超高效液相色谱-串联质谱法快速测定云南野生致命鹅膏菌中的毒伞肽和毒肽毒素

Rapid determination of amanitins and phallotoxins in Yunnan Amanita exitialis by ultra performance liquid chromatography-tandem mass spectrometry

中文关键词:  致命鹅膏菌  毒伞肽  毒肽  超高效液相色谱串联质谱法

英文关键词:Amanita exitialis  amanitins  phallotoxins  ultra performance liquid chromatography-tandem mass spectrometry

中文摘要:

      目的 建立超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS)快速测定野生毒菌中的5种毒伞肽和毒肽毒素的分析方法。方法 将采集的云南野生致命鹅膏菌子实体低温干燥, 用甲醇提取, SPE-C18固相萃取小柱净化, Waters T3色谱柱(2.1 mm×100 mm, 1.8 μm)分离, 串联质谱ESI正离子模式下进行定性定量分析。结果 5种毒肽和毒伞肽在20~1000 μg/kg范围内具有良好的线性关系, 相关系数均大于0.99, 方法检出限均为20 μg/kg。α-毒伞肽、β-毒伞肽、γ-毒伞肽、二羟基毒肽、羟基毒肽在高低2个浓度的平均回收率范围分别为89.2%~110.6%、88.5%~109.3%、90.1%~112.3%、92.6%~107.8%、91.6%~105.2%, 变异系数(coefficient of variation, CV)在3.2%~5.6%之间。在云南野生致命鹅膏菌中检测出α-毒伞肽(α-amanitin)、β-毒伞肽(β-amanitin)、羟基毒肽(phallacidin), 含量分别为794.84、501.23、230.96 mg/kg。结论 该方法使用固相萃取小柱净化样品, 减少了试剂的用量, 串联质谱可同时对毒肽和毒伞肽毒素进行定性定量检测, 提高了定性检测的准确性和定量检测的灵敏度。

英文摘要:

      Objective To establish a method for rapid determination of 5 kinds of amanitins and phallotoxins in wild poisonous mushrooms by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Methods The Amanita exitialis fruiting body samples collected in Yunnan were dried at low temperature, the amanitins and phallotoxins in samples were extracted with methanol, purified by SPE-C18 solid phase extraction column and separated on Waters BEH Amide column (2.1 mm×100 mm, 1.7 μm). The analytes were detected by an electrospray ionization (ESI) source in positive mode. Results The 5 kinds of amanitins and phallotoxins had good linear relationships in the range of 20~1000 μg/kg, and the correlation coefficients were greater than 0.99. The detection limits of 5 kinds of amanitins and phallotoxins were 20 μg/kg. The average recovery rates of α-amanitin, β-amanitin, γ-amanitin, phalloidin, phallacidin at 2 spiked levels were 89.2%~110.6%, 88.5%~109.3%, 90.1%~ 112.3%, 92.6%~107.8% and 91.6%~105.2%, respectively, with the coefficients of variation of 3.2%~5.6%. The content of α-amanitin, β-amanitin and phallacidin in Yunnan Amanita exitialis were 794.84, 501.23, 230.96 mg/kg, respectively. Conclusion This method uses solid phase extraction small column for purifying samples, and reduces the amount of reagents. Tandem quadrupole mass spectrometry can simultaneously qualitatively and quantitatively detect 5 kinds of amanitins and phallotoxins and improve the accuracy of qualitative detection and sensitivity of quantitative detection.