Optimization of ultrasonic extraction by response surface methodology combined with ultrafast liquid chromatography–ultraviolet method for determination of four iridoids in Gentiana rigescens

Gentiana rigescens is a rich source of iridoids and is commonly used as a folk medicine for treatment of hepatitis and cholecystitis for over 1000 years. A rapid ultrafast liquid chromatography–ultraviolet method was developed for simultaneous determination of four major iridoid glycosides in G. rigescens. Response surface methodology based on the Box–Behnken design was applied to optimize the extraction conditions of iridoid glycosides. Using the Shim-Pack XR-ODS III, four iridoid glycosides were efficiently separated with an acetonitrile:0.1% formic acid aqueous solution gradient at a flow rate of 0.25 mL/min for 8 minutes. All the regression equations revealed a good linear relationship (R2 > 0.9995). The intraday and interday variations were <1.95%. The recoveries ranged from 99.7% to 103.2%. The optimal extraction conditions were as follows: methanol concentration, 82%; the ratio of liquid to solid material, 68:1 (mL/g); and extraction time, 32 minutes. The yield of the four iridoid glycosides under the optimal process was found to be 63.08 mg/g, which was consistent with the predicted yield. In addition, the total content of 50 cultivated samples from Lincang, Yunnan, China, was within the range of 33.6–113.26 mg/g, which provides a more reasonable foundation for utilization of G. rigescens.