Reversed-phase high-performance liquid chromatographic determination of 7 alpha-hydroxy-4-cholesten-3-one in human...

A method for the determination of 7 alpha-hydroxy-4-cholesten-3-one in human serum has been developed. The method is based upon solid-phase (C18) extraction of serum after addition of 7-beta-hydroxy-4-cholesten-3-one as internal standard. The extract is analysed by reversed-phase HPLC using acetonitrile-water as a mobile phase. The effluent is monitored at 241 nm. The overall recoveries of the method range from 96% to 105%. The coefficient of variation for the within-run precision is 3.2% (n = 20, mean = 13.0 ng/ml) and for the between-run precision 3.8% (n = 32, mean = 13.3 ng/ml). The limit of detection is 3 ng at a signal-to-noise ratio of 3:1, which corresponds to 1 ng/ml using 3 ml of serum. The median value of 7 alpha-hydroxy-4-cholesten-3-one found in blood donors (n = 27) was 8.9 ng/ml (range 2-35 ng/ml).