Simultaneous Fluorometric Determination of Cortisol and Corticosterone in Human Plasma

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Abstract
A fluorometric method is described for the simultaneous determination of cortisol and corticosterone in 2 ml of plasma. The method incorporates oxime derivative formation which alters cortisol and corticosterone fluorescence and thereby provides a measure of plasma blank fluorescence. The technique consists of the extraction of alkalinized plasma with methylene chloride, interaction of a dried down aliquot with 1% hydroxylamine, hexane wash and carbon tetrachloride partition for corticosterone followed by methylene chloride extraction for cortisol. Fluorescence is developed with sulfuric acid in both sample and blank tubes. Of various concentrations of fluorescence reagent tested, 10% ethanol in 85% aqueous sulfuric acid has been found to provide the greatest stability with the lowest blank values. Fluorescence may be read at leisure approximately 1 hr after adding the acid. Data on sensitivity, reproducibility, accuracy and specificity are presented. Recoveries of cortisol and corticosterone added to plasma averaged greater than 80%. The method is sensitive to concentrations of cortisol as low as 1–2 μg/100 ml and corticosterone 0.4–0.6 μg/100 ml. Results obtained for normal individuals, pregnant women in the third trimester and patients with pituitary and adrenal hypofunction are presented. The data compare well with results obtained from the literature using much more complicated procedures involving chromatography or labeled tracers. A good technician can complete up to 20 determinations of cortisol and corticosterone comfortably in one 8-hr working day.