ABSTRACT
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， 1.8 μm） was used for analysis with 0.05% formic acid and 2.5 mmol·L<sup>-1</sup> ammonium formate in water （A）-0.05% formic acid and 2.5 mmol·L<sup>-1</sup> 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 mL·min<sup>-1</sup>， the column temperature was 40 ℃， and the injection volume was 1 μL. 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 <italic>N</italic>-oxide， lycopsamine<italic> N</italic>-oxide， echimidine<italic> N</italic>-oxide and echimidine were good （<italic>r</italic>≥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， 1.016-50.80 µg·L<sup>-1</sup>， 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.