Simultaneous measurement of prednisone, prednisolone and 6β-hydroxyprednisolone in urine by high-performance liquid chromatography coupled with a radioactivity detector

We describe the first method for routine measurement of prednisone, prednisolone and 6β-hydroxyprednisolone concomitantly in urine. Urine (3 ml) is extracted with ethyl acetate, washed with base and separated by high-performance liquid chromatography on a silica column with a solvent system of hexane—diethyl ether—ethanol—tetrahydrofuran—glacial acetic acid (59.9:31:2.3:6.5:0.3, v/v). The steroids are detected at 254 nm. Because no conventional internal standard was found, 6β-³H]hydroxycortisol and ³H]prednisolone are added to urine prior to extraction; ³H is monitored by a radioactivity detector coupled with the chromatograph. The assay exhibits linearity from 200 to 7500 ng and an inter-day variability of < 11.4% (C.V.).