Study On Quality Control Of Yinchen-Qingjin Granules

Background and purpose:Diabetic foot is a serious diabetic complication which seriously affects the quality of patients’health as a result of high rate of disability and mortality.Yinchen-Qingjin granules are obtained by adding the herbal medicines of Yinchenhao decoction which was famous TCMs formulae.Yinchen-Qingjin granules is used to treat diabetic foot gangrene and relieve the symptoms of foot fascia infection.For the purpose of the quality control,this study established the quality control method of Yinchen-Qingjin granules by researching chemical composition and pharmacokinetics.It will ensure the safe use of this preparation.Methods:（1）An analysis method based on UPLC-LTQ-Orbitrap MS/MS was established to identify the chemical composition of Yinchen-Qingjin granules in positive and negative ion mode.（2）An analysis method based on UPLC-Qq Q-MS/MS was established to quantitatively analyze the components of Yinchen-Qingjin granules.This method was applied to the pharmacokinetic study of rats after oral administration of Yinchen-Qingjin granules.（3）The crude drug in Yinchen-Qingjin granules were identify by TLC.The assay of gallic acid,chlorogenic acid,geniposide and caffeic acid in Yinchen-Qingjin granules was determined by HPLC.the content of gallic acid,caffeic acid,chlorogenic acid,liquiritin,isoliquiritin,neochlorogenic acid,geniposide and rhein were determined by UPLC-Qq Q-MS/MS.Results:（1）Through literature,Mass frontier 7.0 software and reference standards,a total of 117chemical components in Yinchen-Qingjin granules were tentatively identified,31 of which were identified by reference standards.（2）A UPLC-Qq Q-MS/MS method was established to determine the content of gallic acid,neochlorogenic acid,chlorogenic acid,caffeic acid,geniposide,rutin,genipin,hyperoside,liquiritin,isoliquiritin,liquiritigenin,isoliquiritigenin and rhein in rat plasma.SD rats were given 2 g/kg Yinchen-Qingjin granules respectively.T_max of chlorogenic acid,gallic acid,neochlorogenic acid,geniposide,liquiritin,isoliquiritin,caffeic acid,rhein,liquiritigenin and genipin were 0.50±0.00,0.50±0.00,0.50±0.00,1.75±1.15,0.21±0.15,0.33±0.13,0.26±0.12,0.32±0.16,2.75±1.77,5.67±0.75 h respectively.t_1/2were 5.53±2.41,4.78±1.35,5.00±2.59,8.58±0.74,8.67±4.02,4.73±1.38,2.91±1.73,8.46±1.34,2.54±0.84,10.95±3.93 h respectively.（3）The TLC for seven crude drugs of Artemisiae scopariae herba,Sedi herba,Gardeniae fructus,Bistortae rhizoma,Rhei radix et rhizoma,Glycyrrhizae radix et rhizoma,Semen coisic and Taraxaci herba were established to identify the traditional Chinese medicines of Yinchen-Qingjin granules.Results showed that the established method had clear characteristic spots with good separation and no interference in negative samples,and can be used for the qualitative identification of seven crude drugs in Yinchen-Qingjin granules.The content of gallic acid,chlorogenic acid,geniposide and caffeic acid in Yinchen-Qingjin granules was determined by HPLC,and the results showed that gallic acid,chlorogenic acid,geniposide and caffeic acid have good linear relationship in the range of 10.16-101.6,24.94-249.4,39.69-396.9 and 1.38-13.84μg/m L;RSD is 0.83%,0.92%,0.65%and 1.2%on the precision,respectively;RSD is 1.3%,1.2%,1.4%and 1.6%on the repeatability,respectively;RSD were 0.59%,0.79%,0.77%and 1.3%on the 24 h stability;the average recoveries were 100.13%,99.08%,98.24%and 98.63%,and the RSD were 1.3%,1.2%,1.4%and 1.9%;The contents of gallic acid,chlorogenic acid,geniposide and caffeic acid in 3 batches of Yinchen-Qingjin granules were 1.453-1.505,3.319-3.337,8.416-8.582 and 0.233-0.241 mg/g,respectively.The content of gallic acid,caffeic acid,chlorogenic acid,liquiritin,isoliquiritin,neochlorogenic acid,geniposide and rhein in Yinchen-Qingjin granules was determined by UPLC-Qq Q-MS/MS,and the results showed that gallic acid,caffeic acid,chlorogenic acid,liquiritin,isoliquiritin,neochlorogenic acid,geniposide and rhein have good linear relationship in the range of 50.6-5060.0,9.8-980.0,53.2-5320,48.3-4825.0,11.0-1096,51.0-5100.0,98.0-9800.0 and 9.9-988 ng/m L,and the limits of quantification are 7.6,2.5,3.3,4.8,4.1,1.8,2.5 and 3.1 ng/m L,the limits of detection are 3.8,1.3,1.7,1.2,2.1,0.9,1.3 and 1.5 ng/m L,respectively;RSD of the precision and accuracy of all components is within the limits of 0.71-1.2%;RSD of the repeatability of all components is within the limits of 0.4-2.0%;RSD of the 24 hour stability of all components is within the limits of0.49-1.3%;the average recovery is in the range of 96.83-100.1%,and the RSD is 1.2-2.6%;The content of gallic acid,caffeic acid,chlorogenic acid,glycyrrhizin,isoliquiritin,neochlorogenic acid,shanzhiside,geniposide,glycyrrhizic acid,and rhein in 6 batches of Yinchen-Qingjin granules were 1.507-1.678,0.230-0.252,3.219-3.417,0.733-0.796,0.228-0.258,2.371-2.643,8.375-8.712 and 0.263-0.293 mg/g,respectively.Conclusion:In this study,based on the research of chemical composition and pharmacokinetics of Yinchen-Qingjin granules,we will carry out further pharmacodynamic research on Yinchen Qingjin granules related to diabetic foot.By establishing methods for TLC and contents determination of characteristic components,we have improved the quality standards of Yinchen-Qingjin granules,and ensured the safe use of Yinchen-Qingjin granules.