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目的建立超高效液相色谱串联三重四级杆质谱(UPLC-MS/MS)同时测定积雪草中7种有效成分(积雪草苷B、羟基积雪草苷、积雪草苷、羟基积雪草酸、积雪草酸、槲皮素和山柰酚)的含量。方法色谱柱:ACQUITYBEHC_(18)(2.1mm×100mm,1.7μm),流动相:乙腈-5mmol·L~(-1)醋酸铵,梯度洗脱,流速:0.4mL·min~(-1),柱温:35℃,正离子多反应监测模式。考察该方法的专属性、标准曲线和定量下限、精密度和回收率、稳定性和重复性。结果异构体积雪草苷B和羟基积雪草苷达到基线分离。定量分析的7种有效成分:积雪草苷B、羟基积雪草苷、积雪草苷、槲皮素、山柰酚、羟基积雪草酸和积雪草酸分别在考察的浓度范围内:20.00~400.00ng·mL~(-1),20.00~400.00ng·mL~(-1),20.00~400.00ng·mL~(-1),10.00~200.00ng·mL~(-1),10.00~200.00ng·mL~(-1),50.00~1000.00ng·mL~(-1),50.00~1000.00ng·mL~(-1),呈良好的线性关系(r>0.9902);回收率和精密度RSD分别在1.76%~5.69%和1.03%~4.67%内。稳定性和重复性良好,RSD范围分别为0.86%~3.21%和0.94%~3.45%。结论本研究所建立的同时测定雪草中7种有效成分(5个五环三萜和2个黄酮)的UPLC-MS/MS定量分析方法简便、快捷、准确,可为综合评价积雪草的质量提供参考。
OBJECTIVE To establish a method for the simultaneous determination of seven active constituents in Asteraceae by UPLC-MS / MS (asiaticoside B, madecassoside, asiaticoside, Hyaluronic acid, Asiatic acid, quercetin, and kaempferol). Methods The mobile phase consisted of ACQUITYBEHC_ (18) (2.1 mm × 100 mm, 1.7 μm) and acetonitrile-5 mmol·L -1 ammonium acetate with gradient elution at a flow rate of 0.4 mL · min -1. Column temperature: 35 ℃, positive ion multi-reaction monitoring mode. The specificity, standard curve and quantitation limits, precision and recovery, stability and reproducibility of this method were examined. Results Isostile tooside B and madecassoside achieved baseline separation. Quantitative analysis of the seven active ingredients: asiaticoside B, madecassoside, asiaticoside, quercetin, kaempferol, asiatic acid and asiatic acid in the investigated concentration range: 20.00 ~400.00 ng · mL -1, 20.00 ~ 400.00 ng · mL -1, 20.00 ~ 400.00 ng · mL -1, 10.00 ~ 200.00 ng · mL -1, 10.00 ~ 200.00 (-1), 50.00 ~ 1000.00 ng · mL -1 and 50.00 ~ 1000.00 ng · mL -1, respectively (r> 0.9902). The recovery and precision of RSD Respectively in 1.76% ~ 5.69% and 1.03% ~ 4.67%. The stability and reproducibility were good, with RSD ranges of 0.86% -3.21% and 0.94% -3.45%, respectively. Conclusion The UPLC-MS / MS method for the simultaneous determination of seven active constituents (five pentacyclic triterpenoids and two flavonoids) in Selaginellariae found in this study is simple, rapid and accurate. It can be used for the comprehensive evaluation of Centella asiatica Quality for reference.