HPLC⁃MS/MS法同时测定白头翁汤中13种成分及其配伍前后含量变化

Simultaneous determination of thirteen constituents in Baitouweng Decoction by HPLC-MS/MS and their content changes before and after compatibility

目的建立高效液相色谱-串联质谱(HPLC-MS/MS)法同时测定白头翁汤中白头翁皂苷B4、白头翁皂苷B5、黑海常春藤皂苷A1、白头翁皂苷D、白头翁皂苷A3、α-常春藤皂苷、秦皮素、秦皮甲素、秦皮乙素、黄柏碱、巴马汀、黄连碱、小檗碱的含量,并考察配伍前后其变化。方法白头翁、白头翁汤、缺白头翁阴性汤50%甲醇提取液的分析采用ACQUITY UPLC BEH C 18色谱柱(2.1 mm×50 mm,1.7μm);流动相甲醇-0.1%甲酸,梯度洗脱;体积流量0.2 mL/min;柱温24.5℃;电喷雾离子源;正负离子扫描;多反应监测模式。结果13种成分在各自范围内线性关系良好(R 2>0.9900),平均加样回收率94.4%~106.2%,RSD 1.1%~4.2%。在白头翁汤中,双糖类皂苷(白头翁皂苷B4、白头翁皂苷B5)、单糖类皂苷(白头翁皂苷A3、α-常春藤皂苷、黑海常春藤皂苷A1、白头翁皂苷D)含量低于白头翁中,香豆素(秦皮素、秦皮甲素、秦皮乙素)含量高于缺白头翁阴性汤中,生物碱(小檗碱、黄柏碱)含量低于缺白头翁阴性汤中。结论白头翁汤中生物碱可能与皂苷结合而形成其他化合物,导致两者含量降低。

AIM To establish an high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method for the simultaneous content determination of anemoside B4,anemoside B5,hederacolchiside A1,pulsatilla saponin D,pulchinenoside A3,α-hederin,fraxetin,esculin hydrate,esculetin,phellodendrine,palmatine,coptisine and berberine in Baitouweng Decoction,and to investigate their changes before and after compatibility.METHODS The analysis of 50%methanol extracts of Pulsatillae Radix,Baitouweng Decoction and negative decoction without Pulsatillae Radix was performed on a 24.5℃thermostatic ACQUITY UPLC BEH C 18 column(2.1 mm×50 mm,1.7μm),with the mobile phase comprising of methanol-0.1%formic acid flowing at 0.2 mL/min in a gradient elution manner,and electron spray ionization source was adopted in positive and negative ion scanning with multiple reaction monitoring mode.RESULTS Thirteen constituents showed good linear relationships within their own ranges(R 2>0.9990),whose average recoveries were 94.4%-106.2%with the RSDs of 1.1%-4.2%.In Baitouweng Decoction,the contents of disaccharide saponins(anemoside B4,anemoside B5)and monodesmosidic saponins(pulchinenoside A3,α-hederin,hederacolchiside A1,pulsatilla saponin D)were lower than those in Pulsatillae Radix,the contents of coumarins(fraxetin,esculin hydrate,esculetin)were higher than those in negative decoction without Pulsatillae Radix,and the contents of alkaloids(berberine,phellodendrine)were lower than those in negative decoction without Pulsatillae Radix.CONCLUSION In Baitouweng Decoction,alkaloids may combine with pulsatilla saponins to form other compounds,leading to the decrease of their contents.