HPLC-MS法同时测定白蒲黄片中7种五环三萜皂苷类化学成分

Simultaneous determination of seven components in Baipuhuang tablets by HPLC-MS

目的：建立同时测定白蒲黄片中7种五环三萜皂苷类成分白头翁皂苷A3、白头翁皂苷B4、23-羟基白桦脂酸、白头翁皂苷B、白头翁皂苷C、刺人参苷S和齐墩果酸的HPLC-MS法。方法：采用Diamonsil ODS C18（250 mm×4.6 mm,5μm）色谱柱,流动相为含0.1%甲酸的水（A）-含0.1%甲酸的甲醇（B）,梯度洗脱程序为25%A（0 min）,5%A（0-6 min）,5%A（6-14min）,25%A（保持6 min）,流速0.8 m L·min-1,柱温30℃,进样量10μL;采用正离子和负离子模式同时监测,正离子和负离子监测模式下源喷射电压分别为5.5 k V和-4.5 k V,离子源温度为650℃,雾化气（Gas 1）344 k Pa,加热气（Gas 2）412 k Pa,接口持续加热,帘气172 k Pa,全程氮气通入状态。结果：在14 min内白蒲黄片中7种有效成分白头翁皂苷A3、白头翁皂苷B4、23-羟基白桦脂酸、白头翁皂苷B、白头翁皂苷C、刺人参苷S和齐墩果酸被完全分离;峰面积与其浓度呈良好的线性;平均回收率范围为95.11%-107.3%,RSD为0.86%-3.26%。结论：本文建立的方法经验证简便、重现性好、专属性高,可为白蒲黄片质量控制提供参考。

Objective：To establish a method based on high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry（HPLC-ESI-MS/MS） for the simultaneous determination of anemoside A3,anemoside B4,23-hydroxybetulinic acid,pulsatilloside B,pulsatilloside C,cirenshenoside S and oleanolic acid in Baipuhuang tablets.Methods：The samples were separated on a Diamonsil ODS C18column（250 mm × 4.6 mm,5 μm） by a mobile phase of 0.1% formic acid aqueous solution（A）-methanol containing 0.1% formic acid（B） at the flow rate of 0.8 m L·min-1.The gradient elution program was 25% A（0 min）,5% A（0-6 min）,5% A（6-14min）,and then back to the original 25% A held for 6 min.The column temperature was 30 ℃,and the injection volume was 10 μL.Multiple-reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run.The ion spray voltage for the two modes was set to 5.5 k V and-4.5 k V,respectively.The turbo spray temperature was maintained at 650 ℃.Nebulizer gas（Gas1） and heater gas（Gas 2） was set at 344 and 412 k Pa,respectively.The curtain gas was kept at 172 k Pa and the interface heater were kept operating.Nitrogen was used throughout the course.Results：The complete separation was obtained within 14 min for the seven compounds（anemoside A3,anemoside B4,23-hydroxybetulinic acid,pulsatilloside B,pulsatilloside C,cirenshenoside S and oleanolic acid）.Seven regression equations showing linear relationships between peak area and content of each compound were obtained.The recoveries（n = 6） of the compounds were95.11%-107.3% and RSDs were 0.86%-3.26%,respectively.Conclusion：The method is proved to be simple,accurate and with good reproducibility by the method validation,and can be used for the determination of seven triterpenoid saponins in Baipuhuang tablets.