Sensitive Analysis of Prednisolone and Prednisone in Human Plasma by Reverse Phase High-Performance Liquid Chromatography with Ultraviolet Detection

Abstract

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 C18 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 ≥ 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 1 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.