Highly sensitive and simple determination of cholesterol and cholestanol in human serum by high-performance liquid chromatography with fluorescence detection.
A simple and highly sensitive high-performance liquid chromatographic method for the determination of cholesterol and cholestanol in human serum is described. After extraction of serum with n-hexane, these compounds and 1-eicosanol (internal standard) are converted into the corresponding fluorescent carbamic esters by treatment with 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxo-quinoxaline-2-carbonyl azide in benzene. The derivatives are separated within 32 min on a reversed-phase column (YMC Pack C8) with acetonitrile-methanol-water (81:9:10, v/v/v) as eluent and detected fluorimetrically. The detection limits are 2 pg (5 fmol) and 3 pg (7 fmol) for cholesterol and cholestanol, respectively, at a signal-to-noise ratio of 2 in a 20-microliters injection volume. This sensitivity permits precise determination of cholesterol and cholestanol in 1-5 microliters of normal human serum.
Faculty of Pharmaceutical Sciences, Fukuoka University, Japan.
T
T
Iwata
M
M
Yamaguchi
M
M
Nakamura
D002777:Cholestanols / Q000097:blood*; D002784:Cholesterol / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007202:Indicators and Reagents; D013050:Spectrometry, Fluorescence; D013696:Temperature
D002777:Cholestanols; D007202:Indicators and Reagents; D002784:Cholesterol