PDF(Pubmed)
Abstract：
The discovery and identification of mushroom toxins has long been an important area in the fields of toxicology and food safety. Mushrooms are widely favored for their culinary and medicinal value; however, the presence of potentially lethal toxins in some species poses a substantial challenge in ensuring their safe consumption. Therefore, the development of a robust and sensitive analytical method is necessary for accurately identifying the risks associated with mushroom consumption. The study of mushroom toxins, which are characterized by their diversity and substantial variations in chemical structures, present a considerable challenge for achieving precise and high-throughput analysis. To address this issue, the present study employed a robust approach combining a solid-phase extraction (SPE) purification technique with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to establish an analytical method for the detection and quantification of five amatoxins and two tryptamines (psilocybin and bufotenine) present in some mushrooms. Several optimization procedures were undertaken, including optimizing the chromatographic conditions, mass spectrometric parameters, and sample extraction and purification. The procedure involved the extraction of dry mushroom powder with methanol containing 0.3% formic acid, followed by purification using a strong cation exchange cartridge (SCX). The analytes were separated on a T3 chromatographic column (100 mm×2.1 mm, 1.8 μm) using mobile phases of acetonitrile and 5 mmol/L ammonium acetate solution containing 0.1% formic acid. The multiple reaction monitoring (MRM) mode was employed for data acquisition. Amatoxins were quantified using matrix-matched standard calibration curves, whereas isotopic internal standards were used to quantify tryptamine. The results showed that all seven toxins exhibited good linearities (r2>0.99) within the optimized concentration range. The limits of detection (LODs) for bufotenine, psilocybin, and amatoxins were determined as 2.0, 5.0, and 10 μg/kg, respectively, while the limits of quantification (LOQs) were determined as 5.0, 10, and 20 μg/kg, respectively. The LOD and LOQ values further underscore the ability of the method to detect minute quantities of toxins, making it particularly well suited for screening food samples for potential contamination. Using dried shiitake mushroom powder as the matrix, the recoveries of the two tryptamines ranged from 80.6% to 117%, with relative standard deviations (RSDs) ranging from 1.73% to 5.98%, while the recoveries of amatoxins ranged from 71.8% to 115%, with RSDs varying from 2.14% to 9.92% at the three concentration levels. The consistent and satisfactory recoveries of amatoxins and tryptamines demonstrated the ability of this method to accurately quantify the target analytes even in a complex matrix. Comparison with the results of supplementary test method recognized by State Administration for Market Regulation for food (BJS 202008) demonstrated comparable results, indicating no significant differences (p>0.05) in amatoxin contents. The newly developed method is rapid, accurate, precise, meets the required standards, and is suitable for the detection of seven toxins in wild mushrooms. As part of the application of this method, a comprehensive investigation of the distribution of toxins in wild mushrooms from Fujian Province was undertaken. In this study, 59 wild mushroom samples from nine cities were collected in the Fujian province. Species identification was conducted using rDNA-internal transcribed space (rDNA-ITS) molecular barcode technology, which revealed the presence of toxins in the two samples. Notably, one specimen named Amanita fuligineoides contained α-amanitin, β-amanitin, and phalloidin in quantities of 607, 377, and 69.0 mg/kg, respectively. Additionally, another sample, identified as Tricholomataceae, had a psilocybin concentration of 12.6 mg/kg.
摘要：
蘑菇毒素的发现和鉴定长期以来一直是毒理学和食品安全领域的重要领域。蘑菇因其烹饪和药用价值而受到广泛青睐；然而，某些物种中潜在致命毒素的存在对确保其安全食用构成了重大挑战。因此,为了准确识别与蘑菇消费相关的风险，有必要开发一种稳健而灵敏的分析方法。蘑菇毒素的研究，它们的特点是化学结构的多样性和实质性变化，对实现精确和高通量分析提出了相当大的挑战。为了解决这个问题，本研究采用了一种稳健的方法，将固相萃取（SPE）纯化技术与高效液相色谱-串联质谱（HPLC-MS/MS）相结合，建立了一种分析方法，用于检测和定量存在于某些蘑菇中的五种阿马霉素和两种色胺（psilocybin和bufotenine）。进行了几个优化程序，包括优化色谱条件，质谱参数,和样品提取和纯化。该程序涉及用含0.3％甲酸的甲醇提取干蘑菇粉，随后使用强阳离子交换柱(SCX)纯化。分析物在T3色谱柱上分离（100mm×2.1mm，1.8μm），使用乙腈和含0.1％甲酸的5mmol/L乙酸铵溶液的流动相。采用多反应监测(MRM)模式进行数据采集。使用基质匹配的标准校准曲线定量阿马毒素，而同位素内标用于定量色胺。结果表明，在优化的浓度范围内，所有7种毒素均表现出良好的线性度（r2>0.99）。Bufotenine的检测限（LODs），psilocybin,和阿马毒素测定为2.0、5.0和10μg/kg，分别,而定量限（LOQs）确定为5.0、10和20μg/kg，分别。LOD和LOQ值进一步强调了该方法检测微量毒素的能力，使其特别适用于筛选食品样品的潜在污染。使用干香菇粉作为基质，两种色胺的回收率为80.6%至117%，相对标准偏差(RSD)在1.73%至5.98%之间，而阿马毒素的回收率在71.8%至115%之间，在三个浓度水平下，RSD从2.14%到9.92%不等。阿马毒素和色胺的一致和令人满意的回收率证明了该方法即使在复杂的基质中也能准确定量目标分析物的能力。与国家食品市场监管总局认可的补充测试方法（BJS202008）的结果进行比较，证明结果具有可比性，表明amatoxin含量没有显着差异（p>0.05）。新开发的方法是快速的，准确,精确，符合规定的标准,适用于野生蘑菇中七种毒素的检测。作为该方法应用的一部分，对福建省野生蘑菇中毒素的分布进行了全面调查。在这项研究中,收集了福建省9个城市的59个野生蘑菇样品。使用rDNA-内部转录空间（rDNA-ITS）分子条形码技术进行物种鉴定，这表明两个样本中存在毒素。值得注意的是,一个名为Amanitafuligineoides的标本含有α-amanitin，β-amanitin,和phalloidin的数量分别为607、377和69.0mg/kg，分别。此外,另一个样本,被鉴定为口蹄科，具有12.6mg/kg的psilocybin浓度。
