Development and validation of a bioanalytical hplc-dad method for ketoprofen quantification in rat plasma and brain tissue: application to pharmacokinetic studies and tissue distribution analysis
Abstract
Ketoprofen (KTP) is used as an anti-inflammatory agent and has been studied as a potential treatment for neurodegenerative diseases. This study developed and validated a sensitive high-performance liquid chromatography with diode-array detection method for quantifying KTP in rat plasma and brain tissue. Sample preparation was performed via solvent extraction assisted by protein precipitation, using acetonitrile acidified with 10% HCl in plasma or brain homogenate (1:10, v/v). Chromatographic separation was carried out on a Waters C18 reversed-phase column (150 × 4.6 mm, 5 μm) with an isocratic mobile phase composed of water, 0.3% triethylamine, acetonitrile, and methanol (50:40:10, v/v/v). The aqueous phase was adjusted to pH 3.5 with orthophosphoric acid, and the flow rate was set at 1 mL/min. Detection wavelengths were set at 255 nm for KTP and 355 nm for the internal standard, with a total run time of 5 min. The method demonstrated lower limits of quantification of 0.5 μg/mL for plasma and 0.25 μg/mL for brain tissue, with linearity ranges of 0.5–20 μg/mL and 0.25–8 μg/mL, respectively, and correlation coefficients above 0.999. Intra- and interday accuracy and precision ranges were within ±15% for both matrices, in accordance with the official guidelines of Brazil’s National Health Surveillance Agency and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. The method also demonstrated adequate selectivity, an absence of endogenous interferences, and consistent recovery in plasma and brain tissue. Stability studies confirmed the analyte content under different storage and processing conditions. Hence, the validated method proved sensitive, precise, accurate, and selective, supporting its application in preclinical pharmacokinetic and tissue-distribution research on KTP.