Determination of cortisol in human plasma by reversed-phase high-performance liquid chromatography

A method is described for the measurement of cortisol in human plasma using 45% aqueous methanol eluent on a 120 mm x 4.5 mm I.D. Hypersil octadecylsilane column with UV detection at 239 nm after a simple dichloromethane extraction and evaporation with a prednisone internal standard. The sample preparation time and chromatography time are each about 15 min and linear correlations have been obtained with plasma samples assayed by the Mattingly fluorimetric technique and a commercial-kit competitive protein binding method. Concentration down to 30 nmol/l may be measured and the method can be used when fluorimetry is invalidated by interference, particularly from spironolactone.