Determination of plasma and urinary cortisol by high-performance liquid chromatography using fluorescence derivatization with dansyl hydrazine

A method is described for the determination of cortisol in human plasma and urine by high-performance liquid chromatography using fluorophotometric detection. After extraction with methylene chloride, cortisol is labelled with dansyl hydrazine, and then separated by high-performance chromatography. The eluate is monitored by a fluorophotometer at 350 nm (excitation) and 505 nm (emission). The optimum conditions for the determination, such as HCl and dansyl hydrazine concentrations, reaction time and reaction temperature, and for the eluent of high-performance liquid chromatography, are discussed. Linearity of the fluorescence intensity (peak height) with the amount of cortisol was obtained between 0.5 and 60 ng. The recoveries for 50 and 100 ng of added cortisol were 98.7 and 95.4% for plasma, and 96.4 and 90.6% for urine, respectively. Comparison with a radioimmunoassay gave a correlation coefficient of 0.978. The proposed method is suitable for the routine analysis of cortisol in plasma and urine.