Fang Li, Qiu Fengmei, Yu Xinwei
Key Laboratory of Health Risk Factors for Seafood of Zhejiang Province (Zhoushan Municipal Center for Disease Control and Prevention), Zhoushan 316021, China.
Daishan Center for Disease Control and Prevention, Daishan 316200, China.
Se Pu. 2021 Mar;39(3):338-345. doi: 10.3724/SP.J.1123.2020.06005.
Amanita peptide toxins are cyclic polypeptide mushroom toxins that can cause acute liver damage. The fatality rate associated with these toxins is very high. Monitoring the concentration of amanita peptide toxins in human urine can provide valuable information for early clinical diagnosis and treatment. Therefore, a TurboFlow online clean-up-liquid chromatography-triple quadrupole mass spectrometry (TF-LC-MS/MS) method was established for the simultaneous quantitative determination of five amanita peptide toxins (-amanitin, -amanitin, -amanitin, phallacidin, and phalloidin) in human urine. After pre-treatment with high-speed centrifugation, urine samples were analyzed using TF-LC-MS/MS. The main factors influencing purification efficiency, including the TF column, loading solution, eluting solution, transfer flow, and transfer time, were optimized in this study. Under the optimized experimental conditions, the analytes were purified using a TurboFlow Cyclone column (50 mm×0.5 mm) and separated on a Hypersil GOLD C column (100 mm×2.1 mm) using the mobile phases of methanol and 4 mmol/L aqueous ammonium acetate solution with gradient elution. The analytes were detected in selected reaction monitoring (SRM) mode via positive electrospray ionization. Matrix-matched external standard calibration was used for quantitation. The linear range of the method ranged from 1.0 μg/L to 50.0 μg/L for all five amanita peptide toxins, with correlation coefficients () higher than 0.997. The limits of detection were 0.15-0.3 μg/L and the limits of quantification (LOQs) were 0.5-1.0 μg/L for the five amanita peptide toxins in urine. The intra-day and inter-day recoveries of amanita peptide toxins were 87.0%-108.6% and 86.8%-112.7%, respectively, at the spiked levels of 2.0, 5.0, and 10.0 μg/L. The intra-day and inter-day relative standard deviations (RSDs) were less than 14.5%. The method is accurate, rapid, sensitive, easy to operate, and can satisfy the requirements of public health emergency testing or clinical poisoning testing.
鹅膏肽类毒素是一类可导致急性肝损伤的环肽类蘑菇毒素。与这些毒素相关的死亡率非常高。监测人尿液中鹅膏肽类毒素的浓度可为早期临床诊断和治疗提供有价值的信息。因此，建立了一种TurboFlow在线净化-液相色谱-三重四极杆质谱法（TF-LC-MS/MS），用于同时定量测定人尿液中的五种鹅膏肽类毒素（α-鹅膏菌素、β-鹅膏菌素、γ-鹅膏菌素、鬼笔环肽和鬼笔毒环肽）。尿液样品经高速离心预处理后，采用TF-LC-MS/MS进行分析。本研究对影响净化效率的主要因素，包括TF柱、上样溶液、洗脱溶液、转移流速和转移时间进行了优化。在优化的实验条件下，使用TurboFlow Cyclone柱（50 mm×0.5 mm）对分析物进行净化，并在Hypersil GOLD C柱（100 mm×2.1 mm）上进行分离，流动相为甲醇和4 mmol/L乙酸铵水溶液，采用梯度洗脱。通过正电喷雾电离在选择反应监测（SRM）模式下对分析物进行检测。采用基质匹配外标校准进行定量。该方法对所有五种鹅膏肽类毒素的线性范围为1.0 μg/L至50.0 μg/L，相关系数（r）高于0.997。尿液中五种鹅膏肽类毒素的检测限为0.15 - 0.3 μg/L，定量限（LOQ）为0.5 - 1.0 μg/L。在2.0、5.0和10.0 μg/L的加标水平下，鹅膏肽类毒素的日内和日间回收率分别为87.0% - 108.6%和86.8% - 112.7%。日内和日间相对标准偏差（RSD）小于14.5%。该方法准确、快速、灵敏、操作简便，能够满足突发公共卫生事件检测或临床中毒检测的要求。
