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). Copyright © 1979 Wiley‐Liss, Inc., A Wiley Company