超高效液相色谱-四极杆/静电场轨道阱高分辨质谱法测定毒蘑菇中5种鹅膏肽类毒素

Determination of five amanita peptide toxins in poisonous mushrooms by ultra performance liquid chromatography-quadrupole electrostatic field orbitrap high resolution mass spectrometry

鹅膏肽类毒素是毒蘑菇中毒事件中最常见的致死毒素,因此研究建立了超高效液相色谱-四极杆/静电场轨道阱高分辨质谱检测毒蘑菇中5种鹅膏肽类毒素的方法。样品经纯水提取后,以乙腈-5 mmol/L乙酸铵水溶液(含0.1%甲酸)为流动相进行梯度洗脱,用HSS T3色谱柱(100 mm×2.0 mm, 2.1μm)对待测组分进行色谱分离;采用可加热电喷雾电离源(HESI),全扫描/数据依赖二级质谱扫描(Full mass-ddMS^(2))模式对待测物进行定性分析;在目标离子选择性扫描(Targeted-SIM)模式下,以外标法对待测物进行定量测定。结果显示,5种鹅膏肽类毒素在1.0~20.0μg/L范围内均呈现良好的线性关系,相关系数均大于0.99,检出限均为0.006 mg/kg,加标回收率为81.8%~102.4%,相对标准偏差为3.2%~8.3%。方法提供了丰富的化合物特征信息,可根据提取离子流色谱图结合同位素分布信息锁定可疑化合物,根据一级质谱和二级质谱碎片离子的精确质荷比,在没有相关标准品的情况下可对未知化合物进行结构推断和确证。方法样品前处理简单,定性分析特异性强,定量测定灵敏度高,可满足突发公共卫生事件快速定性定量的检测要求,同时也为开展此类毒素中毒快速筛查及未知毒素的结构锁定提供了可靠的技术支撑。

Food poisoning by toxic mushrooms occurs frequently worldwide. It is one of the most common food poisoning events and the main cause of death. Amanita peptide toxins are the most common lethal toxins in poisonous mushrooms. Presently, a novel method based on ultra performance liquid chromatography-quadrupole electrostatic field orbitrap high resolution mass spectrometry(UPLC-Q/Orbitrap HRMS) was developed for the determination of five amanitapeptide toxins(α-amanitin, β-amanitin, γ-amanitin, phalloidin, and phallacidin). Because the isotope summit of α-amanitin affects the detection of β-amanitin, it cannot be distinguished by low resolution mass spectrometry. Therefore, experimental conditions including chromatography and mass spectrometry were explored in detail. The five peptide toxins were extracted from poisonous mushrooms with pure water and filtered through a 0.22 μm teflon microporous membrane. The procedure was rapid, simple, and environmentally friendly. Chromatographic separation was performed on a strong polarity HSS T3 column(100 mm×2.0 mm, 2.1 μm) with gradient elution using acetonitrile and 5 mmol/L ammonium acetate containing 0.1%(v/v) formic acid as mobile phases at a flow rate of 0.3 mL/min. The column temperature was set to 40 ℃. The analytes were ionized using a heating electrospray ionization source and collected in positive ion mode. Full scanning/data-dependent secondary mass spectrometry(Full mass-ddMS^(2)) mode was used for qualitative analysis of the targets within 10 min. The target ion selective scan(Targeted-SIM) mode was used for quantification by external standard calibration. The measured and theoretical values of the exact mass and the MS^(2) fragment ions of the five compounds were within an error of 5×10^(-6). Method validation was performed according to the criteria recommended by the Chinese National Standard. All the compounds showed an excellent linear relationship in the range of 1.0-20.0 μg/L. The correlation coefficients(r) ranged from 0.997 4 to 0.998 9. The limit of detection was 0.006 mg/kg for all five compounds. Recoveries ranged from 81.8% to 102.4%. There was no matrix effect in the blank mushroom sample for the five compounds, and the relative standard deviations ranged from 3.2% to 8.3%. This method provides abundant compound characteristic mass information, such as retention time, exact mass, fragment ions, and other information. The data can be used to identify suspected compounds based on the extracted ion flow diagram and isotope distribution information. Comparison between the actual exact mass and the theoretical exact mass, combined with the fragment ions enables identification of the structures of unknown compounds and collision methods, which can be confirmed in the absence of standard materials. In this study, the isomer of γ-amanitin was identified as amaninamide. The novel method is simple, accurate, specific, and sensitive. The method permits the rapid qualitative and quantitative detection of compound in public health emergency settings and will provide reliable technical support for the rapid screening of such toxic compounds and the structural locking of unknown toxins in the future.