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Fluorometric high-pressure liquid chromatographic determination of hydrocortisone in human plasma.

A highly sensitive and specific method for hydrocortisone determination in plasma is described. In this method, an internal standard (Δ4-pregnen-17α,20α,21-triol-3,11-dione) was added to plasma, which was then extracted with a mixture of methylene chloride—ether (60:40). After separation and evaporation of the organic phase, derivatization was carried out with dansylhydrazine. Upon completion of the reaction, the excess dansylhydrazine was reacted with pyruvic acid and a second extraction and evaporation step was performed. The residue was taken up in lOO μl of the high-pressure liquid chromatographic mobile phase, and 5 μl was injected onto a microparticulate silica column. Elution was carried out with an ethylene dichloride-butanol-water (91:8.5:0.5) mobile phase. The effluent was monitored with a fluorescence detector (excitation 240 nm; emission 470-nm cutoff filter). A linear calibration curve was found from 5 to 150 ng/ml with the precision estimated to be ±7% (CV).