Studies on Steroids. CLXXXIL. Determination of 6β-Hydroxycortisol in Urine by High-Performance Liquid Chromatography with Fluorescence Detection

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Abstract
A new sensitive method for the determination of 6.beta.-hydroxycortisol in urine by high-performance liquid chromatography with fluorescence detection was developed. 6.beta.-Hydroxycortisol and its C-6 epimer (internal standard) were transformed quantitatively into the 21-(9-anthroyl) derivatives when treated with 9-anthroyl nitrile in the presence of triethylamine in acetonitrile. The resulting fluorescent esters were readily separated on a Cosmosil 5SL column using ethyl acetate/hexane (2:1) as a mobile phase with a detection limit of 25 pg. The efficient cleanup was achieved by the combined use of Bond Elut and Clin Elut cartridges. The present method is applicable to the quantification of 6.beta.-hydroxycortisol in human urine with satisfactory accuracy and precision.