Study On Quality Control Of Shiweiehuang Granule

Objective:To establish a TLC identification method of Shiweiehuang granule;To establish the methods of GC-MS fingerprints of volatile oil in Shiweiehuang granule intermediate and HPLC-UV fingerprints of Shiweiehuang granule;To establish a HPLC-ELSD method for the simultaneous determination of the contents of saponins in Shiweiehuang granules and a HPLC-UV method for the simultaneous determination of the contents of eight components in Shiweiehuang granules.This study aims to develop a specific and reproducible quality control method for Shiweiehuang granules and provide experimental foundation and scientific basis for its establishment of quality standards.Methods:（1）TLC was used to distinguish the Angelica dahurica,Saposhnikoviae divaricata,Magnoliae biondii,Bupleurum chinense,Astragalus membranaceus and Centipeda minima.（2）The GC-MS method was used to establish fingerprints of volatile oil in Shiweiehuang granule intermediate,the analysis was achieved with an Agilent HP-5MS（30m×250μm,0.25μm）capillary column MS chromatogram column with high purity helium（99.999%）as carrier gas at the flow rate of 1.0m L/min,with temperature programming.The injector temperature was250℃,the injection volume was 1μL,the split ratio was 5:1.Ionization mode was as follows as electron bombardment ion source,ion source temperature was 230℃,and interface temperature was 300℃,electronic energy of 70 e V,solvent delay time of 3.5 min,mass charge ratio（m/z）of50～400.Using the chromatograph peak of Paeonol as reference peak,MS spectrums of 10 batches of samples were determined.After GC-MS dates were transformation processed,similarity evaluation was conducted by using TCM fingerprint chromatogram similarity evaluation system（2012.1 editions）to determine the common peaks,and the chemical composition and structures were determined by GC-MS date analysis.The HPLC-UV was used to establish fingerprints in Shiweiehuang granule,the analysis was achieved with an Agilent ZORBAX Eclipse XDB-C₁₈（4.6×250mm,5μm）column by gradient elution of acetonitrile-water at 30℃.The flow rate of 1.0 m L/min,and the determination wavelength was set at 230 nm.Using the chromatograph peak of 5-O-methylvisammioside as reference peak,10batches of Shiweiehuang granule were analyzed.The mutual mode of fingerprint was set up for Shiweiehuang granule.（3）The HPLC-ELSD method was used for quantitative determination of saponins in Shiweiehuang granules.the separation was carried on an Agilent ZORBAX Eclipse XDB-C₁₈（4.6×250mm,5μm）column with mobile phase consisting of acetonitrile-water in a gradient elution manner,at the flow rate of 1.0 m L/min.The column temperature was 30℃.A Evaporative light-scattering detector was used,at the drift tube temperature was 80℃,the gas flow rate was 2.5 L/min and the gain value is 4.The HPLC-UV method was used for the simultaneous determination of the contents of such Paeoniflorin eight components in Shiweiehuang granules as with the method which using in the part of establish HPLC-UV fingerprints in Shiweiehuang granule.Results:（1）The TLC result show that petroleum ether（60～90℃）-ethyl acetate（7:1）,chloroform-methanol（7:1）,chloroform-diethyl ether（10:1）,the lower solution of chloroform-ethyl acetate-methanol-water（7:15:5:2）（below10℃）,the lower solution of chloroform-methanol-water（13:5:2）（below10℃）,petroleum ether（60～90℃）-dichloromethane（1:2）were used as developing solvent,respectively,which applied on a silica G plate.The method which was set can identify Angelica dahurica,Saposhnikoviae divaricata,Magnoliae biondii,Bupleurum chinense,Astragalus membranaceus and Centipeda minima of Shiweiehuang granules and there no interfere with the negative reference.（2）The GC-MS was used to establish fingerprints of volatile oil in Shiweiehuang granule intermediate,there are 17 common peaks in the 10batches of samples,and were identified.The common pattern of fingerprints of volatile oil in Shiweiehuang granule intermediate was established.Compared with the common pattern of fingerprints,the similarities of 10batches of samples were at 0.995～0.998.The HPLC was used to establish fingerprints of Shiweiehuang granule,10 batches of Shiweiehuang granule were determined,and the common pattern of fingerprints was established,the11 common peaks were defined,8 of which were identified and assigned.Compared with the common pattern of fingerprints,the similarities of 10batches samples were greater than 0.96.（3）The HPLC-ELSD method was establish to simultaneous determination of the contents of Astragaloside IV,Saikosaponin A and Magnolin showed good linear relationships within the ranges of 81.60～816.0μg/m L（r=0.9990）,19.10～191.0μg/m L（r=0.9989）,51.80～518.0μg/m L（r=0.9991）,whose average recoveries were 100.5%,103.9%and 99.4%with the RSDs of 0.49%,0.55%and 0.35%,respectively.The HPLC-UV method was set up to simultaneous determination of the contents of such Paeoniflorin eight components in Shiweiehuang granules.The Paeoniflorin,Prim-O-glucosylcimifugin,Calycosin-7-glucoside,Cimifugin,5-O-methylvisammioside,Calycosin,Paeonol and Magnolin showed good linear relationships within the ranges of 40.00～400.0μg/m L,12.04～120.4μg/m L,9.020～90.20μg/m L,2.620～26.20μg/m L,23.54～235.4μg/m L,4.240～42.40μg/m L,22.34～223.4μg/m L,44.22～442.2μg/m L,whose average recoveries were102.5%,101.0%,103.5%,99.0%,101.7%,103.5%,101.8%,101.0%with the RSDs of 0.87%,0.62%,1.0%,1.8%,1.5%,0.71%,1.0%,1.8%,respectively.Conclusion:The TLC method was established is simple and characteristic,and could be used for quality control in Shiweiehuang granule.The common pattern of GC-MS fingerprints of Shiweiehuang granule intermediate and HPLC fingerprints of Shiweiehuang granule are characteristic,reproducibility and stability,which could be used for qualitative identification in Shiweiehuang granule.The HPLC-ELSD method and HPLC-UV method were established for the simultaneous determination of the contents of saponins and such Paeoniflorin eight components in Shiweiehuang granules,which can comprehensively control the contents of various components in Shiweiehuang granule.Qualitative identification of TLC and fingerprint analysis combine with simultaneous determination of multicomponent can more comprehensively control the quality of Shiweiehuang granules to ensure its quality and safety in clinical treatment and provide reliable method and basis.