Determination of cortisol in plasma and urine by thin-layer chromatography and fluorescence derivatization with isonicotinic acid hydrazide

Abstract

A simple, inexpensive and reliable method is described for the measurement of cortisol in guinea-pig plasma and urine by thin-layer chromatography (TLC) and densitometry. Plasma and urine were extracted with dichloromethane and the extracts chromatographed on aluminium-backed silica gel TLC plates with concentrating zones. The plates were then dipped into the isonicotinic acid hydrazide (INH) reagent (3 g INH, 5 g trichloroacetic acid, 300 mL ethanol). The fluorescence of the cortisol hydrazone was further increased by dipping the plates into chloroform-liquid paraffin (9:1,v/v). The fluorescence was determined densitometrically (excitation: 366 nm, cut-off filter: >460 nm). The fluorescence intensity was linearly dependent on the amount of cortisol between 1 ng and 200 ng; the coefficients of variation ranged between 6.3% (1 ng) and 1.4% (200 ng). The sensitivity of this method (<1 ng) enables the measurement of cortisol in the plasma or urine of saline-, ACTH- or cortisol-treated guinea-pigs. Comparison of the cortisol concentration measured by this method or by a TLC-radioimmunoassay technique gave correlation coefficients ofr=0.98 (plasma) andr=0.97 (urine). Thus, in conjunction with the isolation of cortisol by TLC, the densitometric measurement of the fluorescent hydrazone of cortisol is a sensitive and rapid routine means of determination of the concentration of cortisol in biological materials such as plasma or urine.