Chiral liquid chromatography resolution and stereoselective pharmacokinetic study of pioglitazone enantiomers in rats

A selective chiral high performance liquid chromatographic (HPLC) method was developed and validated to separate and quantify the pioglitazone enantiomers in rat plasma. After extraction of the plasma samples with ethyl acetate, the separation of pioglitazone enantiomers and internal standard (I.S., dexamethasone acetate) was achieved on a cellulose tris (3,5-dichlorophenylcarbamate) column known as Chiralpak IC with a mobile phase of hexane–isopropanol (70:30, v/v) at a flow rate of 1.0 mL/min. The ultraviolet (UV) detection wavelength was set at 225 nm. Baseline separation of pioglitazone enantiomers and I.S., free from endogenous interferences, was achieved in less than 25 min. Ratio of peak area of each enantiomer to I.S. was used for quantification of plasma samples. Linear calibration curves were obtained over the range of 0.25–50 μg/mL in plasma for both enantiomers (R2 > 0.9990) with quantitation limit of 0.25 μg/mL. The mean extraction recoveries were 82.37–91.38% for pioglitazone enantiomers and 95.76% for I.S. from rat plasma. The mean relative error (R.E. %) of accuracy and the mean relative standard deviation (R.S.D. %) of intra-day and inter-day precision for both enantiomers were <10%. The method was validated with accuracy, precision, recovery and stability and used to determine the pharmacokinetics of pioglitazone enantiomers, after a single oral administration of racemic pioglitazone (30 mg/kg). The differences between the pharmacokinetic parameters Cmax, AUC0–24, AUC0–∞, CL/F of (+)-pioglitazone and (–)-pioglitazone were significant, suggesting that the disposition of pioglitazone in rats may be enantioselective. Moreover, the plasma levels of (+)- and (–)-pioglitazone in female rats were apparently higher than that in male rats, respectively.