目的: 以藏柴胡特征成分尼泊尔柴胡皂苷k为指标,建立超高效液相色谱-质谱法检测全粉投料中成药制剂中柴胡掺伪藏柴胡成分。方法: 采用Agilent Eclipse Plus C18(100 mm×3 mm,1.8 μm)色谱柱,以水(A)-乙腈(B)为流动相,二元梯度洗脱(0~3 min,75%A→70%A;3~12 min,70%A→68%A;12~12.5 min,68%A→10%A;12.5~15.5 min,10%A),流速0.3 mL·min-1,柱温40 ℃,进样量1 μL。采用电喷雾负离子化,多反应监测(MRM)模式,选择m/z 943.5→797.5、943.5→781.5和943.5→635.4为检测离子对。结果: 尼泊尔柴胡皂苷k质量浓度在0.05~5 μg·mL-1范围与峰面积呈现出良好的线性关系(r=0.996 5),仪器精密度RSD(n=6)为1.4%,掺伪10%藏柴胡参比溶液重复性RSD(n=6)为1.8%,掺伪检测限为1%。对代表性品种逍遥丸、加味逍遥丸、补中益气丸、龙胆泻肝丸进行筛查,结果9批逍遥丸中有2批,8批加味逍遥丸中有4批,10批补中益气丸中有1批,10批龙胆泻肝丸中有2批含尼泊尔柴胡皂苷k超过拟定限度。结论: 本方法经过方法学验证,专属性强,灵敏度高,可应用于全粉投料中成药制剂中柴胡掺杂藏柴胡成分的检查。
Objective: To establish an UPLC-MS/MS method for the detection of Radix Bupleuri adulterated B. marginatum var. stenophyllum in Chinese patent medicine fed with whole powder, using nepasaikosaponin k, a characteristic component of B. marginatum var. stenophyllum, as an index. Methods: Agilent Eclipse Plus C18 column (100 mm×3 mm, 1.8 μm) was adopted with the mobile phase of water(A)-acetonitrile(B) in gradient mode(0-3 min, 75%A→70%A; 3-12 min, 70%A→68%A; 12-12.5 min, 68%A→10%A; 12.5-15.5 min, 10%A), the flow rate was 1.0 mL·min-1, the column temperature was 40 ℃ and the injection volume was 1 μL. The target compound was analyzed under multiple reaction monitoring(MRM) mode with an ESI- source operating in negative ion mode,m/z 943.5→797.5, m/z 943.5→781.5, m/z 943.5→635.4 were selected as the detection ion pairs. Results: The linear range of nepasaikosaponin k was 0.05-5 μg·mL-1 and r was 0.996 5, the precision RSD(n=6) of apparatus was 1.4%, the repeatability RSD(n=6) of 10% B. marginatum var. stenophyllum reference solution were 1.8%, the detection limit of adulteration was 1%. The results indicated that 2 out of 9 batches of Xiaoyao pills, 4 out of 8 batches of Jiawei Xiaoyao pills, 1 out of 10 batches of Buzhong Yiqi pills, 2 out of 10 batches of Longdan Xiegan pills contained nepasaikosaponin k exceeding the proposed limit. Conclusion: The method has been verified by methodology,is specific and sensitive, and can be applied to detect the B. marginatum var. stenophyllum which is illegally incorporated in Chinese patent medicine fed with whole powder.
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