免疫亲和层析及其在真菌毒素检测中应用

该文简介免疫亲和层析工作原理,载体要求与选择,免疫亲和柱制备与使用,重点综述免疫亲和层析在黄曲霉毒素,赭曲霉毒素,玉米赤霉烯酮,脱氧雪腐镰刀菌烯醇,伏马毒素,T-2毒素等真菌毒素检测中应用最新进展。     

基于UPLC-MS-MS技术定量确证小麦中10种真菌毒素

建立了小麦中黄曲霉毒素B_1、黄曲霉毒素B_2、黄曲霉毒素G_1、黄曲霉毒素G_2、脱氧雪腐镰刀菌烯醇、雪腐镰刀菌烯醇、3-乙酰脱氧雪腐镰刀菌烯醇、脱氧雪腐镰刀菌烯醇-3-葡萄糖苷、15-乙酰脱氧雪腐镰刀菌烯醇和玉米赤霉烯酮等10种真菌毒素的超高液相色谱-三重四级杆串联质谱法检测的方法。小麦样品经乙腈-水-乙酸溶液震荡超声提取后,采用多功能净化柱净化。待测物经Kinetex SB-C_(18)色谱柱分离,甲醇-0.1%甲酸为流动相,梯度洗脱,正负模式同时扫描,三重串联四级杆质谱多反应离子监测(MRM)方式检测,基质标准校正,外标法定量。结果表明,在一定的浓度范围内,10种真菌毒素线性关系良好,相关系数均大于0.999,方法的检出限为0.015~1μg/kg,空白样品中添加高中低不同浓度的真菌毒素的回收率为65.12%~89.16%,RSD%(n=6)在4.26%~9.46%之间。该方法分析速度快、重复性好、灵敏度高,适合小麦中多种真菌毒素的高灵敏度快速测定。     

免疫亲和柱净化-HPLC法测定小麦粉中的黄曲霉毒素B_1、玉米赤霉烯酮和脱氧雪腐镰刀菌烯醇的含量

建立了免疫亲和柱净化-高效液相色谱法测定小麦粉中黄曲霉毒素B1(AFB1)、玉米赤霉烯酮(ZEN)、脱氧雪腐镰刀菌烯醇(DON)的方法。小麦粉样品经乙腈-水振荡提取,三合一免疫亲和柱净化后,采用双通道荧光检测器串联光化学衍生器同时测定黄曲霉毒素B1和玉米赤霉烯酮,二极管阵列检测器测定脱氧雪腐镰刀菌烯醇。样品中各真菌毒素平均加标回收率为74.8%~105.2%,相对标准偏差为1.96%~6.26%。此方法可用于实验室开展小麦中3种真菌毒素含量的检测。     

超高效液相色谱串联质谱法检测谷物中8种真菌毒素

建立了液质联用法同时测定脱氧雪腐镰刀菌烯醇、3-乙酰基脱氧雪腐镰刀菌烯醇、15-乙酰基脱氧雪腐镰刀菌烯醇、黄曲霉毒素B_1、B_2、G_1、G_2、玉米赤霉烯酮共8种真菌毒素谷物中的含量。样品经乙腈-水溶液提取,通过多功能净化柱净化、Waters Acquity UPLC? BEH C18色谱柱分离、超高效液相色谱串联质谱系统测定,脱氧雪腐镰刀菌烯醇及其衍生物同位素内标法定量,黄曲霉毒素和玉米赤霉烯酮外标法定量。结果表明,8种真菌毒素在线性范围内线性相关性R~2≥0.999,检出限在0.3~1.0μg/kg之间,加标回收率在72.2%~101.7%之间,相对标准偏差在1.22%~9.69%之间。该方法操作简单、灵敏度高、回收率好,适用于谷物及其制品中多种真菌毒素的同时检测。     

大米中16种真菌毒素同时检测分析

目的建立大米中黄曲霉毒素、伏马毒素、雪腐镰刀菌烯醇、脱氧雪腐镰刀菌烯醇类毒素、玉米赤霉烯酮、赭曲霉毒素A、T-2毒素、HT-2毒素以及杂色曲霉毒素等16种真菌毒素便捷准确的液相色谱-串联质谱法。方法大米样品经粉碎均质后采用70%甲醇水溶液浸泡提取,通过PriboFastRM226多功能净化柱净化,利用液相色谱串联质谱的多反应监测模式进行测定分析,采用内标法定量。结果 16种真菌毒素均具有良好的线性关系(r0.99),3个加标水平回收率为85.2%～102.8%,相对标准偏差范围为2.05%～4.75%。在61件大米检测中,共有5件样品检出真菌毒素,检测到的真菌毒素有5种,其中雪腐镰刀菌烯醇、伏马毒素B_1、伏马毒素B_2、15-乙酰基脱氧雪腐镰刀菌烯醇各有1件样品检出,黄曲霉毒素B_1有3件检出,检出率为8.33%,检出的5种真菌毒素含量在0.69～71.47μg/kg,均未超过国家食品安全标准规定的真菌毒素限量标准。结论大米中真菌毒素的检出率低,含量符合国家标准要求,该检测方法准确、可靠、便捷,可适用于大米中多种真菌毒素的检测。     

2022年昭通市新收获玉米真菌毒素污染情况分析

目的：调查分析2022年云南省昭通市新收获玉米真菌毒素污染情况。方法：采集2022年昭通市各县区100份玉米样品,分别采用高效液相色谱-柱后衍生法、免疫亲和层析净化高效液相色谱法、液相色谱法测量黄曲霉毒素B₁、脱氧雪腐镰刀菌烯醇、玉米赤霉烯酮。结果：71份玉米样本未检测出真菌毒素,29份样本检测出真菌毒素,所有样本真菌毒素均未超标,脱氧雪腐镰刀菌烯醇总体检出率最高。盐津县有1份检出3种真菌毒素的玉米样本,巧家县有3份检出2种真菌毒素的玉米样本,且脱氧雪腐镰刀菌烯醇与玉米赤霉烯酮检出率最高,巧家县和绥江县所收样本脱氧雪腐镰刀菌烯醇检出率最高,巧家县和水富市所收玉米样本赤霉烯酮检出率最高。结论：昭通市2022年新收获玉米真菌毒素污染情况在合理范围内,盐津县、巧家县新收获玉米易受真菌毒素污染,需加强关注。     

基于Meta分析对全球小麦及小麦粉中多种真菌毒素污染特征的比较研究

目的 针对小麦及小麦粉中脱氧雪腐镰刀菌烯醇（DON）、黄曲霉毒素（AFs）、玉米赤霉烯酮（ZEN）、雪腐镰刀菌烯醇（NIV）、T-2毒素（T2）、HT-2毒素（HT2）、赭曲霉毒素A(OTA)、伏马菌素（FBs）、交链孢酚（AOH）、交链孢酚单甲醚（AME）、腾毒素（TEN）及交链孢菌酮酸（TeA）等真菌毒素的全球污染情况进行Meta分析。方法 数据主要来自PubMed、Web of Science、知网及万方数据库。结果 通过文献筛选,共纳入69篇文献22 308个样本。小麦及小麦粉中以上真菌毒素的全球总体污染率为58%(95%CI:51%～66%),其中,TeA为99%、TEN为88%、DON为85%、AFs为57%、ZEN为42%、T2为39%、AOH为30%、AME为29%、NIV为28%、HT2为25%、OTA为21%及FBs为16%;小麦及小麦粉中真菌毒素的全球总体污染水平为32.80μg/kg(95%CI:24.96～43.10μg/kg),DON在小麦及小麦粉中的含量最高,为317.53μg/kg,其次为TeA 117.37μg/kg及FBs 45.09μg/kg。DO...     

高效液相色谱串联质谱法测定花生及制品中的五种真菌霉素

建立了高效液相色谱-串联质谱(HPLC-MS/MS)测定花生及制品中黄曲霉毒素B1、黄曲霉毒素M1、脱氧雪腐镰刀菌烯醇、赭曲霉毒素A、玉米赤霉烯酮五种真菌霉素的快速分析方法。用甲醇-水(55:45,V/V)对样品进行提取,采用真菌毒素免疫亲和柱萃取,在ESI+模式下采用多反应监测(MRM)模式进行检测。目标物在C18色谱柱上实现了有效分离,在6 min内完成一个样品的分析,相关系数(r2,n=6)大于0.999,检测结果稳定、灵敏。黄曲霉毒素B1、黄曲霉毒素M1、脱氧雪腐镰刀菌烯醇线性范围0.5~50.0μg/L,检出限为0.05μg/kg(LOD,S/N=3),赭曲霉毒素A、玉米赤霉烯酮线性范围5.0~100.0μg/L,检出限为0.5μg/kg(LOD,S/N=3),方法回收率为86.8~102.7%,精密度RSD为0.36~4.79%。该方法快速、灵敏,适用于花生及制品中黄曲霉毒素B1、黄曲霉毒素M1、脱氧雪腐镰刀菌烯醇、赭曲霉毒素A、玉米赤霉烯酮五种真菌霉素的检测与确证。     

肉制品中真菌毒素污染现状与控制研究进展

简要介绍了肉制品中常见真菌毒素(黄曲霉毒素、赭曲霉毒素、脱氧雪腐镰刀菌烯醇、玉米赤霉烯酮、展青霉素、桔霉素)的来源及其危害,重点介绍了肉制品中常见真菌毒素的污染现状及其控制方法,并展望了肉制品中真菌毒素未来的研究方向。     

粮油食品中真菌毒素的LC-MS法检测

粮油食品中常见的真菌毒素有:黄曲霉毒素、赭(棕)曲霉毒素、展青霉毒素、玉米赤霉烯酮、串珠镰刀菌毒素及脱氧雪腐镰刀菌烯醇等,因含量较低,用常规方法检测不出来。用LC-MS检测,灵敏度高、前处理比较简单,结果准确可靠,在检测食品中真菌毒素上前景广阔。     

Simultaneous detection of 12 mycotoxins in cereals using RP-HPLC-PDA-FLD with PHRED and a post-column derivatization system

A new method for the simultaneous quantification of 12 mycotoxins was developed and optimized using reverse phase high performance liquid chromatography (RP-HPLC) with a photodiode array (PDA) and fluorescence detector (FLD), a photochemical reactor for enhanced detection (PHRED) and post-column derivatization. The mycotoxins included aflatoxins (AFB(1), AFB(2), AFG(1), and AFG(2)), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB(1), FB(2), and FB(3)), T-2 and HT-2 toxins. A double sample extraction with a phosphate-buffered saline solution (PBS) and methanol was used for co-extraction of mycotoxins, and a multifunctional immunoaffinity column was used for cleanup. Optimum conditions for separation of the mycotoxins were obtained to separate 12 mycotoxins in FLD and PDA chromatograms with a high resolution. The method gave recoveries in the range 72-111% when applied to spiked corn samples. The limits of detection (LOD) were 0.025 ng/g for AFB(1) and AFG(1), 0.012 ng/g for AFB(2) and AFG(2), 0.2 ng/g for OTA, 1.5 ng/g for ZEA, 6.2 ng/g for FB(1), FB(3) and HT-2 toxin, 9.4 ng/g for FB(2) and T-2 toxin, and 18.7 ng/g for DON. In addition, the limits of quantification (LOQ) ranged from 0.04 ng/g for AFB(2) and AFG(2) to 62 ng/g for DON. The method was successfully applied to the determination of these mycotoxins in 45 cereal samples obtained from the Malaysian market. The results indicated that the method can be applied for the multi-mycotoxin determination of cereals.     

Determination of chloramphenicol and zeranols in pig muscle by immunoaffinity column clean-up and LC-MS/MS analysis

An immunoaffinity column clean-up and LC-MS/MS method was successfully developed for simultaneous determination of chloramphenicol, zearalanone, α-zearalanol, β-zearalanol, zearalenone, α-zearalenol and β-zearalenol in pig muscle. The sample was extracted with diethyl ether after enzymatic digestion by β-glucuronidase/sulfatase. The extracted solution was evaporated to dryness and the residue was then dissolved in 1 ml of 50% acetonitrile solution. After filtration and dilution with phosphate buffer solution (PBS), the reconstituted solution was cleaned-up with an IAC-CZ immunoaffinity column and then analysed by HPLC-MS/MS. The established method were validated by linearity (r ≥ 0.9990), precision (RSD ≥ 2.9%), average recovery (74.5–105.0%) and limit of detection (0.04–0.10 μg kg^–1). The developed method is rapid, reliable, sensitive, accurate and has good applicability for real samples.     

重庆地区辣椒、花椒、八角中真菌毒素污染状况分析

目的 了解重庆地区辣椒、花椒和八角中真菌毒素污染状况。方法 采用随机抽样方法,在重庆地区盘溪、菜园坝和江津三个市场,抽取180批次香辛料样品,采用HPLC-MS/MS检测黄曲霉毒素（AFB1、AFB2、AFG1和AFG2）、赭曲霉毒素A（OTA）和伏马毒素（FB1和FB2）。结果 重庆地区辣椒、花椒和八角中的真菌毒素总检出率分别为60.0%、100%、96.7%。AFs和OTA是三种香辛料最主要的污染毒素,其中AFG2是最主要的黄曲霉代谢产物。盘溪市场的AFs污染总体情况稍好,菜园坝市场AFs污染最严重;江津市场的OTA污染最为严重。结论 香辛料中存在真菌毒素污染,有必要建立香辛料中真菌毒素的限量标准。     

免疫亲和净化-超高效液相色谱-串联质谱法测定食品中玉米赤霉烯酮类真菌毒素

建立食品中6种玉米赤霉烯酮类(α-玉米赤霉醇、β-玉米赤霉醇、α-玉米赤霉烯醇、β-玉米赤霉烯醇、玉米赤霉酮、玉米赤霉烯酮)真菌毒素的免疫亲和净化-超高效液相色谱-串联质谱检测的实验方法。样品经80%乙腈溶液提取,通过免疫亲和柱净化富集,用2 mL乙腈洗脱,氮吹至近干,0.5 mL 50%乙腈溶液复溶,采用超高效液相色谱-串联质谱进行测定。在ACQUITY UPLC HSS T3反相柱上分离,梯度洗脱,流动相为乙腈和水,质谱采集模式为电喷雾负离子多反应监测模式。6种目标物的线性范围为0.1~100μg/L,相关系数(R~2)均大于0.992,检出限为0.05μg/kg,定量限为0.2μg/kg,3个不同水平的加标平均回收率为73.0%~119.1%,相对标准偏差不大于10%。该方法具有操作简单、重复性好、灵敏度高、杂质干扰小等特点,可以用于食品中玉米赤霉烯酮类真菌毒素的检测。     

Simultaneous Determination of Ractopamine,Chloramphenicol, and Zeranols in Animal-Originated Foods by LC-MS/MS Analysis with Immunoaffinity Clean-up Column

A novel analytical method employing immunoaffinity column (IAC) clean-up coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of ractopamine, chloramphenicol, and zeranols (α-zearalanol, β-zearalanol, zearalanone, α-zearalenol, β-zearalenol, and zearalenone) in animal-originated foods. The sample was first digested by β-glucuronidase/sulfatase and then extracted with ethyl acetate-diethyl ether (9:1, v/v). The extracted solution was evaporated to dryness and then the residue was dissolved by 2 mL of 50% acetonitrile solution. After filtration, 1 mL filtrate was diluted to 10 mL with PBS. The reconstituted solution was cleaned up with immunoaffinity column and then analyzed by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The established method was shown to be sensitive efficient and reliable as indicated by the linearity (r ² ≥ 0.9994), precision (RSD ≤ 1.7%), average recovery (72.3–103.2%), and the limit of detection (0.05–0.10 μg/kg). The method can be used for determination of trace residues of ractopamine, chloramphenicol, and zeranols in animal-originated foods.     

LC–MS/MS multi-method for mycotoxins after single extraction,with validation data for peanut,pistachio, wheat,maize, cornflakes,raisins and figs

Mycotoxin analysis is usually carried out by high performance liquid chromatography after immunoaffinity column cleanup or in enzyme-linked immunosorbent assay tests. These methods normally involve determination of single compounds only. EU legislation already exists for the aflatoxins, ochratoxin A and patulin in food, and legislation will come into force for deoxynivalenol, zearalenone and the fumonisins in 2007. To enforce the various legal limits, it would be preferable to determine all mycotoxins by routine analysis in different types of matrices in one single extract. This would also be advantageous for HACCP control purposes. For this reason, a multi-method was developed with which 33 mycotoxins in various products could be analysed simultaneously. The mycotoxins were extracted with an acetonitrile/water mixture, diluted with water and then directly injected into a LC–MS/MS system. The mycotoxins were separated by reversed-phase HPLC and detected using an electrospray ionisation interface (ESI) and tandem MS, using MRM in the positive ion mode, to increase specificity for quality control. The following mycotoxins could be analysed in a single 30-min run: Aflatoxins B₁, B₂, G₁ and G₂, ochratoxin A, deoxynivalenol, zearalenone, T-2 toxin, HT-2 toxin, α-zearalenol, α-zearalanol, β-zearalanol, sterigmatocystin, cyclopiazonic acid, penicillic acid, fumonisins B₁, B₂ and B₃, diacetoxyscirpenol, 3- and 15-acetyl-deoxynivalenol, zearalanone, ergotamin, ergocornin, ergocristin, α-ergocryptin, citrinin, roquefortin C, fusarenone X, nivalenol, mycophenolic acid, alternariol and alternariol monomethyl ether. The limit of quantification for the aflatoxins and ochratoxin A was 1.0 µg kg^?1 and for deoxynivalenol 50 µg kg^?1. The quantification limits for the other mycotoxins were in the range 10–200 µg kg^?1. The matrix effect and validation data are presented for between 13 and 24 mycotoxins in peanuts, pistachios, wheat, maize, cornflakes, raisins and figs. The method has been compared with the official EU method for the determination of aflatoxins in food and relevant FAPAS rounds. The multi-mycotoxin method has been proven by the detection of more than one mycotoxin in maize, buckwheat, figs and nuts. The LC–MS/MS technique has also been applied to baby food, which is subject to lower limits for aflatoxin B₁ and ochratoxin A, ergot alkaloids in naturally contaminated rye and freeze-dried silage samples.     

Determination of mycotoxins in biscuits,dried fruits and fruit jams: an assessment of human exposure

ABSTRACT

A reliable, fast and simple method using UHPLC-MS/MS was developed for the determination of aflatoxins B₁ (AFB1), G₁ (AFG1), B₂ (AFB2) and G₂ (AFG2), ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEA), HT-2 toxin and T-2 toxin in crude extracts of biscuits with fruit filling, cookies, dried fruits and fruit jams. The method was successfully demonstrated on 39 samples of biscuits with fruit filling, 34 cookies, 14 dried fruits and 10 fruit jams. The mycotoxins detected in biscuits samples were ZEA, OTA, T-2 and AFB1 with an average concentrations of positive samples of 2.64, 4.10, 8.13 and 1.32 µg kg^?1, respectively; while the mycotoxins detected in jam samples were AFB1, OTA, T-2 and AFB2 with an average concentrations of positive samples of 2.00, 17.7, 4.37 and 1.15 µg kg^?1, respectively. The results showed that the majority of samples were in compliance with relevant regulations. However in eight samples of biscuits and three samples of fig jam the contents of OTA were higher than the existing OTA limits. The combined dietary exposure of selected mycotoxins was estimated for the first time for children, adolescents and adults. The estimated combined dietary exposures were all lower than the proposed value assumed to predict a possible risk scenario.     

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。     

Simultaneous Determination of Aflatoxins and Ochratoxin A in Bee Pollen by Low-Temperature Fat Precipitation and Immunoaffinity Column Cleanup Coupled with LC-MS/MS

A simple and sensitive method has been developed for simultaneous determination of aflatoxins (AFB1, AFB2, AFG1, and AFG2) and ochratoxin A (OTA) in bee pollen. The analytes in the sample were extracted with a mixture of acetonitrile/water (60:40, v/v), using low temperature for fat precipitation, followed by immunoaffinity column cleanup of extracts. The mycotoxins were quantified using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with an electrospray ionization (ESI) and a triple quadrupole (QqQ) analyzer. Matrix effect, accuracy, and precision were evaluated and achieved good results calculated by matrix-matched calibration standards which reduced the influence of matrix effect. Recoveries at three levels were in the range of 74.3–96.5 % with RSD less than 10.0 %. The correlation coefficients (r ²) of the five mycotoxins were higher than 0.997. The method showed high sensitivity with LOD below 0.05 μg/kg.     

Limited survey for the presence of aflatoxins in foods from local markets and supermarkets in Valencia, Spain

Aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) were extracted by matrix solid-phase dispersion with C18 silica and acetonitrile as the eluting solvent, analysed by liquid chromatography with fluorescence detection and confirmed by liquid chromatography with mass spectrometry using an electrospray interface in 58 samples grouped as cereals, dried fruits, herbs and spices, pulses, snacks, and nuts and nut products collected from local markets and supermarkets in Valencia, Spain. All samples analysed by the proposed method were previously studied with an enzyme-linked immunosorbent assay as a screening protocol for the fast detection of mycotoxins. The samples containing residues (3/58) were hazelnut (0.42 and 0.52 μg kg^-1 for AFB1 and AFG1, respectively), nut cocktail (0.29 and 0.47 μg kg^-1 for AFB1 and AFG1, respectively) and pinhol (0.30 μg kg^-1 for AFG1). Such values were below the legislated maximum residue levels for the European Union.