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目的:建立HPLC-MS/MS法同时测定山银花中7个有机酸(新绿原酸、绿原酸、隐绿原酸、咖啡酸、3,4-二-O-咖啡酰奎宁酸、3,5-二-O-咖啡酰奎宁酸和4,5-二-O-咖啡酰奎宁酸)的含量,并对其在3种不同植物来源的药材中的含量进行比较。方法:采用Shiseido Capcell Pak-C_(18)色谱柱(4.6 mm×250 mm,5μm),以0.1%甲酸水溶液(A)-乙腈(B)为流动相,梯度洗脱,流速为1 mL·min~(-1),加柱后分流器,使约30%洗脱液进入质谱仪,柱温30℃,进样量5μL;质谱采用ESI-多反应监测(MRM)模式扫描,以水杨酸为内标,脱溶剂温度350℃,脱溶剂气流(N_2)600 L·h~(-1),雾化器压力0.2 MPa,毛细管电压4 kV。结果:新绿原酸、绿原酸、隐绿原酸、咖啡酸、3,4-二-O-咖啡酰奎宁酸、3,5-二-O-咖啡酰奎宁酸和4,5-二-O-咖啡酰奎宁酸质量浓度分别在0.032~32.224μg·mL~(-1)(R~2=0.999 5)、0.168~16.800μg·mL~(-1)(R~2=0.999 4)、0.038~15.104μg·mL~(-1)(R~2=0.999 8)、0.010~4.064μg·mL~(-1)(R~2=0.999 2)、0.013~5.372μg·mL~(-1)(R~2=0.999 5)、0.079~39.840μg·mL~(-1)(R~2=0.999 5)和0.041~16.208μg·mL~(-1)(R~2=0.999 3)范围内线性关系良好;定量限(LOQ)为10.0~19.9 ng·mL~(-1),平均回收率在94.01%和105.0%之间。样品测定结果显示,山银花中绿原酸和3,5-二-O-咖啡酰奎宁酸含量较高,分别为1.262%~7.408%和0.636%~4.803%;咖啡酸含量较低,为0.003%~0.022%。通过比较不同植物来源的样品,发现其在有机酸的含量和比例方面存在较大差异。结论:本实验建立的方法灵敏、准确,可用于山银花药材中有机酸的含量测定,测定结果可为该药材质量标准的完善提供依据。
OBJECTIVE: To establish a HPLC-MS / MS method for the simultaneous determination of seven organic acids (chlorogenic acid, chlorogenic acid, cryptocholic acid, caffeic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid), and compared their contents in three kinds of medicinal plants of different plant origin. Methods: A gradient elution was carried out on a Shiseido Capcell Pak-C 18 column (4.6 mm × 250 mm, 5 μm) using 0.1% formic acid in water (A) -acetonitrile as mobile phase at a flow rate of 1 mL · min ~ (-1). After the column was loaded with a splitter, about 30% of the eluent was eluted into the mass spectrometer. The column temperature was 30 ℃ and the injection volume was 5 μL. The mass spectra were scanned with MRM mode. As internal standard, the desolvation temperature was 350 ℃, desolvation flow (N 2) was 600 L · h -1, atomizer pressure was 0.2 MPa and capillary voltage was 4 kV. Results: New chlorogenic acid, chlorogenic acid, cryptocholic acid, caffeic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid and 4,5- The mass concentration of di-O-caffeoylquinic acid was in the range of 0.032-32.224 μg · mL -1 (R 2 = 0.999 5), 0.168-16.800 μg · mL -1 (R 2 = 0.999 (R ~ 2 = 0.999 8), 0.010 ~ 4.064μg · mL -1 (R ~ 2 = 0.999 2), 0.013 ~ 5.372μg · mL ~ (-1) (R 2 = 0.999 5), 0.079 ~ 39.840 μg · mL -1 (R 2 = 0.999 5) and 0.041 ~ 16.208 μg · mL -1 (R 2 = 0.999 5) 3). The limit of quantification (LOQ) ranged from 10.0 to 19.9 ng · mL -1. The average recoveries ranged from 94.01% to 105.0%. The results of sample measurement showed that the content of chlorogenic acid and 3,5-di-O-caffeoylquinic acid in Shan-hua flower was higher from 1.262% to 7.408% and from 0.636% to 4.803%, respectively. The content of caffeic acid was lower, Is 0.003% to 0.022%. By comparing samples from different plant sources, it is found that there is a large difference in the content and proportion of organic acids. Conclusion: The method established in this experiment is sensitive and accurate and can be used to determine the content of organic acids in the medicinal materials of Shannxihua. The determination results can provide basis for improving the quality standard of the medicinal materials.