基于HPLC-Q-TOF-MS/MS技术咳喘宁颗粒化学成分分析及多指标定量测定

Chemical composition analysis and multi-index content determination of Kechuanning Granules based on HPLC-Q-TOF-MS/MS technology

目的采用高效液相色谱-四极杆飞行时间串联质谱技术(HPLC-Q-TOF-MS/MS)对咳喘宁颗粒化学成分进行分析,并建立HPLC多成分含量测定方法。方法采用HPLC-Q-TOF-MS/MS技术,色谱柱Agilent ZORBAX C_(18)(150 mm×4.6 mm,5μm),柱温35℃,体积流量1.0 mL·min^(−1),进样量10μL,流动相A:0.1%甲酸,流动相B:乙腈,梯度洗脱;电喷雾离子源(ESI),正、负离子模式扫描,结合对照品和文献数据鉴定咳喘宁颗粒中的化学成分,并建立HPLC法同时测定新绿原酸、绿原酸、3,5-O-二咖啡酰基奎宁酸、异绿原酸B、4,5-O-二咖啡酰基奎宁酸、柚皮苷的含量。色谱条件:以十八烷基硅烷键合硅胶为填充剂(Waters AtlantisTM T3色谱柱,250 mm×4.6 mm,5μm),以甲醇-0.1%甲酸为流动相,梯度洗脱:0~20 min,3%~15%甲醇;20~25 min,15%~18%甲醇;25~26 min,18%~23%甲醇;26~45 min,23%甲醇;45~46 min,23%~38%甲醇;46~60 min,38%甲醇;60~61 min,38%~42%甲醇;61~70 min,42%甲醇;70~80 min,42%~90%甲醇;体积流量1.0 mL·min^(−1),柱温30℃;进样量10μL,检测波长324、283 nm。结果共鉴定咳喘宁颗粒中86个化合物,20个来自生麻黄、15个来自白屈菜、30个来自金银花、22个来自枇杷叶、12个来自金沸草、14个来自化橘红,其中有26个化合物经与对照品比对确认,选择新绿原酸、绿原酸、3,5-O-二咖啡酰基奎宁酸、异绿原酸B、4,5-O-二咖啡酰基奎宁酸和柚皮苷作为含量测定的质控指标,6种成分在各自质量浓度范围内线性关系良好(r≥0.9999),精密度、稳定性及重复性良好,加样回收率为99.48%~103.39%,RSD为1.29%~1.98%。对3批咳喘宁颗粒进行多成分含量测定,方法可行。结论在质谱成分解析的基础上,建立咳喘宁颗粒6种化学成分的含量测定方法,为咳喘宁颗粒的药效物质基础和质量标准研究提供参考。

Objective High performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry(HPLC-QTOF-MS/MS)was used to analyze the chemical constituents of Ke Chuanning Granules,and a HPLC method for the determination of multi-components was established.Methods Using HPLC-Q-TOF-MS/MS technology,the chromatographic column was Agilent ZORBAX C_(18)(150 mm×4.6 mm,5μm),the column temperature was 35℃,the volume flow rate was 1.0 mL·min^(−1),the injection volume was 10µL,the mobile phase A was 0.1%formic acid,and the mobile phase B was acetonitrile,and gradient elution was performed.The chemical constituents in Kechuanning Granules were identified by electrospray ionization source(ESI),positive and negative ion mode scanning,and the contents of neochlorogenic acid,chlorogenic acid,3,5-O-dicaffeoyl quinic acid,isochlorogenic acid B,4,5-O-dicaffeoyl quinic acid and naringin were determined simultaneously by HPLC.Chromatographic conditions:Octadecylsilane bonded silica gel as filler(Waters AtlantisTM T3 column,250 mm×4.6 mm,5μm),methanol-0.1%formic acid as mobile phase,gradient elution:0—20 min,3%—15%methanol;20—25 min,15%—18%methanol;25—26 min,18%—23%methanol;26—45 min,23%methanol;45—46 min,23%—38%methanol;46—60 min,38%methanol;60—61 min,38%—42%methanol;61—70 min,42%methanol;70—80 min,42%—90%methanol.The volume flow rate is 1.0 mL·min^(−1),and the column temperature is 30℃;The sample volume is 10μL,and the detection wavelength is 324 and 283 nm.Results A total of 86 compounds in Kechuanning Granules were identified,including 20 compounds from Ephedrae Herba,15 compounds from Chelidonii Herba,30 compounds from Lonicerae Japonicae Flos,22 compounds from Eriobotryae Folium,12 compounds from Inulae Herba,and 14 compounds from Citri Grandis Exocarpium.Among 26 compounds,neochlorogenic acid,chlorogenic acid,3,5-O-dicaffeoyl quinic acid,isochlorogenic acid B,4,5-O-dicaffeoyl quinic acid and naringin were selected as quality control indicators for content determination after comparison with reference substances.The linear relationship of the six main components was good(r≥0.9999)within the respective concentration ranges,and the precision,stability and repeatability were good.The recovery after sample addition was 99.48%—103.39%,and RSD was 1.29%—1.98%.It is feasible to determine the multicomponent content of three batches of Kechuanning Granules.Conclusion Based on the analysis of mass spectrometry components,a method for the determination of six chemical components in Kechuanning Granules was established,which provided reference for the study of pharmacodynamic substance basis and quality standard of Kechuanning Granules.