HPLC determination of luteolin and apigenin in rat bile and its application

Objective: To establish a RP-HPLC method for the determination of luteolin and apigenin in rat bile, and study the biliary excretion of luteolin and apigenin after rat orally administrated Chrysanthemum morifolium extract (CME). Methods:Bile samples were enzymatic hydrolyzed before analysis by RP-HPLC. SB-C_(18) HPLC column (250 mm×4.6mm, 5μm)was used. The mobile phase was methanol-0.2% phosphoric acid (52:48,v/v) with the flow rate of 1.0mL·min~(-1); the UV detector was set at 350 nm. Results:A good separation was obtained. The standard curves for luteolin and apigenin in bile were linear over the range of 0.1745-6.986μg·mL~(-1) and 0.3693-11.08μg·mL~(-1), respectively. The absolute recovery for luteolin and apigenin was 84.6% to 106.2% and assay recovery was 80.1% to 106.9%. The intra -and inter -day precisions (RSD) for luteolin and apigenin were all less than 14%. The relative biliary excretion rate of intake dose Of luteolin and apigenin during 36 hours after oral administration were 2.05% and 6.34%, respectively. Conclusion:The method developed is accurate, sensitive for the determination of luteolin and apigenin in bile. Enterohepatic circulation of luteolin and apigenin associated with double peaks in the blood concentration-time curves can be proved by the biliary excretion.