高效液相色谱-三重四极杆串联质谱法测定深圳梧桐山野生蘑菇中5种鹅膏肽类毒素的含量

Determination of 5 Amanita Toxoids in Wild Mushrooms from Wutong Mountain in Shenzhen by High Performance Liquid Chromatography-Triple Quadrupole Tandem Mass Spectrometry

针对深圳梧桐山野生蘑菇中毒案件,于事发地采集23种野生蘑菇样品,采用题示方法同时测定其中α-鹅膏毒肽(α-AMA)、β-鹅膏毒肽(β-AMA)、羧基二羟鬼笔毒肽(PCD)、羧基三羟鬼笔毒肽(PSC)和二羟鬼笔毒肽(PHD)等5种鹅膏肽类毒素的含量,并对唯一检出毒素的致命鹅膏不同生长时期的子实体中的5种毒素含量进行考察。在新鲜蘑菇1 g或干蘑菇0.2 g中加入10 mL水,匀浆后超声10 min,离心5 min。分取1 mL上清液过0.22μm水相滤膜,滤液供高效液相色谱-三重四极杆串联质谱法分析。以ACQUITY UPLC HSS T3色谱柱为固定相,以不同体积比5 mmol·L^(-1)乙酸铵溶液和乙腈的混合溶液为流动相进行梯度洗脱,分离后的各目标物用串联质谱仪的电喷雾离子源正离子模式电离,多反应监测模式检测。结果显示:5种鹅膏肽类毒素的质量浓度均在20.0~500.0μg·L^(-1)内与其对应的峰面积呈线性关系,检出限(3S/N)均为6 ng·g^(-1);对空白样品进行加标回收试验,回收率为78.3%~98.3%,测定值的相对标准偏差(n=6)为0.30%~11%。在23种野生蘑菇中,除致命鹅膏外,其他22种野生蘑菇均未检出5种鹅膏肽类毒素。致命鹅膏样品中检出了α-AMA、β-AMA、PCD和PSC,毒素总量在成熟期最高(1.50 mg·g^(-1),湿重),生长期最低(1.05 mg·g^(-1),湿重);β-AMA检出量最高、α-AMA次之。

For the case of wild mushroom poisoning from Wutong Mountain in Shenzhen,23 species of wild mushrooms were collected at the accident site.Simultaneous determination of the 5 Amanita toxoids,includingα-amanitin(α-AMA),β-amanitin(β-AMA),phallacidin(PCD),phallisacin(PSC)and phalloidin(PHD),was made by the title method,and the 5 toxins in the fruiting bodies of Amanita exitialis,the only one in which toxins was detected,at different growth periods were investigated.Water of 10 mL was added into 1 g of fresh mushroom or 0.2 g of dried mushroom,and the mixture was homogenized,sonicated for 10 min,and centrifuged for 5 min.An aliquot(1 mL)of the supernatant was taken and passed through a 0.22μm water phase filtration membrane.The filtrate was analyzed by high performance liquid chromatography-triple quadrupole tandem mass spectrometry.ACQUITY UPLC HSS T3 column was used as stationary phase,and the mixed solution composed of 5 mmol·L^(-1)ammonium acetate solution and acetonitrile at different volume ratios was used as mobile phase for gradient elution.The separated targets were ionized under positive ion mode with electricspray ion source,and detected under multiple reaction monitoring mode in the tandem mass spectrometer.It was shown that the mass concentrations of the 5 Amanita toxoids were all linearly related the respective peak areas in the range of 20.0-500.0μg·L^(-1),with the same detection limits(3 S/N)of 6 ng·g^(-1).The spiked recovery test was made on the blank samples,giving results in the range of 78.3%-98.3%,and RSDs(n=6)of the determined values ranged from 0.30%to 11%.Except for Amanita exitialis,the 5 Amanita toxoids were not detected in the other 22 species among the 23 species of wild mushrooms.α-AMA,β-AMA,PCD and PSC were detected in Amanita exitialis,the total content of toxins was the highest in mature period(1.50 mg·g^(-1),wet weight)and the lowest in growing period(1.05 mg·g^(-1),wet weight),and detection amount ofβ-AMA was the highest,following by that ofα-AMA.