Simultaneous determination of plasma prednisolone, prednisone, and cortisol levels by high-performance liquid chromatography

Abstract

Recipients of organ transplants remain particularly dependent on prednisolone as part of their maintenance immunosuppression. Despite this, the pharmacokinetics of prednisolone have never been fully characterized in these patients, and consequently dosing remains empirical. Accurate monitoring of prednisolone, its primary metabolite prednisone, and endogenous cortisol suppression in such patients may provide a means of improving the clinical outcome by adjusting for variability in prednisolone pharmacokinetics and pharmacodynamics. Measurement of endogenous cortisol may provide an independent marker of prednisolone pharmacodynamics. A simple isocratic reverse-phase high-performance liquid chromatography procedure, using betamethasone as an internal standard, was developed to quantify plasma prednisolone, prednisone, and cortisol simultaneously. The steroids were extracted from 0.5 mL plasma with 3 mL (1:1 v/v) ethyl acetate/tert-methyl butyl ether and 0.1 mL phosphoric acid, washed in 0.1 mol/L NaOH before a final drying step and reconstitution in mobile phase for injection. Separation was achieved using a Supelcosil LC-18-DB, 150 x 4.6-mm, 5-microm particle size, reverse-phase column attached to a Newguard 15 x 3-mm, RP8 guard column maintained at 25 degreesC, with ultraviolet detector set at 254 nm. The mobile phase consisted of 16% isopropanol in water containing 0.1% trifluoroacetic acid, set at a flow rate of 1.2 mL/min. The assay was linear up to 1,002 microg/L for prednisolone, 982 microg/L for prednisone, and 545 microg/L for cortisol. Mean intra-assay and interassay imprecision levels were 6.0% and 7.2%, respectively, for prednisolone, 5.8% and 7.2% for prednisone, and 5.6% and 7.9% for cortisol. Intra-assay inaccuracy was <7% of nominal values for prednisolone, prednisone, and cortisol. The lower limit of quantification was 7 microg/L for prednisolone and prednisone and 10 microg/L for cortisol. Corticosteroid recoveries were 73%, 74%, and 90% for prednisolone, prednisone, and cortisol, respectively. The authors describe a robust, inexpensive, and simple method suitable for therapeutic drug monitoring or pharmacokinetic studies of prednisolone; it may also be used to measure the suppression of endogenous cortisol production.