HPLC Method with Solid-Phase Extraction for Determination of (R)- and (S)-Ketoprofen in Plasma without Caffeine Interference: Application to Pharmacokinetic Studies in Rats

摘要

A fast and reproducible high-performance liquid chromatography method has been developed for the determination of (R)- and (S)-ketoprofen. Ketoprofen enantiomers were determined in plasma samples (50 mL), after solid-phase extraction, using diclofenac as internal standard. Analyses were performed on a (S, S)-Whelk-O 1 stainless steel column (5 mm, 250 3 4.6 mm) using hexane– ethanol–acetic acid (93:7:0.5, v/v/v) as the mobile phase and detection at 254 nm. The method was selective for ketoprofen enantiomers in the presence of caffeine and endogenous plasma compounds. Standard curves were linear (R~2 > 0.999) over the concentration range of 0.25–12.50 and 0.25 mg/mL was taken as the limit of quantification. The intra- and interday precision (relative standard deviation) values were <15.0% and the accuracy (relative error) was within ±12.0% at 1.0, 5.0 and 10.0 mg/mL. Enantiomer recoveries yielded 100.0±15%. No significant differences were determined in plasma samples stored at room temperature for 24.0 h, after two freeze–thaw cycles, and between 0 and 4 weeks at 2208C (P > 0.05). The validated method was successfully applied in determination of (S)-ketoprofen in Wistar rats after oral administration of 3.2 mg/kg of (S)-ketoprofen alone or 3.2 mg/kg of (S)-ketoprofen + 17.8 mg/kg of caffeine.