Published:Journal of Chromatographic Science,
ISSN 0021-9665 Volume
46, Number 1, January 2008, pp. 1-3
Determination of Cortisol in Human Plasma by
Thin-Layer Chromatography and Fluorescence Derivatization with
Isonicotinic Acid Hydrazide
Martin Fenske
Department of Animal Physiology, University of Bayreuth, 95440
Bayreuth, Germany
The present work describes a specific and rapid
determination of cortisol in human plasma. The method includes
liquid–liquid extraction of plasma samples, thin-layer
chromatography (TLC) of ethanolic extracts on aluminium foil-backed
silica gel 60 TLC plates, derivatization of cortisol with isonicotinic
acid hydrazide, and densitometric measurement of the fluorescence
intensity of cortisol hydrazone. The fluorescence was linearly
related to cortisol amounts; the correlation coefficients of
standard curve plots were r > 0.99. The coefficient of variation
ranged between 2.8–7.9% (20 ng, within-assay/between assay
variation) and 1.6–6.8% (80 ng, within-assay/between assay
variation). The recovery of cortisol from plasma spiked with
21-deoxycortisol was 85% ± 4%. Cortisol concentration
in the plasma was 66 ± 32 ng/mL (mean ± standard
deviation, n = 24). The advantage of this method is its simplicity
to separate cortisol from other steroids by TLC, its specificity
(formation of cortisol hydrazone), and the rapid quantitation
of cortisol by densitometry.
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