Liquid-chromatographic measurement of endogenous and exogenous glucocorticoids in plasma

Abstract

The therapeutic response to and side effects of glucocorticoids will be better recognized and the recovery of the adrenals during the tapering of therapy with steroids better evaluated if endogenous and exogenous glucocorticoids are separately assessed. We describe a specific method for simultaneously measuring the concentrations of cortisone, cortisol, prednisone, and prednisolone in plasma by "high-pressure" liquid chromatography. The steroids, together with an internal standard, dexamethasone, are extracted from 1 mL of plasma with methylene chloride-ether, washed with acid and base, and separated isocratically on a normal-phase silica column with a mobile phase consisting of methylene chloride/tetrahydrofuran/methanol/glacial acetic acid (96.85/1/2.1/0.05 by vol) at a flow rate of 1.3 mL/min. The steroids are detected at 254 nm and quantitated by peak-height measurements; their retention times range from 6 to 20 min. The lower limits for routine detection of all four compounds is 10 microgram/L. The analytical recoveries are about 75%; the intra-day variability (CV) is 1 to 9%, and the inter-day variability 2 to 11%. Of 26 drugs and 20 steroids tested, only theophylline presents an interference problem.