Sensitive analysis of prednisolone and prednisone in human plasma by reverse phase high-performance liquid chromatography with ultraviolet detection

This report describes a simple and sensitive reverse phase HPLC method with UV detection for the simultaneous determination of prednisolone and prednisone in human plasma. The analysis utilized a C-18 analytical column and a mobile phase consisting of acetonitrile:0.08% trifluoroacetic acid in deionized water (28:72, v/v). Prednisolone, prednisone and corticosterone (internal standard) were extracted from plasma by one-step extraction with t-butyl methyl ether. Prednisolone, prednisone and the internal standard were eluted at 19.8, 21.4 and 34.3 min, respectively. The standard curves were linear (r greater than or equal to 0.998) for prednisolone and prednisone over the concentration ranges of 2-1000 ng/mL and 5-100 ng/mL, respectively. The intra- and inter-day assay variabilities ranged from 1.8-10.5% and 0.7-9.5%, respectively, for prednisolone, and from 6.3-18.5% and 1.8-4.5%, respectively, for prednisone. The LOD and LOQ were 0.5 and 2 ng/mL, respectively, for prednisolone, and I and 5 ng/mL, respectively, for prednisone using a plasma sample volume of 0.5 mL. This highly sensitive and selective assay method was successfully applied to a pharmacokinetic study after oral administration of 10 mg prednisolone to human volunteers.