目的 建立镇脑宁胶囊中马兜铃酸类成分的超高效液相-串联质谱联用检测分析方法。方法 采用Phenomemex Kinetex (2.1 mm×100 mm,2.6 μm)色谱柱,以甲醇-体积分数0. 1%甲酸溶液(含1mmol·L-1乙酸铵)为流动相梯度洗脱,流速0.3 mL·min-1,以正离子多反应监测模式,建立同时测定镇脑宁胶囊中马兜铃酸Ⅲa、7-羟基马兜铃酸Ⅰ、马兜铃酸Ⅳa、马兜铃酸Ⅱ、马兜铃内酰胺BⅡ、马兜铃内酰胺Ⅰ、马兜铃酸Ⅰ共7种马兜铃酸类成分的液质联用检测方法。结果 马兜铃酸Ⅲa、7-羟基马兜铃酸Ⅰ、马兜铃酸Ⅳa、马兜铃酸Ⅱ、马兜铃内酰胺BⅡ、马兜铃内酰胺Ⅰ、马兜铃酸Ⅰ分别在1.241~1 240.572、1.034~1 034.056、2.109~2 108.709、1.070~1 070.378、1.064~1 064.000、2.154~2 154.022、1.086~1 085.922 ng·mL-1 内线性关系良好; 平均加样回收率分别为101.8% (RSD1.84%)、101.9% (RSD 2.67%)、102.2% (RSD 2.74%)、102.1%(RSD 2.13%)、101.8% (RSD 2.60%)、100.7% (RSD 0.96%)、104.1% (RSD 6.15%)。10批样品均检出马兜铃酸Ⅳa、马兜铃内酰胺Ⅰ和马兜铃酸Ⅰ等马兜铃酸类成分,含量范围分别为3.875~6.913,2.984~6.072 及0.350~1.719 ng·g-1,而马兜铃酸Ⅲa、7-羟基马兜铃酸Ⅰ、马兜铃酸Ⅱ和马兜铃内酰胺BⅡ均未检出。结论 建立的同时测定7种马兜铃酸类成分的液质联用方法准确、可靠。镇脑宁胶囊中检出马兜铃酸Ⅳa、马兜铃内酰胺Ⅰ和马兜铃酸Ⅰ。其中,马兜铃酸Ⅳa、马兜铃内酰胺Ⅰ毒性较低或无明显毒性,而真正可能有毒性的是含硝基基团的马兜铃酸Ⅰ,因部分样品马兜铃酸Ⅰ含量超出限度,提示该制剂中需严格控制马兜铃酸Ⅰ的含量。
Abstract
OBJECTIVE To establish the determination method for aristolochic acid analogues in Zhennaoning Capsules. METHODS A Phenomemex Kinetex (2.1 mm×100 mm, 2.6 μm) column was used with methanol-0.1% formic acid (containing 1 mmol·L-1 ammonium acetate) as mobile phase gradiently eluted at a flow rate of 0.3 mL·min-1. By using multi-reaction monitoring mode (MRM), an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was established for simultaneous determination of seven aristolochic acid analogues including aristolochic acid Ⅲa, 7-hydroxy aristolochic acid Ⅰ, aristolochic acid Ⅳa, aristolochic acid Ⅱ, aristolactam BⅡ, aristolactam Ⅰ, and aristolochic acid Ⅰ. RESULTS The linear relationships for aristolochic acid Ⅲa, 7-hydroxy aristolochic acid Ⅰ, aristolochic acid Ⅳa, aristolochic acid Ⅱ, aristolactam B Ⅱ, aristolactam Ⅰ, and aristolochic acid Ⅰ were good in the ranges of 1.241-1 240.572, 1.034-1 034.056, 2.109-2 108.709, 1.070-1 070.378, 1.064-1 064.000, 2.154-2 154.022, 1.086-1 085.922 ng·mL-1, respectively. And their respective average recovery rates were 101.8% (RSD 1.84%), 101.9% (RSD 2.67%), 102.2% (RSD 2.74%), 102.1% (RSD 2.13%), 101.8% (RSD 2.60%), 100.7% (RSD 0.96%), 104.1% (RSD 6.15%), respectively. Among ten batches of samples, aristolochic acid Ⅳa, aristolactam Ⅰ and aristolochic acid Ⅰ were detected and the content range was 3.875-6.913, 2.984-6.072 and 0.350-1.719 ng·g-1, respectively, meanwhile, there were no aristolochic acid Ⅲa, 7-hydroxy aristolochic acid Ⅰ, aristolochic acid Ⅱ and aristolactam B Ⅱ detected. CONCLUSION The established UHPLC-MS/MS method is accurate and reliable. Aristolochic acid Ⅳa, aristolactam Ⅰ and aristolochic acid Ⅰ were detected in Zhennaoning Capsules. Among them, aristolochic acid Ⅳa and aristolactam Ⅰ have low toxicity or no obvious toxicity, while the toxin substance is aristolochic acid Ⅰ with nitro group. Because the content of aristolochic acid Ⅰ in some samples exceeds the limit, it suggests that the content of aristolochic acid Ⅰ in this preparation needs to be strictly controlled.
关键词
镇脑宁胶囊 /
马兜铃酸类成分 /
超高效液相色谱-串联质谱法
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Key words
Zhennaoning Capsules /
aristolochic acid analogues /
UHPLC-MS /MS
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中图分类号:
R284
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参考文献
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脚注
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基金
国家药品标准制修订研究课题资助(2021Z16-2)
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