Gicquel Thomas, Lepage Sylvie, Fradin Manon, Tribut Olivier, Duretz Bénédicte, Morel Isabelle
Laboratoire de Toxicologie Biologique et Médico-légale, CHU Pontchaillou, F-35033 Rennes, France.
UF Biomarqueurs, CHU Pontchaillou, F-35033 Rennes, France.
J Anal Toxicol. 2014 Jul-Aug;38(6):335-40. doi: 10.1093/jat/bku035. Epub 2014 Apr 28.
Mycotoxin intoxications can result from the consumption of amatoxins like α- and β-amanitin or of phallotoxin, present in several toxic mushrooms like Amanita phalloides. To identify and quantify amatoxins and phallotoidin in biological matrixes, we developed a method using liquid chromatography coupled with an ultra-high-resolution and accurate mass instrument (liquid chromatography-high-resolution-mass spectrometry, LC-HR-MS), Q Exactive™ (Thermo Fisher). The method includes a simple solid-phase extraction of urine samples spiked with flurazepam as internal standard (IS), using Bond Elut Agilent Certify cartridges (C18, 200 mg, 3 mL). LC separation was performed on a C18 Accucore column (100 × 2.1 mm, 2.6 µm) using a gradient of 10 mM ammonium acetate buffer containing 0.1% (v/v) formic acid and of acetonitrile with 0.1% (v/v) formic acid. Separation of analytes was obtained in 7 min, with respective retention times for α-amanitin, β-amanitin, phalloidin and IS of 1.9, 1.7, 3.5 and 3.8 min, respectively. Quantitation on the LC-HR-MS system was performed by extracting the exact mass value of each protonated species using a 5-p.p.m. mass window, which was 919.3614, 920.3455, 789.3257 and 388.1586 for α-amanitin, β-amanitin, phalloidin and IS, respectively. Calibration curves were obtained by spiking drug-free urine at 1-100 ng/mL. Mean correlation coefficients, r(2), were above 0.99 for each amatoxins and phalloidin. According to currently accepted validation procedures, the method was tested for selectivity, calibration, accuracy, matrix effect, precision and recovery. Authentic urine samples from 43 patients suffering from a suspected intoxication with mushrooms were analyzed by LC-HR-MS, and the results were compared with ELISA competitive immunoassay. The LC-HR-MS presented large benefits over immunoassay of being specific, faster and more sensitive, making it suitable for daily emergency toxicological analysis.
食用含有α-鹅膏毒肽和β-鹅膏毒肽等鹅膏毒素或鬼笔毒素的毒蘑菇可导致霉菌毒素中毒，这些毒素存在于如毒蝇伞等多种有毒蘑菇中。为了鉴定和定量生物基质中的鹅膏毒素和鬼笔环肽，我们开发了一种方法，该方法使用液相色谱与超高分辨率和精确质量仪器（液相色谱-高分辨率质谱，LC-HR-MS）联用，即赛默飞世尔科技公司的Q Exactive™。该方法包括使用安捷伦Bond Elut Certify小柱（C18，200 mg，3 mL）对添加氟西泮作为内标（IS）的尿液样品进行简单的固相萃取。液相色谱分离在C18 Accucore柱（100×2.1 mm，2.6 µm）上进行，使用含有0.1%（v/v）甲酸的10 mM醋酸铵缓冲液和含有0.1%（v/v）甲酸的乙腈梯度洗脱。7分钟内实现了分析物的分离，α-鹅膏毒肽、β-鹅膏毒肽、鬼笔环肽和内标的保留时间分别为1.9、1.7、3.5和3.8分钟。在LC-HR-MS系统上进行定量分析时，使用5 ppm的质量窗口提取每个质子化物种的精确质量值进行定量，α-鹅膏毒肽、β-鹅膏毒肽、鬼笔环肽和内标的精确质量值分别为919.3614、920.3455、789.3257和388.1586。通过向不含药物的尿液中添加1-100 ng/mL的毒素来获得校准曲线。每种鹅膏毒素和鬼笔环肽的平均相关系数r(2)均高于0.99。根据目前公认的验证程序，对该方法的选择性、校准、准确性、基质效应、精密度和回收率进行了测试。通过LC-HR-MS对43例疑似蘑菇中毒患者的真实尿液样本进行分析，并将结果与ELISA竞争免疫测定法进行比较。LC-HR-MS在特异性、速度和灵敏度方面比免疫测定法具有更大优势，使其适用于日常紧急毒理学分析。
