Preliminary Study On Quality Control And Pharmacokinetics Of Qinxing Qingre Zhike Granules

ObjectiveIn this study,Qinxing Qingre Zhike Granule for the treatment of pulmonary heat and cough syndrome is used as the research subject.The chemical components of Qinxing Qingre Zhike Granules and the prototype components in plasma of rats after oral administration is identified by UPLC-LTQ-Orbitrap technology.An LC-MS/MS method for the determination of the main components concentrations is built and the way for the measure of plasma concentration of the main components in blood is established.TLC method is used for qualitative identification and HPLC method is for quantitative determination of the main effective component.The quality control method of Chinese compound formula based on pharmacodynamic substance will be established to ensure the internal quality and better serve the clinic.Method1.Blood composition analysis of Qinxing Qingre Zhike Granules based on UPLC-LTQ-Orbitrap technologyUPLC-LTQ-Orbitrap technology was used to analyze and identify the components of Qinxing Qingre Zhike Granules and the prototype components in rat plasma after oral administration.By searching Chinese and foreign literatures and professional chemical database,the information of reported compounds in Scutellariae Radix,Gardeniae Fructus,Mori Folium,Armeniacae Semen Amarum and Glycyrrhizae Radix et Rhizoma was collected.Finally,a database containing 565 chemical components was established,including name,molecular formula,structural formula and accurate molecular weight,etc.2.LC-MS/MS method was used to simultaneously determine the content of 12effective components in Qinxing Qingre Zhike GranulesAgilent 6460 Triple Quad LC/MS,Ultimate XB-C18?4.6 mm×100mm,3?m?chromatographic column was used.Flow rate was 0.4 m L·min^-1;The mobile phase A was pure water containing 0.5‰formic acid,and B was acetonitrile,with gradient elution.Electrospray ion source?ESI?and multiple reaction detection mode?MRM?was utilized for analysis.The samples were ultrasonic extracted with 50%methanol solution.Paeoniflorin and fenbuprofen were the internal standard for negative and positive ion scanning,respectively.The contents of chlorogenic acid,geniposidic acid,amygdalin,geniposide,liquiritin,isoliquiritin,baicalin,wogonoside,baicalein,liquiritigenin,isoliquiritigenin and glycyrrhizic acid in Qinxing Qingre Zhike Granules were determined.3.A preliminary study on the pharmacokinetics of Qinxing Qingre Zhike GranulesThe drug concentration was 0.25 g·m L^-1.Blood was collected at different time points after gavage.Heparin was added for anticoagulant and Vitamin C was for antioxidant in plasma.Protein was precipitated by acetonitrile.Plasma concentration was determined.Pharmacokinetics parameters including half-life(T_1/2),total area under the curve(AUC_0-?),the mean residence time extrapolated to infinity(MRT_0-?),the volume of distribution at terminal phase?Vz?and the clearance rate?Cl?were estimated by using the non-compartmental analysis in Win Nonlin programs.4.TLC identification of Qinxing Qingre Zhike GranulesTLC identification of Scutellariae Radix:A polyamide thin film plate was used.Baicalin,baicalin,and baicalin were used as the reference materials.The developing agent was toluene-ethyl acetate-methanol-formic acid?10:3:1:2?.Examined by UV lamp?365 nm?.TLC identification of Gardeniae Fructus:HSG thin layer plate was used.Geniposide was reference substance.The developing agent was ethyl acetate-acetone-formic acid-water?5:5:2:1?.The color reagent was 10%sulfate ethanol solution.TLC identification of Mori Folium:HSG thin layer plate was used.Control herb Mori Folium was choosen as control.The developing agent was toluene-ethyl acetate-formic acid?5:2:1?with pre-saturated for 10 min.Examined by UV lamp?365 nm?.TLC identification of Armeniacae Semen Amarum:HSG thin layer plate was used.Amygdalin was reference substance.The developing agent was n-butanol-acetic acid-water?4:1:5?.The color reagent was 0.8%phosphomolybdate dissolved in 15%sulfate ethanol solutionTLC identification of Glycyrrhizae Radix et Rhizoma:HSG thin layer plate prepared by 1%sodium hydroxide or HSGF₂₅₄ thin player plate were used.Ammonium glycyrrhetate was reference substance.The developing agent was ethyl acetate-formic acid-acetic acid-water?15:1:1:2?.The color reagent was 10% sulfate ethanol solution,and examined by UV lamp?365 nm?.Otherwise,examine by UV lamp?254nm?without color reagent.5.Determination of multiple components of Qinxing Qingre Zhike Granules by HPLCDetermination of amygdalin content:Ultimate XB-C18 column?4.6 mm×100mm,3?m?was used;Mobile phase was acetonitrile?A?-0.1%phosphoric acid?B?solution?8:92?.Flow rate was 1.0 m L·min^-1;Detection wavelength was 207 nm.Determination of the contents of baicalin,wogonoside,baicalein and wogonin:Diamonsil Plus C18 chromatographic column?4.6 mm×250mm,5?m?was used;Mobile phase A was 0.2%phosphate water,and B was methanol,with gradient elution.Flow rate was 1.0 m L·min^-1;The detection wavelength was 274nm.Determination of the content of geniposide,liquiritin,isoliquiritin and glycyrrhizic acid:Diamonsil Plus C18 chromatographic column?4.6 mm×250mm,5?m?was used;Mobile phase A was 0.05%phosphate water,and B was acetonitrile,gradient elution.Detection wavelength switching:0?25 min,237 nm;25?33 min,360 nm;33?50 min,237 nm.Volume flow rate was 1.0 m L·min^-1.Results1.Blood composition analysis of Qinxing Qingre Zhike Granules based on UPLC-LTQ-Orbitrap technologyA sensitive and efficient identification method was developed.130 chemical constituents were identified,including flavonoids such as baicalin,organic acids such as chlorogenic acid and saponins such as glycyrrhizic acid.35 prototype components in rat plasma after granule administration were distinguished.2.LC-MS/MS method was used to simultaneously determine the content of 12effective components in Qinxing Qingre Zhike GranulesAn LC-MS/MS method was established to simultaneously determine the content of chlorogenic acid,geniposidic acid,amygdalin,geniposide,liquiritin,isoliquiritin,baicalin,wogonoside,baicalein,liquiritigenin,isoliquiritigenin and glycyrrhizic acid in Qinxing Qingre Zhike Granules.The method is stable,rapid and accurate.3.A preliminary study on the pharmacokinetics of Qinxing Qingre Zhike GranulesA efficient and sensitive LC-MS/MS method was built to detect geniposide,liquiritin,isoliquiritin,baicalin,wogonoside,baicalein,liquiritigenin,isoliquiritigenin,and glycyrrhetinic acid.There was no obvious matrix effect.Linearity,precision,accuracy and stability satisfied the requirement of quantitative test for biological samples.It was successfully used in the pharmacokinetic study of these components in rats after oral administration.Bimodal absorption of baicalin,wogonoside,baicalein,liquiritigenin and isoliquiritigenin could be observed in the drug-time curves.Geniposide,liquiritin and isoliquiritin could be rapidly absorbed into the blood.4.TLC identification of Qinxing Qingre Zhike GranulesThe established method showed clear and complete spots,and no interference with negative samples.The method could be utilized for the identification of various herbs in this prescription.5.Determination of multiple components of Qinxing Qingre Zhike Granules by HPLCThe content of amygdalin ranged from 2.112 to 2.241 mg·g^-1.The content of baicalin was measured between 39.35 and 45.38 mg·g^-1,the content of wogonoside was between 4.137 and 5.135 mg·g^-1,the content of baicalein was between 0.6247and 0.8632 mg·g^-1,and the content of wogonin was between 0.3159 and 0.4038 mg·g^-1.The content of geniposide was determined between 10.93 and 12.25 mg·g^-1,the content of liquiritin was between 1.150 and 1.362 mg·g^-1,the content of isoliquiritin was between 0.9970 and 1.156 mg·g^-1,and the content of glycyrrhizic acid was from 3.388 to 4.158 mg·g^-1.The method was proved to be accurate,reliable and reproducible,and could be used for the quality control of Qinxing Qingre Zhike GranulesConclusionThe UPLC-LTQ-Orbitrap technology was simple,rapid and high-throughput in the separation and identification of ingredients and prototype components in preparations and rat plasma.The established LC-MS/MS method was sensitive for the determination of multicomponent in preparations and in plasma.The TLC spots are clear and complete,which can be used to identify the medicines in this compound.The determination of multiple components by HPLC is helpful to control the quality of the formula.