Analytical method development and validation for determination of ketoprofen from human plasma by high performance liquid chromatography using the ultrafiltration technique

In this research, a separation method with ultrafiltration technique was developed for the high-performance liquid chromatography (HPLC) analysis of the non-steroidal anti-inflammatory drug (NSAID) ketoprofen, and the developed method was validated according to validation parameters in the FDA Bioanalytical Method Validation guide and the reliability of the method was proved. In this methodology, all analyzes were performed using an ACE C18 (250 × 4.6 mm i.d., 5 µm) column. As the mobile phase phase, a mixture of 40 mM disodium hydrogen phosphate dihydrate buffer (pH 6.0): acetonitrile was used at a ratio of 70:30 v/v, and the flow rate was set at 1 mL/min. The wavelength of the UV detector was set to 258 nm. As a result of optimization, the retention time of ketoprofen was 5.75 minutes. As the linearity range, it was studied between 1.0 and 25.0 μg/mL. The limit of detection (LOD) was found to be 0.33 μg/mL, and the lower limit of detection (LOQ) was calculated as 1.00 μg/mL. After the developed method was validated, it was applied to the analysis of ketoprofen from plasma in HPLC. Key words: NSAID, ketoprofen, ultrafiltration, HPLC, UV detector