC). After addition of 6-chlorostigmasterol as internal standard, serum is treated with alcoholic potassium hydroxide. Subsequently the cholesterol and internal standard are extracted from the mixture into n-hexane and then derivatized to phenylurethanes for measurement by HPLC with ultraviolet detection. The effective chromatographic separation and the use of an appropriate internal standard make this procedure free from interferences by other serum sterols and precise. The mean cholesterol concentration in Standard Reference Material (SRM) 909 (human serum) assayed by this procedure (4.346 mmol.g-1 x L-1) agreed well with the value assigned by the National Institute of Standards and Technology (4.359 mmol.g-1 x L-1). Within-run and total CVs were 0.56% and 0.78%, respectively. Therefore the performance of this procedure is sufficiently good to allow its use as a candidate reference method for serum cholesterol determination.