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 50mM LiClO(4) as a mobile phase; and an applied potential at 1.9V versus Ag/AgCl. The current peak height was linearly related to the amount of cholesterol injected from 0.5-100 microM (r>0.999). The detection limit (S/N=3) of cholesterol was 0.36 microM (1.8 pmol). Cholesterol at 100 microM 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%.
Hojo
Kazuhiro
K
Department of Analytical Chemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan.
Hakamata
Hideki
H
Ito
Ayumi
A
Kotani
Akira
A
Furukawa
Chisaki
C
Hosokawa
Yu-Ya
YY
Kusu
Fumiyo
F
D002784:Cholesterol / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004563:Electrochemistry / Q000379:methods; D006801:Humans; D012680:Sensitivity and Specificity
D002784:Cholesterol