Development Of Reference Materials For Chlorogenic Acid And Isochlorogenic Acid In Honeysuckle

Honeysuckle is a typical food and food homologous food,which has a wide range of applications in medicine and food.Its active ingredients mainly include chlorogenic acid,isochlorogenic acid A,B,C,etc.Among them,chlorogenic acid content is an important indicator to judge the quality of honeysuckle.Preparation of chlorogenic acid,isochlorogenic acid A,B,C standard substances have important practical significance for judging the quality of honeysuckle on the field.Standard materials such as chlorogenic acid are expensive in the market,unstable in nature,and difficult to extract.In this study,the high-pressure preparation solution was used to separate and purify the active constituents from the honeysuckle,and the liquid phase preparation conditions were followed as:the mobile phase was 0.1%formic acid aqueous solution and acetonitrile(chlorogenic acid),0.1%aqueous trifluoroacetic acid solution and acetonitrile(isochlorogenic acid A,B,C);collected according to different retention times of the target;and purity analysis by high performance liquid chromatography-ultraviolet detector.The standard curve regression equations for the four targets plotted in the study were:y=5225.1x-11037.9(chlorogenic acid);y=98877.6x-92332.1 isochlorogenic acid A);y=36856.4x-360412.7,(isochlorogenic acid B);y=3935.2x-342392.8(isochlorogenic acid C),correlation coefficient R2 were greater than 0.99 chlorogenic acid,isochlorogenic acid A,B,C showed a good linear relationship,the collection of the target Content detection.In this study,the structure of the extract was identified by infrared spectroscopy,ultraviolet spectroscopy,mass spectrometry and nuclear magnetic resonance analysis.Uniformity test,stability test,8 laboratory collaborative determination and uncertainty calculation were carried out on the extract.The variance analysis method was used to judge the uniformity and stability of the target.The final result showed chlorogenic acid and different Chlorogenic acids A,B,and C all showed good uniformity.They were stable in the long-term stability test for six months.In the short-term stability test,chlorogenic acid was at-20℃,4℃,It is stable at room temperature.The content of isochlorogenic acid A,B and C decreased at room temperature,and was stable at-20℃ and 4℃.In the fixed value and uncertainty engineering,the chlorogenic acid and isochlorogenic acid A,B,and C characteristic values were(99.17± 0.76)%,(99.24±0.27)%,and(99.24±0.18)%,(99.27±0.19)%.The results show that the standard substance of chlorogenic acid,isochlorogenic acid A,B,C can be obtained by the method of preparing liquid phase by high pressure,and the purity is above 99%.