Development and validation of RP-HPLC based bioanalytical method for simultaneous estimation of curcumin and quercetin in rat's plasma

Curcumin (CUR) and quercetin (QUE) are the two important flavonoids that possess very good anti-inflammatory, anti-oxidant, antidiabetic, analgesic and anti-cancer activities. Reverse phase high performance liquid chromatography (RP-HPLC) was used to develop a bioanalytical method for simultaneous estimation of both the drugs in the rat's plasma. The method was further validated as per ICH M10 guidelines using C-18 reverse phase column. Fisetin was used as an internal standard. Gradient elution was carried out at a flow rate of 1 mL/min in acetonitrile (ACN) and 2% glacial acetic acid (GAA). The chromatograms of all three phytoconstituents were recorded at detection wavelength of 392 nm. The drugs were extracted from the plasma samples by using protein precipitation method. The retention times (Rts) of fisetin, QUE and CUR were found to be 4.2, 5.5 and 12.1 min respectively. The developed method was found to be linear in the range of 2–10 µg/mL with regression coefficient (r²) of 0.9998 and 0.9993 for QUE and CUR respectively. The percentage recovery of drug more than 95% and percentage relative standard deviation less than 2% among the replicate studies indicated that the method was accurate and precise. Limit of detection (LOD) and limit of quantification (LOQ) in plasma samples were found to be 0.18 and 0.54 µg/mL for QUE and 0.35 and 1 µg/mL for CUR respectively. Further, the stability studies were also carried out at freeze-thaw cycles, short term stability at room temperature. The developed method was found to be robust and passed all the parameters of validation and therefore can be used effectively for estimation of CUR and QUE in the rat plasma.