HPLC determination of cinnamic acid and hippuric acid in rat plasma

Objective:To develop a method for the determination of cinnamic acid and hippuric acid in rat plasma by HPLC.Methods:The separation was carried out by using a SUPELCO Discovery column(5 μm,4.6 mm×250 mm) with a gradient mobile phase of 0.05% phosphoric acid-acetonitrile,at a flow rate of 1.0 mL·min-1,and detection wavelengths were set at 230 nm(hippuric acid) and 278 nm(cinnamic acid).Plasma samples were extracted with hydrochloric acid and ethyl acetate and methyl parahydroxybenzoate was used as an internal standard.Results:The linear range of cinnamic acid was 0.05 to 5.0 μg·mL-1(r=0.9987) with the lower limit of quantification for 0.05 μg·mL-1.The method recovery was 87.5% to 102.4% and the absolute recovery was more than 80%.The intra-day RSD and inter-day RSD were less than 13.1%.For hippuric acid,the linear range was 0.2 to 20.0 μg·mL-1(r=0.9957) with the lower limit of quantification for 0.2 μg·mL-1.The method recovery was 96.2% to 101.6% and the absolute recovery was more than 70%.The intra-day RSD and inter-day RSD were less than 8.4%.Conclusion:The developed method for the quantification of cinnamic acid and hippuric acid in rat plasma is sensitive,accurate,precise,stable,and is suitable for the pharmacokinetic studies of Ramulus Cinnamomi in rats.