液相色谱-串联质谱法快速测定野生菌中9种蘑菇毒素
作者:
作者单位:

浙江省疾病预防控制中心,浙江 杭州 310051

作者简介:

徐小民 男 主任技师 研究方向为食品卫生理化检验 E-mail: chemxuxm@163.com

通讯作者:

韩见龙 男 主任技师 研究方向为食品卫生理化检验 E­mail: jlhan@edc.zj.cn

中图分类号:

R155

基金项目:

浙江省自然科学基金(LGF18H260007)


Fast determination of 9 mushroom toxins in wild fungus by liquid chromatography-tandem mass spectrometry
Author:
Affiliation:

Zhejiang Provincial Center for Disease Control and Prevention, Zhejiang Hangzhou 310051, China

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    摘要:

    目的 建立液相色谱-串联质谱技术(LC-MS/MS)测定野生菌中5种急性肝衰竭型(α-鹅膏毒肽、β-鹅膏毒肽、γ-鹅膏毒肽、羧基二羟鬼笔毒肽和二羟鬼笔毒肽)和4种神经-精神紊乱型(鹅膏蕈氨酸、毒蝇母、毒蝇碱和裸盖菇素)蘑菇毒素的分析方法。方法 样品经干燥后采用0.5%(V/V)甲酸50%(V/V)甲醇-水溶液提取、乙腈沉淀蛋白质、水稀释后,采用XBridgeTM BEH C18柱(150 mm×3.0 mm, 2.5 μm)分离、0.005%(V/V)甲酸水溶液-甲醇流动相体系梯度洗脱,MS/MS多反应监测(MRM)模式测定。结果 通过优化样品提取、稀释倍数和色谱条件,实现基于纯溶剂标准校正的不同野生菌中9种蘑菇毒素的定性和定量测定。样品基质中9种蘑菇毒素的检出限(LOD)和定量限(LOQ)分别为6~15 mg/kg和20~50 mg/kg,线性范围为0.004~2 mg/L,相关系数r在0.997 3~0.999 5之间。50、500和5 000 mg/kg三水平加标平均回收率为78.6%~109.7%,相对标准偏差(RSD)为2.7%~9.0%。多起中毒事件中采集的不同野生菌毒素组成和含量差异大。结论 本方法操作简便、快速,结果准确,已应用于常见有毒野生菌中蘑菇毒素检测和中毒应急事件处置,可以为中毒病因的鉴定和患者的及时救治提供快速、准确的技术支持。

    Abstract:

    Objective To establish an analytical method for 9 mushroom toxins in wild fungus by liquid chromatography-tandem mass spectrometry (LC-MS/MS) including 5 acute liver failure toxins (α-amanitin, β-amanitin, γ-amanitin, phallacidin and phalloidin) and 4 psycho-neurological disorder toxins (ibotenic acid, musimol, muscarine and psilocybin).Methods The dried sample was extracted with 50%(V/V)methanol /water mixture containing 0.5%(V/V)formic acid, protein was precipitated with acetonitrile and diluted with water. The toxins were separated with XBridgeTM BEH C18 column (150 mm×3.0 mm, 2.5 μm), gradient eluted with the mobile phase of 0.005%(V/V) formic acid aqueous solution–methanol, and measured by MS/MS with multiple reaction monitoring (MRM) mode.Results The conditions of sample extraction, dilution ratio and chromatographic separation were optimized. 9 mushroom toxins in different wild fungus could be qualitatively and quantitatively measured based on the pure solvent standard calibration. The limits of detection (LODs) and quantification (LOQs) in sample matrix were 6-15 mg/kg and 20-50 mg/kg, respectively. The linear ranges were 0.004-2 mg/L with correlation coefficients (r) of between 0.9973-0.9995. The average recoveries at three spiking levels (50 mg/kg, 500 mg/kg and 5000 mg/kg) were 78.6%-109.7% with relative standard deviations (RSDs) of 2.7%-9.0%. The toxin compositions and contents varied greatly in the wild mushrooms sampled in some poisoning cases.Conclusion The method is simple, rapid and accurate. It has been applied to the analysis of mushroom toxins in common poisonous wild mushrooms and the disposal of poisoning emergencies. It can provide rapid and accurate technical support for the etiology identification of poisoning cases and the timely rescue of patients.

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徐小民,郑熠斌,黄百芬,许娇娇,蔡增轩,韩见龙.液相色谱-串联质谱法快速测定野生菌中9种蘑菇毒素[J].中国食品卫生杂志,2022,34(2):262-269.

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  • 收稿日期:2021-09-05
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  • 在线发布日期: 2022-05-18
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