Development and validation of RP-HPLC method for estimation of fisetin in rat plasma

Abstract Fisetin is an important phyto-flavonoid that possesses very good anti-oxidant, anti-Parkinson's and anti-cancer activities. A bioanalytical method was developed and validated for the estimation of fisetin in the rat's plasma using reverse phase ultra-fast liquid chromatography combined with a C-18 reverse phase column. Quercetin was used as an internal standard. The mobile phase was composed of acetonitrile and orthophosphoric acid (0.2% v/v) in the ratio of 30:70 v/v. Flow rate was set at 1 mL/min and chromatogram of both compounds was detected at a wavelength of 362 nm. Protein precipitation method was used to extract drug from plasma samples. The retention times for fisetin and quercetin were found at 8.3 min and 16.9 min, respectively. The developed method was found to be linear in the range of 25–125 ng/mL with regression coefficient (r2) of 0.9996. The method was validated as per the ICH Q2 (R1) guidelines. The percentage recovery was in the range of 95–105%, which indicated that method was accurate. The percentage relative standard deviation was found to be less than 2% which indicates that method was precised. Limit of detection (LOD) and limit of quantification (LOQ) were found to be 3.18 ng/mL and 9.66 ng/mL, respectively. The developed method was found to be robust as there was no any significant change in response with change in flow rate and composition of mobile phase. Obtained results indicated that the developed method has passed all the validation test parameters and can be applied successfully for estimation of fisetin in the rat plasma.