目的: 建立采用超高效液相色谱-串联质谱(UPLC-MS/MS)检测北细辛中马兜铃酸A、B、C、D、Ⅲ及Ⅶa,并对待测成分在入药部位(根或根茎)的分布进行考察。方法: 采用Waters C18 (100 mm×2.1 mm,1.8 μm)色谱柱,以乙腈-0.1%甲酸水溶液为流动相进行梯度洗脱,流速0.3 mL·min-1,柱温30 ℃;电喷雾离子源,多反应监测(MRM)模式,正离子检测,定量离子对为m/z 359.0→298.0(马兜铃酸A)、m/z 329.0→268.0(马兜铃酸B)、m/z 345.0→284.0(马兜铃酸C)、m/z 375.0→312.0(马兜铃酸D)、m/z 359.0→296.0(马兜铃酸Ⅲ)、m/z 375.0→314.0(马兜铃酸Ⅶa)。结果: 马兜铃酸A、B、C、D、Ⅲ及Ⅶa的线性范围分别为5.269 5~526.95、5.543 3~554.33、10.056~502.79、4.723 1~472.31、4.938 2~493.82、5.042 7~504.27 ng·mL-1,相关系数均＞0.999 6;平均加样回收率分别为98.2%、100.2%、103.1%、101.7%、96.2%、104.3%,RSD分别为4.3%、4.2%、3.7%、5.2%、3.0%、2.5%。12批样品的根或根茎中均未检出马兜铃酸B、C、Ⅲ。马兜铃酸A在根、根茎中含量范围分别为0.039 9~0.280 1、0.216 1~1.459 1 μg·g-1;马兜铃酸D在根、根茎中含量范围分别为4.355 2~11.187 9、3.126 2~8.014 4 μg·g-1;马兜铃酸Ⅶa在根、根茎中含量范围分别为0~0.017 3、0.008 4~0.070 3 μg·g-1。马兜铃酸D含量最高。结论: 该方法快速,专属性好,对样品测定结果有明显规律性,为细辛合理应用及其马兜铃酸成分限量测定提供依据。
Objective: To establish an UPLC-MS/MS method for simultaneous detection of 6 aristolochic acids (aristolochic acids A, B, C, D, Ⅲ and Ⅶa) in Asari Radix et Rhizoma. The distribution of the components in different parts (root or rhizome) was investigated. Methods: The chromalographic separation was proformed on a C18 (100 mm×2.1 mm, 1.8 μm) column by gradient program with acetonitrile-0.1% formic acid solution as mobile phase. The flow rate was set at 0.3 mL·min-1 and the column temperature was 30 ℃. Detection and quantification were performed by mass spectrometry in multiple reaction monitoring(MRM) mode with m/z 359.0→298.0 for aristolochic acid A, m/z 329.0→268.0 for aristolochic acid B, m/z 345.0→284.0 for aristolochic acid C, m/z 375. 0→312.0 for aristolochic acid D, m/z 359.0→296.0 for aristolochic acid Ⅲ, and m/z 375.0→314.0 for aristolochic acid Ⅶa. Results: The calibration curves were linear within the ranges of 5.269 5-526.95 ng·mL-1, 5.543 3-554.33 ng·mL-1, 10.056-502.79 ng·mL-1, 4.723 1-472.31 ng·mL-1, 4.938 2-493.82 ng·mL-1, 5.042 7-504.27 ng·mL-1 for aristolochic acids A, B, C, D, Ⅲ and Ⅶa, respectively. All ingredients showed a good linear relationship (r≥0.999 6). The average recoveries were 98.2%, 100.2%, 103.1%, 101.7%, 96.2%, 104.3% with RSDs of 4.3%, 4.2%, 3.7%, 5.2%, 3.0%, 2.5%, respectively. Aristolochic acid B, C and Ⅲ were not detected in the roots or rhizoids of 12 batches of samples. The content of aristolochic acid A were 0.039 9-0.280 1 μg·g-1, 0.216 1-1.459 1 μg·g-1in the roots and rhizoids, respectively. The contents of aristolochic acid D in roots and rhizomes were 4.355 2-11.187 9 μg·g-1, 3.126 2-8.014 4 μg·g-1, respectively. The content of aristolochic acid Ⅶa in roots and rhizomes were 0-0.017 3 μg·g-1 and 0.008 4-0.070 3 μg·g-1, respectively. And the content of aristolochic acid D was the highest. Conclusion: This method is proved to be accurate and specific. The samples are with significantly regular pattern in quality and quantity of aristolochic acid analogues. It provides a more comprehensive scientific basis for the rational application of Asari Radix et Rhizoma and the limited determination of aristolochic acid components.
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