High performance liquid chromatographic assay for the determination of quercetin in plasma

A sensitive, reproducible, simple, and accurate high performance liquid chr omatographic (HPLC) method for the quantitative determination of quercetin in plasma has been developed and validated. Sample preparation involves sim ultaneous precipitation of plasma proteins and extraction of quercetin and kaempferol (the internal standard) from 0.1 mL plasma. The separation was p erformed in a stainless steel Ultrasphere-ODS column with a mobile phase co nsisted of a mixture of 30% acetonitrile and 70% of 5% acetic acid in HPLC water. The mobile phase was pumped at a flow rate of 1.5 mL/min and the eff luent was monitored at 375 nm. The retention times for the drug and the int ernal standard were found to be 3.5 and 6.2 minutes, respectively. Peak-are a ratios of the drug to the internal standard were used for quantitation of quercetin in plasma samples. The limit of detection of drug in plasma was found to be 0.06 mu g/mL. The intra-day coefficient of variation (CV) range d from 4.35% to 9.47%, and the inter-day CV ranged from 1.77% to 6.38% at t hree different concentrations. Mean absolute recoveries ranged from 96.52% to 101.19% and the relative rec overies ranged from 92.64% to 111.03% at three different concentrations. Pr eliminary stability tests showed that quercetin is stable for at least 8 we eks in plasma after freezing at -20 degrees C. The method was applied for t he determination of the pharmacokinetic parameters of quercetin after intra venous administration to three rats.