马鞭草中6个肝毒性吡咯里西啶生物碱的含量测定及风险评估

Determination and Risk Assessment of Six Hepatotoxic Pyrrolizidine Alkaloids in Verbenae Herba

目的:建立超高效液相色谱-串联质谱法(UPLC-MS/MS)对马鞭草中6个肝毒性吡咯里西啶生物碱的含量进行测定,并根据测定结果进行初步的风险评估。方法:选择Waters ACQUITY UPLC HSS T3色谱柱(2.1 mm×100 mm,1.8μm),以含0.05%甲酸和2.5 mmol·L^(-1)甲酸铵的水溶液(A)及含0.05%甲酸和2.5 mmol·L^(-1)甲酸铵的乙腈溶液(B)为流动相进行梯度洗脱(0~12 min,3%~8%B;12~25 min,8%~15%B;25~26 min,15%~3%B;26~30 min,3%B),流速0.3 mL·min^(-1),柱温40℃,进样量1μL;电喷雾离子源(ESI),正离子模式下多反应监测模式,通过优化三重四极杆的MS参数及6个分析物的MS参数进行定性、定量分析。根据含量测定结果,采用暴露限值(MOE)结合热水提取转移率进行风险评估。结果:通过仪器精密度、线性范围、重复性、稳定性和加样回收试验等方法学验证,其结果均符合定量分析的要求。印美定、石松胺、印美定氮氧化物、石松胺氮氧化物、蓝蓟定氮氧化物和蓝蓟定的线性范围分别为0.984~49.20,0.994~49.70,1.012~50.60,1.032~51.60,1.004~50.20,1.016~50.80μg·L^(-1),这6个成分在线性范围内峰面积与质量浓度均具有良好线性关系(r≥0.9990);平均加样回收率在87.2%~94.2%,RSD均<4.0%;MOE均>1万。结论:建立的UPLC-MS/MS稳定可行,可为马鞭草中肝毒性吡咯里西啶生物碱的质量控制和安全性评价提供科学依据。

Objective:To establish ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) for simultaneous determination of six hepatotoxic pyrrolizidine alkaloids in Verbenae Herba,and to carry out preliminary risk assessment according to the research results.Method:An ACQUITY UPLC HSS T3 column(2.1 mm×100 mm,1.8μm)was used for analysis with 0.05%formic acid and 2.5 mmol·L^(-1)ammonium formate in water(A)-0.05%formic acid and 2.5 mmol·L^(-1)ammonium formate in acetonitrile(B)as mobile phase for gradient elution(0-12 min,3%-8%B;12-25 min,8%-15%B;25-26 min,15%-3%B;26-30 min,3%B),the flow rate was 0.3 m L·min^(-1),the column temperature was 40℃,and the injection volume was 1μL.MS system was operated by electrospray ionization(ESI)in the positive ion mode with multiple reaction monitoring mode.MS parameters of triple quadrupole and six analytes were optimized for qualitative and quantitative analysis.According to the determination results,the risk assessment was carried out by using margin of exposure(MOE)combined with transfer rate of hot water extraction.Result:Based on the instrument precision,linear range,repeatability,stability,recovery and other methodological validations,the results were in conformity with relevant standards of quantitative analysis.The linear ranges of intermedine,lycopsamine,intermedine N-oxide,lycopsamine N-oxide,echimidine N-oxide and echimidine were good(r≥0.999 0)between peak area and mass concentration in the ranges of 0.984-49.20,0.994-49.70,1.012-50.60,1.032-51.60,1.004-50.20,1.016-50.80μg·L^(-1),respectively.The average recoveries of these six analytes were 87.2%-94.2%with relative standard deviation(RSD)<4.0%.Their MOE values were>10 000.Conclusion:The UPLC-MS/MS established in this study is stable and feasible,which can provide scientific basis for the quality control and safety evaluation of hepatotoxic pyrrolizidine alkaloids in Verbenae Herba.