Ultrasensitive Differential Measurement of Cortisol and Cortisone in Biological Samples using Fluorescent Ester Derivatives in Normal Phase HPLC

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Abstract
We report a method for differentially measuring the steroid hormones cortisol (F) and cortisone (E) in biological samples containing small quantities of either compound (50 pg F, 70 pg E). the method involves the formation of fluorescent ester derivatives of the anylates by reaction at the 21-hydroxy position with 9-anthroyl nitrile. Reversed-phase chromatography is used as a pre-derivatization sample cleanup, and solid-phase extraction on microcolumns of beta-cyclodextrin is used as a cleanup step before final separation and quantitation of the esters by normal phase chromatography with fluorescence detection. the synthetic steroid prednisolone is used as an internal standard. the derivatization consistently proceeds to a reproducible endpoint, and linearity, sensitivity, and specificity of the analysis are excellent.