基于HPLC-Q-TOF-MS/MS加味清络颗粒生物碱类化学成分分析及多指标含量测定

Chemical composition analysis and multi-index content determination of alkaloids in Jiawei Qingluo Granules by HPLC-Q-TOF-MS/MS

目的对加味清络颗粒中生物碱类成分进行定性鉴别分析,建立加味清络颗粒生物碱类多成分含量测定方法。方法采用HPLC-Q-TOF-MS/MS技术对加味清络颗粒进行正、负离子模式扫描,利用MassHunter软件计算化合物准确相对分子质量,结合质谱二级碎片信息及对照品比对,对加味清络颗粒中生物碱类成分进行定性鉴别,并总结化合物裂解规律;利用HPLC测定加味清络颗粒中6个生物碱成分的含量,检测条件:采用Phenomenex Gemini C18色谱柱(250 mm×4.6 mm,5μm);以乙腈为流动相A,磷酸盐缓冲溶液(pH 5.8)为流动相B,梯度洗脱,柱温30℃,体积流量0.9 mL/min,进样量10μL,检测波长210 nm。结果加味清络颗粒中共鉴定出50个生物碱成分,分别来源于青风藤、苦参、延胡索、知母,其中喹诺里西啶类生物碱16个、苄基四氢异喹啉类生物碱31个、其他类型生物碱3个;建立了HPLC同时测定加味清络颗粒中苦参碱、槐果碱、青藤碱、原阿片碱、延胡索乙素、延胡索甲素的多指标含量测定方法,该方法中6个待测成分在检测质量浓度范围内线性关系良好(r≥0.9999),精密度、重复性、稳定性RSD均小于4%,样品中苦参碱、槐果碱、青藤碱、原阿片碱、延胡索乙素、延胡索甲素的平均加样回收率分别为94.32%、96.89%、97.83%、109.53%、99.32%、87.13%,RSD值分别为3.24%、4.99%、4.95%、4.85%、2.52%、5.95%。结论建立的方法可准确、灵敏地对加味清络颗粒中的生物碱类化学成分进行定性及定量分析,为加味清络颗粒后续物质基础研究、质量控制、制剂开发建立了基础。

Objective To qualitatively identify and analyze the alkaloids in Jiawei Qingluo Granules(加味清络颗粒),and establish a method for the determination of alkaloids in Jiawei Qingluo Granules.Methods HPLC-Q-TOF-MS/MS technology was used to scan Jiawei Qingluo Granules in positive and negative ion mode,the precise molecular weight of the compound was calculated by MassHunter software,combined with secondary fragment information of mass spectrometry and comparison of reference substance,to identify the alkaloids in Jiawei Qingluo Granules qualitatively,and the cracking law of the compound was summarized;Determination of six alkaloids in Jiawei Qingluo Granules by HPLC,detection condition:Phenomenex Gemini C18 column(250 mm×4.6 mm,5μm);Acetonitrile as mobile phase A and phosphate buffer solution(pH 5.8)as mobile phase B,gradient elution(0—25 min,5%A;25—35 min,5%—7%A;35—50 min,7%—15%A;50—60 min,15%—19%A;60—80 min,19%—20%A;80—90 min,20%—35%A;90—115 min,35%A;15—120 min,35%—44%A;120—145 min,44%—48%A),the column temperature was 30℃,the flow rate was 0.9 mL/min,the sample size was 10μL,and the detection wavelength was 210 nm.Results A total of 50 alkaloids were identified in Jiawei Qingluo Granules,which were derived from Qingfengteng(Sinomenii Caulis),Kushen(Sophorae Flavescentis Radix),Yanhusuo(Corydalis Rhizoma)and Zhimu(Anemarrhenae Rhizoma),including 16 quinolizidine alkaloids,31 benzyl tetrahydroisoquinoline alkaloids and three other types alkaloids;A multi-index HPLC method for simultaneous determination of matrine,sophocarpine,sinomenine,protopine,tetrahydropalmatine and corydaline in Jiawei Qingluo Granules was established,the six components in the method had good linear relationship in the concentration range(r≥0.9999),the relative standard deviations(RSD)of precision,repeatability and stability were all less than 4%,the average recoveries of matrine,sophocarpine,sinomenine,protopine,tetrahydropalmatine and corydaline were 94.32%,96.89%,97.83%,109.53%,99.32%,87.13%,and RSD values were 3.24%,4.99%,4.95%,4.85%,2.52%,5.95%,respectively.Conclusion The established method can accurately and sensitively analyze the chemical components of alkaloids in Jiawei Qingluo Granules,which lays a foundation for the follow-up material basic research,quality control and preparation development of Jiawei Qingluo Granules.