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

Abstract

Key WordsThin-layer chromatography Cortisol Isonicotinic acid hydrazide Plasma and urine SummaryA 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 i 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 TLCradioimmunoassay technique gave correlation coefficients of r = 0.98 (plasma) and r = 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.

Determination of cortisol and cortisone in human morning and overnight urine by thin-layer chromatography and fluorescence derivatisation with isonicotinic acid hydrazide

“…The linear relationship of increasing amounts of cortisol and cortisone and INH-derived fluorescence is summarised in Figure 6. In agreement with previous work [9,10] fluorescence intensity of isonicotinyl hydrazones was significantly increased by dipping the chromatograms into paraffin/chloroform (1:2, v/v) and scanning them 10 min after the last dipping. The sensitivity was 12ng spot 1 for cortisol and 16ngspot 1 for cortisone (INH, without dipping into paraffin/chloroform) and 3 ng spot 1 for cortisol and 6 ng spot 1 for cortisone (INH, with dipping into paraffin/chloroform).…”

“…Both steroids were well separated from the bulk of other lipophilic substances which ascended 1 25mm note that the width of the concentrating zone ( furthermore shows the presence of a nonsteroidal compound in urinary chromatograms (Rv = 0.27) that appears to be caffeine because of the same mobility as authentic caffeine in seven different solvent systems, because of the same spectral absorbance curve as authentic caffeine, and because of the linear correlation of caffeine amount ingested in-vivo and its amounts excreted in timed urinary specimens. When plates were dipped into the INH reagent, isonicotinyl hydrazones of cortisol and cortisone formed within 1 h [9]. On the other hand caffeine did not react with INH, thus its presence did not interfere with the determination of steroids ( Figure 3).…”

“…[28] demonstrated the need for TLC prior to HPLC analysis, since TLC not only removed additional non-specifc UV light absorbing chromogens, but also eliminated unknown compounds co-eluting with the steroids of interest. Taking into account these disadvantages, it seemed worth testing whether the INH method established for cortisol determination in guinea pig urine [9] could be used to measure cortisol and/or cortisone in human urine. Initially, it was thought easy to adapt this method to human urine.…”

“…Urinary free cortisol and cortisone were determined using a previously described method [9] with some modifications. In brief, after thawing the frozen samples, 20mL urine were passed through kieselguhr-filled columns.…”

Measurement of Cortisol in Human Plasma and Urine by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry

“…RIA in general has high sensitivity but often lack selectivity due to cross-reactivity with related substances [10,11]. To improve selectivity, Takao et al [12] and Fenske [13] reported methods based on derivatization of cortisol with a fluorescent reagent, followed by HPLC or thin-layer chromatography, respectively. LC coupled with mass spectrometry (MS) or tandem MS/MS is a powerful and selective technique that has been used to determine steroid hormone levels [14][15][16]; however, most of the reported assays used d-4 cortisol as internal standard (IS), which may not be readily available [15,16].…”