Determination of total cholesterol in serum by high-performance liquid chromatography with electrochemical detection

A simple and sensitive HPLC method that does not require derivatization for determining cholesterol has been developed. Investigation of voltammetric behavior of cholesterol showed that cholesterol could be oxidized on a glassy carbon electrode in non-aqueous solvents. This was applied to the development of a method by HPLC with electrochemical detection (HPLC-ED). The HPLC-ED was optimized using the separation of cholesterol and oxysterols including 26-hydroxycholesterol and 24S-hydroxycholesterol. The separation was carried out with a Develosil C30-UG-3 column; acetonitrile-2-propanol (9:1, v/v) containing 50 mM LiClO4 as a mobile phase; and an applied potential at 1.9 V versus Ag/AgCl. The current peak height was linearly related to the amount of cholesterol injected from 0.5–100 μM (r > 0.999). The detection limit (S/N = 3) of cholesterol was 0.36 μM (1.8 pmol). Cholesterol at 100 μM was directly detected with a relative standard deviation (RSD) of less than 1.0% (n = 8). Total cholesterol and free cholesterol in control human serum were determined by the present method with the recovery of more than 90% and the RSD (n = 6) of less than 3.0%.