QuEChERS-HPLC-MS/MS法检测谷物源性运动食品中杂色曲霉毒素和黄曲霉毒素

建立一种快速、高效的QuEChERS-高效液相色谱-串联质谱（QuEChERS-HPLC-MS/MS）法测定谷物源性运动食品中的杂色曲霉毒素和黄曲霉毒素的方法。样品采用乙腈-水（85∶15,V/V）提取,经SiO2+C18净化后检测。以乙腈-0.15%甲酸为流动相,在质谱检测器的多反应监测模式下进行分析。结果表明,5种真菌毒素在0.1～10.0 ng/mL质量浓度范围内线性关系良好,相关系数（R2）为0.999 2～0.999 7,检出限为0.03～0.14 μg/kg,定量限为0.10～0.49 μg/kg,加标回收率为85.8%～99.4%,精密度试验结果的相对标准偏差（RSD）为2.72%～5.23%。该方法具有前处理简单、净化效果好、灵敏度高的优点,适用于谷物源性运动食品中杂色曲霉毒素和黄曲霉毒素的分析和定量检测。     

超高效液相色谱串联质谱法测定啤酒中伏马毒素的含量

该文建立了免疫亲和柱净化-超高效液相色谱串联质谱法测定啤酒中FB1、FB2和FB3的含量。与液相色谱相比较,无需衍生化,避免了化学衍生法受衍生产物的不稳定性、衍生剂的浓度、温度和反应时间等衍生效率的影响。采用多反应监测（MRM）方式进行检测,3种物质在2.5～100 ng/mL范围具有良好的线性关系,相关系数（R2）均大于0.999,在空白样品中加入1.0 μg/kg和8.0 μg/kg两个浓度水平的伏马毒素,平均回收率为88.3%～95.1%,相对标准偏差（RSD）在4.5%～8.1%,3种物质定量限均低于0.6 μg/kg,精密度试验结果相对标准偏差（RSD）为0.9%～1.7%。该方法准确度高、精密度好,适用于啤酒中伏马毒素含量的测定,可为啤酒中伏马毒素的风险评估数据来源提供参考依据。     

T-2毒素快速定量检测试纸条的研究

为研制一种快速定量检测玉米中T-2毒素含量的胶体金试纸条,对T-2毒素单克隆抗体进行标记并对胶体金试纸条性能进行研究。用胶体金标记T-2毒素单克隆抗体,通过测定加入不同量碳酸钾后吸光度值的变化,确定标记的最佳pH值,通过与抗原进行正交试验确定最佳组合条件,应用胶体金定量读数仪通过检测线和对照线的对比,检测试纸条的性能。结果表明,单克隆抗体标记的最佳pH值为8.5,最佳标记浓度为20μg/mL,抗原的包被浓度为0.5mg/mL,羊抗鼠IgG的包被浓度为1.0mg/mL,试纸条检测T-2毒素标准品的检测限为5μg/kg。T-2毒素胶体金快速定量检测试纸条检测方法可得出具体数据,与常见真菌毒素无交叉反应,回收率在77%~117.6%之间,批内和批间重复性检测变异系数小于10%,玉米中T-2毒素的检测结果与高效液相法检测结果一致。T-2毒素胶体金快速定量检测试纸条可用于玉米中T-2毒素含量的快速定量检测。     

免疫亲和柱净化-液相色谱-串联质谱法同时测定水产品中8种霉菌毒素

目的:建立免疫亲和柱净化-液相色谱-串联质谱法(LC-MS/MS)同时测定水产品中8种霉菌毒素残留。方法:样品经乙腈水溶液(84:16,V:V)提取,多毒素免疫亲和柱净化。色谱柱为Agilent Proshell 120 SB·C₁₈(2.1 mm×100 mm,2.7μm),流动相为甲醇-5 mmol/L乙酸铵(含0.1%甲酸),梯度洗脱,流速为0.4 mL/min,柱温为40℃。采用电喷雾离子源正负离子同时扫描,多反应监测模式(MRM)进行质谱检测。结果:8种霉菌毒素在1.0～50.0 ng/mL范围内线性关系良好(R²>0.992),方法检出限在0.05～0.50μg/kg之间,定量限在0.17～1.65μg/kg之间;8种霉菌毒素加标回收率在75.6%～106.3%之间,相对标准偏差(RSD)为3.5%～9.3%。结论:该方法操作便捷、灵敏度高、准确可靠,能满足水产品中多种霉菌毒素残留快速检测需求。     

基于毒性效应通路荧光细胞传感模型的T-2毒素毒性筛查

为实现T-2毒素的可视化毒性筛查,利用T-2毒素毒性通路中AP-1的关键靶点应答元件TRE与红色荧光蛋白（mCherry）的启动子构建pcDNA3.1-TRE-mCherry荧光质粒,建立荧光HEK293细胞传感筛查模型;同时,为验证该细胞传感模型的适用性,对实际样品中残留的T-2毒素和T-2毒素的主要代谢产物HT-2毒素进行了毒性监测。结果表明,T-2毒素刺激模式细胞8 h时,细胞内的红色荧光强度达到最高值并趋于稳定。当T-2毒素质量浓度在1~25 ng/mL时,其与荧光强度呈线性相关,线性方程为y=1.149 38x+64.72,R²=0.969。根据剂量曲线得出T-2毒素的EC₅₀为16.27 ng/mL,检测限为0.691 ng/mL。该细胞传感模型用于样品加标实验,平均加标回收率为86.13%~126.20%,对HT-2毒素进行毒性筛查得到EC₅₀为27.65 ng/mL。     

水产品及螺旋藻片中微囊藻毒素检测方法研究

建立了超高效液相色谱-串联质谱法检测水产品及螺旋藻片中微囊藻毒素的高灵敏度色质联用快速检测方法。样品经溶剂超声提取、固相萃取净化,氮气吹至近干并定容后,通过超高效液相色谱-串联四极杆质谱仪检测。分离柱为Waters Acquity BEH C18柱(1.7μm,2.1 mm×100 mm);流动相为0.3%甲酸水溶液-乙腈;流速0.4 mL/min。4种不同基质样品中有害毒素微囊藻毒素MCYST-RR和MCYST-LR的检测限分别为0.7μg/kg和0.3μg/kg。4种不同基质样品中微囊藻毒素的回收率为80.0%～103.6%,相对标准偏差(RSD)为2.2%～14.6%(n=5),在0.01μg/mL～10μg/mL浓度范围内呈良好的线性关系,线性回归系数r2均大于0.999。     

免疫亲和层析净化-超高效液相色谱法测定生鲜牦牛乳中黄曲霉毒素M_1及M_2

使用免疫亲和层析净化-超高效液相色谱法同时测定了生鲜牦牛乳中的黄曲霉毒素M_1(Aflatoxin M_1,AFM_1)及黄曲霉毒素M_2(Aflatoxin M_2,AFM_2)。黄曲霉毒素M_1仪器线性范围0.400-100μg/mL,仪器定量限0.400ng/mL,仪器检出限0.100ng/mL,方法检出限0.005μg/kg,回收率85.2-95.5%;黄曲霉毒素M_2仪器线性范围0.100-100μg/mL,仪器定量限0.100ng/mL,仪器检出限0.03ng/mL,方法检出限0.0015μg/kg,回收率82.1-93.8%。使用该方法对来自若尔盖地区的10个生鲜牦牛乳进行了测定,结果表明生鲜牦牛乳中黄曲霉毒素M_1和M_2安全可控。     

高效液相色谱法同时检测粮食中常见8 种真菌毒素的含量

建立免疫亲和柱净化-高效液相色谱法同时测定粮食中黄曲霉毒素B1（aflatoxins,AFB1）、黄曲霉毒素B2（AFB2）、黄曲霉毒素G1（AFG1）、黄曲霉毒素G2（AFG2）、赭曲霉毒素A（ochratoxin A,OTA）、玉米赤霉烯酮（zearalenone,ZEN）、呕吐毒素（deoxynivalenol,DON）和T-2毒素的检测方法。样品经乙腈-水提取后,用免疫亲和柱净化,Agilent Elipse Plus C18（100 mm×4 mm,3.5 μm）色谱柱分离,以甲醇-乙腈-水-乙酸为流动相,流速1 mL/min,柱温35 ℃,进样量20 μL,检测系统为可变波长检测器串联光化学衍生器串联荧光检测器。根据信噪比为3的峰响应值,确定各真菌毒素的检出限为：AFB1 0.446 ng/mL、AFB2 0.152 ng/mL、AFG1 0.523 ng/mL、AFG2 0.334 ng/mL、ZEN 7 ng/mL、OTA 0.7 ng/mL、DON 200 ng/mL、T-2毒素100 ng/mL。样品中各真菌毒素的平均加标回收率,玉米为80.0%～104.5%,小麦为83.2%～102.8%,方法精密度为2.6%～10.2%。从样品前处理到分析整个过程耗时约2 h。本方法简便、快速、灵敏度高,适用于粮食中多种真菌毒素的快速测定。     

分散固相萃取-气相色谱法测定水产品中多氯联苯

建立气相色谱批量测定水产品中7 种多氯联苯残留的分析方法。样品前处理采用超声波提取,经浓硫酸磺化后引入分散固相技术进行净化,考察不同条件下多氯联苯的提取分离效果并考虑水产品基质干扰的因素,利用气相色谱-电子捕获检测器测定7 种多氯联苯含量,采用内标校正。结果表明：7 种多氯联苯在0.005～0.2 μg/mL质量浓度范围内具有良好的线性关系,相关系数0.999 86～0.999 97;样品中添加量为2～100 μg/kg时,回收率为80.4%～118.0%,相对标准偏差为1.9%～7.3%,检出限为（RSN=3）0.4～0.8 μg/kg,定量限（RSN=10）1.3～2.7 μg/kg。该方法操作简单、方便快速、重复性好,适用于批量监测水产品中多氯联苯残留的分析。     

高效液相色谱-串联质谱法快速检测粮油制品中黄曲霉毒素B1的含量

目的建立高效液相色谱-串联质谱法快速测定粮油制品中黄曲霉毒素B_1含量的分析方法。方法样品中的黄曲霉毒素B_1经提取、免疫亲和柱净化、浓缩等步骤,以5 mmol/L乙酸铵溶液、乙腈-甲醇溶液(50:50,V:V)为流动相,按照梯度洗脱程序,通过液相色谱-串联质谱法进行测定,外标法定量。结果黄曲霉毒素B_1在0.2～20ng/mL浓度范围内与峰面积呈现良好的线性关系,线性方程为Y=68577.3X-20845,r0.998,加标回收率在85.0%～94.1%之间,相对标准偏差(relative standard deviation, RSD)5.5%,方法检出限和定量限分别为0.07和0.2μg/kg。结论该方法测定结果准确、可靠,可用于快速测定粮油制品中黄曲霉毒素B_1的含量。     

低剂量T-2/HT-2毒素生物转化产物的遗传毒性研究

本文为了研究低剂量T-2和HT-2毒素经弯曲假单胞菌(Pseudomonas geniculata)生物转化至无游离态毒素检出时,降解产物是否具有遗传毒性。通过连续7 d给小鼠灌胃不同剂量T-2/HT-2毒素降解产物,检测小鼠骨髓嗜多染红细胞微核率和精子致畸率。结果表明雄性小鼠产生的微核率均高于雌性小鼠,各剂量组的微核率与阴性组相比差异显著(P0.05),10/20 ng/m L和25/50 ng/m L的雄性小鼠微核率与阳性组相比差异不显著(P0.05)。2.5/5 ng/m L T-2/HT-2组的小鼠精子畸形与阴性组相比差异不显著(P0.05),畸形精子主要表现为胖头和折尾,随着T-2、HT-2剂量的增加,小鼠精子畸形率呈线性增加。说明经过弯曲假单胞菌(Pseudomonas geniculata)转化的T-2和HT-2毒素仍具有遗传毒性,而且雄性比雌性易感性更强,同时为T-2和HT-2毒素生物转化物中存在结合态的T-2毒素提供佐证。     

T-2 and HT-2 toxins in cereals and cereal-based products in South Korea

A total of 214 samples, consisting of brown rice, barley, mixed grains, corn, wheat and wheat flour were analysed for T-2 and HT-2 toxins using high-performance liquid chromatography with fluorescence detection. Recovery and repeatability were 79.9%–107.5% and 4.9%–14.5% for T-2, and 74.0%–106.1% and 5.0%–17.9% for HT-2, respectively. T-2 toxin was detected in 11 (5.1%) of all samples. The highest incidence was found in corn (21.7%) followed by mixed grains and brown rice. Mean of all samples was 1.5–4.1?µg?kg^?1, the maximum level being 41.5?µg?kg^?1 in corn. HT-2 toxin was detected in 126 (58.9%) of all samples, and the mean values were 26.4–59.2?µg?kg^?1. The estimated daily intakes for the sum of T-2 and HT-2 toxins were 2.56, 3.22, 2.53, 0.03, 0.01 and 2.45?ng (kg?bw)^?1?day^?1 in brown rice, barley, mixed grains, corn, wheat and wheat flour, respectively.     

A Sensitive and Validated Method for Determination of T-2 and HT-2 Toxin Residues in Shrimp Tissues by LC-MS/MS

Accurate analyses of T-2 and HT-2 toxin in aquatic organisms including shrimp are important as these two toxins are increasingly detected in aquatic cereal-based feed. Therefore, the potential for these toxins to enter the human food chain from contaminated fish and shrimp products is very real. A rapid, sensitive, and validated method for simultaneous determination of T-2 and HT-2 toxins was developed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method following extraction of the two toxins from shrimp tissues with ethyl acetate. This method is simple in that additional solid-phase extraction is not required to isolate and purify the toxins. LC was performed on an analytical Hypersil GOLD column. The mobile phase consisted of methanol and 5 mM ammonium acetate containing 0.1 % formic acid. The MS/MS ion transitions for both the T-2 toxin (484.20?→?214.87) and HT-2 toxin (442.20?→?214.96) were monitored. And the most intense transition ion product (m/z) of T-2 and HT-2 used for quantification on the SRM mode of a mass spectrometer was 304.95 and 262.91, respectively. The results linearly correlated with coefficients >0.9990. The limits of quantification ranged from 0.02 to 0.51 ng·g^?1 and from 0.17 to 4.48 ng·g^?1 for T-2 and HT-2, respectively, depending on the shrimp tissue type. The overall extraction recovery for both toxins ranged between 84 and 111 % with RSD values less than 15.0 %, indicative of good replication. Furthermore, the recovery and precision levels were within the predefined limits (≤15 %) at all concentrations. The application of this method to study the accumulation of T-2 toxin in shrimp showed that it can be successfully used to monitor even very low tissue toxin concentrations. Research is in progress to extend this method for the measurement of T-2 and HT-2 in aquatic foods that enter the human food chain.     

Validation of a gas chromatography-electron capture detection of T-2 and HT-2 toxins in Chinese herbal medicines and related products after immunoaffinity column clean-up and pre-column derivatization

A sensitive, reproducible and accurate gas chromatography-electron capture detection (GC-ECD) method was developed for simultaneous determination of T-2 and HT-2 toxins in Chinese herbal medicines (CHMs) and related products after immunoaffinity column (IAC) clean-up and pre-column derivatization with N-heptafluoro-butyryl imidazole (HFBI). Then, gas chromatography-spectrometry spectrometer (GC-MS) was applied to confirm the positive results and interfering peaks. The limits of detection (LODs) for T-2 and HT-2 toxins were 1.88 and 0.47 ng/g, and the recoveries for different CHMs ranged from 89.2% to 99.1% with relative standard deviation (RSD) <6.0% for T-2 and from 85.9% to 99.0% with RSD <8.8% for HT-2 toxin, respectively. The validated method was successfully applied for the determination of T-2 and HT-2 toxins in 89 Chinese herbal medicines and 10 related products from various sources, where it was found that T-2 and HT-2 toxins were not detected in any of the tested samples. These results were reliable by confirmation using GC-MS. Some unknown peaks were interfering peaks not the target toxins.     

Measurement of T-2 and HT-2 toxins in eggs by high-performance liquid chromatography with fluorescence detection

T-2 toxin is a mycotoxin produced by several species of common fungi capable of infesting human food and animal feeds. Lower-quality feeds given to chickens may be contaminated with T-2 toxin, which may affect their health. The literature suggests that T-2 toxin is transmitted from the hen to the eggs. This article describes the development of a liquid chromatographic assay for T-2 and the related mycotoxin HT-2 in eggs. T-2 and HT-2 toxins were isolated from spiked eggs with a tandem charcoal-alumina-Florisil column and immunoaffinity column cleanup. The isolated toxins were derivatized with the fluorophore 1-anthroyl nitrile, separated by high-performance liquid chromatography, and quantitated by fluorescence. The limit of detection of the method was 1 ng ml(-1) (parts per billion) of T-2 and HT-2 in whole (with shell removed) eggs. The limit of quantitation for both toxins was 5 ng ml(-1). Recoveries from spiked eggs over the range from 5 to 50 ng ml(-1) averaged 89.2% for T-2 and 100.3% for HT-2, with coefficients of variation of 3.5 and 8.2%, respectively. This method is sensitive enough to be used to check for the presence of T-2 or HT-2 toxins in eggs.     

Study of the natural occurrence of T-2 and HT-2 toxins and their glucosyl derivatives from field barley to malt by high-resolution Orbitrap mass spectrometry

This paper reports a new method for the determination of T-2 and HT-2 toxins and their glucosylated derivatives in cereals, and some survey data aimed at obtaining more comprehensive information on the co-occurrence of T-2 and HT-2 toxins and their glucosylated derivatives in naturally contaminated cereal samples. For these purposes, barley samples originating from a Northern Italian area were analysed by LC-HRMS for the presence of T-2, HT-2 and relevant glucosyl derivatives. Quantitative analysis of T-2 and HT-2 glucosides was performed for the first time using a recently made available standard of T-2 glucoside. The glucosyl derivative of HT-2 was detected at levels up to 163 µg kg^–1 in 17 of the 18 analysed unprocessed barley grains, whereas the monoglucosyl derivative of T-2 toxin was detected in only a few samples and at low µg kg^–1 levels. The ratio between glucosylated toxins (sum of T-2 and HT-2 glucosides) and native toxins (sum of T-2 and HT-2) ranged from 2% to 283%. Moreover, taking advantage of the possibility of retrospective analysis of full-scan HRMS chromatograms, samples were also screened for the presence of other type-A trichothecenes, namely neosolaniol, diacetoxyscirpenol and their monoglucosyl derivatives, which were detected at trace levels. A subset of nine different samples was subjected to micro-maltation in order to carry out a preliminary investigation on the fate of T-2, HT-2 and relevant glucosides along the malting process. Mycotoxin reduction from cleaned barley to malt was observed at rates ranging from 4% to 87%.     

Thermal stability of T-2 and HT-2 toxins during biscuit- and crunchy muesli-making and roasting

ABSTRACT

The mycotoxins T-2 and HT-2 toxin are frequently occurring food contaminants which are produced by Fusarium species. Humans and animals are mainly exposed to these substances by the consumption of contaminated oats, maize and wheat. For the production of crunchy muesli, bread and bakery products, these cereals undergo multiple processing steps, including baking, roasting and extrusion cooking. However, the influence of food processing on T-2 and HT-2 toxin levels is to date poorly understood. Thus, the effects of baking and roasting on both mycotoxins were evaluated during biscuit-, crunchy muesli- and toasted oat flakes-production under precise variation of various parameters: heating time and temperature as well as recipe formulation were varied in the range they are applied in the food processing industry. Therefore, oatmeal or flaked oats were artificially contaminated individually with both toxins and processed at the laboratory scale. T-2 toxin generally showed a higher degradation rate than HT-2 toxin. During biscuit-making up to 45% of T-2 toxin and 20% of HT-2 toxin were thermally degraded, showing a dependency on water content, baking time and temperature. The preparation of crunchy muesli yielded no significant toxin degradation which is probably due to the low temperatures applied. Roasting led to a degradation of 32% of T-2 toxin and 24% of HT-2 toxin. Taken together, both mycotoxins are partially degraded during thermal food processing; the degradation rates are influenced by the food composition and processing parameters.     

紫外光辐照对脱氧雪腐镰刀菌烯醇和T-2毒素的去除作用

目的：研究紫外光辐照对脱氧雪腐镰刀菌烯醇（deoxynivalenol,DON）和T-2毒素的去除作用。方法：测定不同辐照时间、不同辐照距离、不同pH值条件下紫外光辐照对DON和T-2毒素影响,采用高效液相色谱-质谱联用技术测定毒素及其衍生物,外标法定量。结果：经紫外光辐照后,溶液中DON、T-2毒素的质量浓度均随着辐照时间的延长而不断减小,随着辐照距离和pH值的减小而不断减小。pH 7的1.0 μg/mL DON、T-2毒素溶液,在紫外灯功率20 W、辐照距离150 mm条件下辐照60 min后,DON、T-2毒素去除率分别为（84.90±2.52）%、（74.60±2.74）%。紫外光辐照后,毒素溶液中不含有已知的毒素衍生物,可能被转化成新的未知产物。结论：在非碱性条件下,紫外光辐照对DON、T-2毒素具有明显的去除作用。     

Determination of trichothecenes in cereals and cereal-based products by liquid chromatography-tandem mass spectrometry

A sensitive, accurate and precise method for the simultaneous determination of nivalenol (NIV), deoxynivalenol (DON), T-2 toxin (T-2) and HT-2 toxin (HT-2) in different food matrices, including wheat, maize, barley, cereal-based infant foods, snacks, biscuits and wafers, has been developed. The method, using liquid chromatography coupled with atmospheric pressure chemical ionization triple quadrupole mass spectrometry (LC-APCI-MS/MS), allowed unambiguous identification of the selected trichothecenes at low µg per kg levels in such complex food matrices. A clean-up procedure, based on reversed phase SPE Oasis® HLB columns, was used, allowing good recoveries for all studied trichothecenes. In particular, NIV recoveries significantly improved compared to those obtained by using Mycosep® #227 columns for clean-up of the extracts. Limits of detection in the various investigated matrices ranged 2.5-4.0 µg kg^-1 for NIV, 2.8-5.3 µg kg^-1 for DON, 0.4-1.7 µg kg^-1 for HT-2 and 0.4-1.0 µg kg^-1 for T-2. Mean recovery values, obtained from cereals and cereal products spiked with NIV, DON, HT-2 and T-2 toxins at levels from 10 to 1000 µg kg^-1, ranged from 72 to 110% with mean relative standard deviation lower than 10%. A systematic investigation of matrix effects in different cereals and cereal products was also carried out by statistically comparing the slopes of standard calibration curve with matrix-matched calibration curve for each of the four toxins and the eight matrices tested. For seven of the eight matrices tested, statistically significant matrix effects were observed, indicating that, for accurate quantitative analysis, matrix-matched calibration was necessary. The method was applied to the analysis of 57 samples of ground wheat originated from South Italy and nine cereal food samples collected from retail markets.     

Occurrence of type A trichothecenes in conventionally and organically produced oats and oat products

Among cereals, oats are known to be very frequently contaminated with type A trichothecenes and so they can play a major role in the exposition of the consumer to these mycotoxins. Seventy representative oat samples of both conventional and organic production were drawn at mills and at wholesale stage according to Commissions Regulation (EC) No 401/2006 and analyzed for nine type A trichothecenes by LC-MS/MS. High contamination rates were found for most of the toxins in conventional as well as in organic products (e. g. 100% for T-2 toxin or 99% for HT-2 toxin). The mean concentration of T-2/HT-2 (sum of the toxins) was 17 +/- 18 microg/kg (mean +/- SD) in all samples, 27 +/- 21 microg/kg in conventional, and 7.6 +/- 4.6 microg/kg in organic products, respectively. The highest T-2/HT-2 level has been determined in conventionally produced oat flakes (85 microg/kg). The mean level of T-2 tetraol (9.5 +/- 7.7 microg/kg) in all samples was found to be even higher than that of T-2 (5.1 +/- 6.0 microg/kg), whereas levels of T-2 triol, 4,15-diacetoxyscirpenol, 15-monoacetoxyscirpenol, and neosolaniol were considerably lower. For oats and oat products from organic farming contamination levels of T-2, HT-2, T-2 triol, T-2 tetraol, and neosolaniol were significantly lower. The results are discussed with respect to possible health risks for the consumer.