A rapid, sensitive and economic method for the detection, quantification and confirmation of aflatoxins

Summary A rapid, sensitive and economic method for the detection, quantification and confirmation of aflatoxins is described. Aflatoxins B₁, B₂, G₁, and G₂, are extracted by methanol/water (85+15) and partitioned into methylene dichloride. The methylne dichloride solution is cleaned up on a polypropylene column, filled with 0.5 g silica gel 60. The aflatoxins are eluted with cloroform-acetone (90:10) and are detected using bidirectional thin-layer chromatography (TLC) with aluminium silica gel foil. The mean recovery of aflatoxins B₁, B₂, G₁, and G₂, in corn samples was 73, 78, 80, and 64%, respectively; the limit of detection was 0.5 g/kg. The results can also be confirmed by derivative formation using trifluoroacetic acid on the TLC plate. The method has been applied to a wide range of foods with good results.

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Eine schnelle, empfindliche und kostengünstige Methode zum Nachweis, zur Bestimmung und Bestätigung von Aflatoxinen

Zusammenfassung Es wird eine schnelle, empfindliche und kostengünstige Methode zum Nachweis, Bestimmung und Bestätigung von Aflatoxinen beschrieben. Die Aflatoxine B₁, B₂, G₁ und G₂ werden mit Methanol/Wasser (85+15) extrahiert und in Dichlormethan überführt. Der Dichlormethanextrakt wird auf einer mit 0,5 g Kieselgel 60 gefüllten Polypropylensäule gereinigt. Die Aflatoxine werden mit Chloroform/Aceton (90+10) eluiert und mit zweidimensionaler DC auf Kieselgel-Alufolien nachgewiesen. Die mittleren Wiederfmdungsraten für die Aflatoxine B₁, B₂, G₁ und G₂ in Maismehl betragen 73, 78, 80 und 64%, die Nachweisgrenzen liegen durchschnittlich bei 0,5 g/kg. Zur Bestätigung verdächtiger Befunde kann auf der Platte mit Trifluoressigsäure derivatisiert werden. Die Methode ist bisher an einer Vielzahl von verschiedenen Lebensmitteln mit gutem Erfolg getestet worden.

     

Comparison of six methods of analysis for the determination of aflatoxin B1 in feeding stuffs containing citrus pulp

Six published methods of analysis for the determination of aflatoxin B1 have been compared for their suitability to determine aflatoxin B1 in feeding stuffs containing citrus pulp. These methods are the official European Community (EC) procedure, four procedures proposed in the European Community to replace this method and a new procedure developed by the authors of this article. In all procedures chloroform is used for initial extraction. Various clean-up systems are then applied. For the ultimate separation and detection, use is made of high performance liquid chromatography (HPLC) in three procedures and two-dimensional thin layer chromatography (TLC) in two procedures. One method allows either HPLC or TLC. All experiments were carried out with samples of a batch of feeding stuff containing citrus pulp, artificially contaminated with aflatoxins B1, B2, G1 and G2 at levels of ca 13, 5, 10 and 4 micrograms/kg respectively. Three methods were found to be suitable: a procedure in which gel permeation clean-up and two-dimensional TLC are used; a procedure in which TLC clean-up and reverse phase HPLC with postcolumn derivatization are used: a procedure in which cartridge clean-up and either HPLC or TLC are used. The latter method is preferred because its efficient clean-up yields a very clean extract, allowing the application of various systems of HPLC or TLC. Published recovery data of these three methods for aflatoxin B1 vary from 85-90% at a level of ca 10 micrograms/kg feeding stuff.     

2种超高效液相色谱-柱后衍生法对植物油中黄曲霉毒素的测定与分析

目的建立超高效液相色谱-柱后衍生法测定植物油中黄曲霉毒素G_2、G_1、B_2、B_1的含量,并通过柱后光化学衍生法及柱后碘衍生法进行比对确认。方法样品采用70%的甲醇水提取,经多功能净化柱净化,利用在线超高效液相色谱-荧光检测器-柱后光化学衍生及柱后碘衍生进行测定分析。结果黄曲霉毒素通过柱后光化学衍生后,在0.014～0.241 ng/mL范围内线性关系良好,通过柱后碘衍生在0.02～0.804 ng/m L范围内线性关系良好,相关系数均在0.999以上。参与2016年花生油中黄曲霉毒素的能力验证,经过2种方法的检测,各黄曲霉毒素检测值均在能力验证的参考值允许范围内,考核结果为满意结果,质控样的检测值均在参考值允许范围内,表明2种方法均准确稳定。结论 2种衍生方法的测定结果比较接近,均有较好的重复性和准确性,通过2种衍生方法的比较,光化学衍生法较之有灵敏度高、操作简便、绿色环保、性价比较高的优点,适用于实验室对植物油中黄曲霉毒素的检测。     

Rapid analysis of aflatoxins in raw peanuts,corn, buckwheat and red pepper by a new mini-column cleanup and HPLC using post-column photochemical derivatization system

A rapid and clean method for the analysis of aflatoxins (AFs) was developed by using a new column and post-column photochemical derivatization HPLC with fluorescence detection. The new cleanup column consisted of magnesia and basic alumina poured on the top of a commercial multi-functional mini-column. It was extremely effective for the cleanup of AFs from raw peanut, corn, buckwheat and red pepper. Fluorescent substances, which interfered with the analysis of AFs from corn, were completely absorbed at the top of the magnesia layer. Recoveries of AFs (B1, B2, G1, G2) added to raw peanuts, corn, buckwheat and red pepper were over 80% at two levels of fortification (higher level: 10, 3, 10, 3 ng/g, respectively, lower level: 1.0, 0.3, 1.0, 0.3 ng/g, respectively). Coefficients of variation were smaller than 12%, except the lower fortified level for red pepper. Limits of detection for AFs in raw peanuts, corn and buckwheat were 0.3 ng/g for B1 and G1, and 0.1 ng/g for B2 and G2. Those in red pepper were 0.5 ng/g for B1, B2, G1 and G2.     

Production of aflatoxin B1 and G1 by Aspergillus parasiticus on silica gels.

Silica gels were prepared by acidifying alkaline silicate solutions with phosphoric or tartaric acid. Various combinations of glucose, sucrose, yeast extract, and salts were included in the gels an nutrients. Maximum production of aflatoxins B1 and G1 occurred when silica gel (0.4 to 0.5 cm deep in a petri dish) containing 20% sucrose and 2% yeast extract, and gelled with tartaric acid, was inoculated with approximately 120 to 12000 spores of Aspergillus parasiticus per plate; and plates were incubated at 28 degrees C for 10 days.     

液相色谱串联质谱检测食品中的黄曲霉毒素

为提高黄曲霉毒素的检测灵敏度,建立快速液相色谱串联质谱(LC-MS/MS)法对食品中的4 种黄曲霉毒素B1、B2、G1、G2 进行定性定量分析。样品粉碎后用体积比为84:16 的乙腈- 水混合液提取,过滤后通过真菌毒素净化柱进样,采用C18 柱分离,0.1% 甲酸溶液和甲醇做流动相,以60:40 比例等度洗脱,质谱在多反应监测(MRM)的正离子模式下进行分析。4 种组分在5min 内完全分离,而且此方法线性关系良好,黄曲霉毒素B1、B2、G1、G2 的检出限分别是0.012、0.009、0.013、0.007μg/kg,平均加标回收率在80%～95% 之间,相对标准偏差小于5%。该方法快速灵敏、准确可靠,其检出限可满足欧盟地区严格的黄曲霉毒素限量标准。     

Validation of a UHPLC-FLD method for the simultaneous quantification of aflatoxins,ochratoxin A and zearalenone in barley

A fast and simple UHPLC-FLD method has been developed for the simultaneous determination in barley of aflatoxins (B1, G1, B2 and G2), ochratoxin A (OTA) and zearalenone (ZEA), some of the most important mycotoxins due to their toxicity and occurrence. The procedure is based on the extraction of the six mycotoxins with a mixture of acetonitrile and water, and the purification of the extract with immunoaffinity columns before analysis. Detection of AFB1 and AFG1 is improved using a photochemical reaction. The method has been validated with satisfactory results. Limits of detection were 340 ng kg⁻¹ for ZEA, 13 ng kg⁻¹ for OTA and varied from 0.5 to 15 ng kg⁻¹ for aflatoxins. Recovery percentages were between 78.2% and 109.2%. After being validated, the method has been successfully applied to 20 barley samples cultivated in a region of northern Spain (Navarra).     

光化学衍生-高效液相色谱法测定 粮谷类样品中黄曲霉毒素

目的建立光化学衍生-高效液相色谱荧光法测定粮谷类样品中的黄曲霉毒素(AFT)含量。方法样品经Romer Labs免疫亲和柱净化,经SW-3光化学柱后衍生,经高效液相色谱分离和荧光检测器测定,分析其中黄曲霉毒素B_1、B_2、G_1、G_2的含量。同时对免疫亲和柱洗脱条件、流动相的洗脱程序进行了优化。结果在0.5~10 ng/mL(AFT B_1,G_1)和0.15~3.0 ng/mL(AFT B_2,G_2)线性范围内,所得回归方程的相关系数均大于0.999。黄曲霉毒素B_1、G_1方法检出限为0.15 ng/g,黄曲霉毒素B_2、G_2方法检出限为0.05 ng/g,加标回收率为89.5%~107%,精密度为1.4%~7.2%。采集粮谷类样品222件,其中有5件样品检出AFT,但均未超过国家限值标准。结论该方法灵敏度和准确度较高,可适用于粮谷类食品中黄曲霉毒素的检测。     

Survey of breakfast and infant cereals for aflatoxins B1, B2, G1 and G2.

Three hundred and forty-nine breakfast and infant cereal samples were collected at retail level across Canada from 2002 to 2005. They included rice-, soy-, barley-based and mixed-grain infant cereals, corn-, wheat-, rice-based and mixed-grain breakfast cereals, and were analysed for aflatoxins B1, B2, G1 and G2 using a modified AOAC International official method. An immunoaffinity column was used for the cleanup and purification of extracts. Determination of aflatoxins was by LC using post-column derivatization with pyridinium hydrobromide perbromide and fluorescence detection. Results indicated that 50% of both breakfast and infant cereals had detectable levels (limit of detection = 0.002 ng g-1) of aflatoxin B1, which is the most toxic of the four toxins. The levels found varied from 0.002 to 1.00 ng g-1 for aflatoxin B1, from 0.002 to 0.14 ng g-1 for aflatoxin B2, from 0.008 to 0.27 ng g-1 for aflatoxin G1, and from 0.008 to 0.048 ng g-1 for aflatoxin G2. Only 4% of the breakfast cereals and 1% of the infant cereals had aflatoxin B1 levels exceeding 0.1 ng g-1, which is the European Union maximum limit for aflatoxin B1 in baby foods and processed cereal-based foods for infants and young children.     

Simple and high-throughput method for the multimycotoxin analysis in cereals and related foods by ultra-high performance liquid chromatography/tandem mass spectrometry

A rapid, reliable and sensitive method was developed to determine 12 mycotoxins (deoxynivalenol, aflatoxins B1, B2, G1, G2 and M1, fumonisins B1 and B2, ochratoxin A, HT-2 and T-2 toxin and zearalenone) simultaneously in maize, walnuts, biscuits and breakfast cereals. The method is based on a single extraction step using acetonitrile/water mixture (80/20 v/v) followed by ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS). The selectivity of the MS/MS detection allowed the elimination of further clean up steps. Extraction, chromatographic and detection conditions were optimised in order to increase sample throughput and sensitivity. Matrix-matched calibration was used for quantification and recoveries of the extraction process ranged from 70.0% and 108.4%, with relative standard deviations lower than 25% in all the cases, when samples were fortified at 5 and 50 μg/kg. Limits of detection ranged from 0.01 to 2.1 μg/kg and limits of quantification ranged from 0.03 to 6.30 μg/kg, which were always below the tolerance levels of mycotoxins set by European Union in the matrices evaluated. Several samples were analysed and aflatoxins B1, B2, G1, G2 and T-2 toxin were detected in one maize sample, with concentrations lower than 6.0 μg/kg and deoxynivalenol was detected in a breakfast cereal at 42.1 μg/kg.     

麦芽糖月桂酸酯的分离纯化与结构鉴定

采用薄层层析(TLC)和硅胶柱层析分离纯化了脂肪酶催化合成的麦芽糖月桂酸酯,然后用高效液相色谱(HPLC)、质谱(MS)、红外光谱(IR)和核磁共振(NMR)法鉴定了纯化组分的分子结构。TLC分离麦芽糖月桂酸酯的合理条件:点样量5μl,以氯仿/甲醇(4:1,V/V)展开15min,然后用5%硫酸乙醇溶液喷雾、120℃显色20min。硅胶柱层析分离麦芽糖月桂酸酯的适宜条件:10ml反应液上12mm×1000mm硅胶(100～200目)层析柱,流动相为氯仿/甲醇(4:1,V/V),流速18ml/h,按1管/10min收集洗出液。两种分离组分分别为6’-O-麦芽糖月桂酸酯和6,6’-O-麦芽糖月桂酸二酯。     

Application and improvement of aflatoxin analysis in foods using a multifunctional column and HPLC

In an earlier report, we developed a rapid, sensitive and clean method consisting of non-chloroform extraction, clean-up on a commercial multifunctional cartridge column and HPLC with fluorescence detection for the analyses of aflatoxins. In this report, we applied this method to analyze aflatoxins in nuts, giant corn, cereals, spice and black teas. The method was effective for macadamia nuts, walnuts, hazelnuts, brazil nuts, giant corn, rice, wheat and buckwheat, and the recoveries of aflatoxins B1, B2, G1 and G2 spiked in them at the level of 10 ng/g were 85-106%. However, in the chromatograms of spices and black tea, many background peaks were observed. Therefore, we added a purification step with an affinity column to the clean-up of these samples with the multifunctional cartridge column. After the additional purification, most of the background peaks were gone. The recoveries of aflatoxins B1, B2 and G1 spiked at the level of 10 ng/g were 71-112% except for the case of B2 in white pepper (48%). The recoveries of G2 were 49-95%.     

Survey of the natural occurrence of zearalenone in maize from northern Iran by thin-layer chromatography densitometry

During September 2000, forty samples of preharvest maize from the province of Mazandaran, north Iran, were randomly collected. Samples were analysed for zearalenone (ZEA) by a thin-layer chromatograpy (TLC) method (AOAC Official Method). ZEA was extracted with chloroform, purified through a chromatographic column containing silica gel, separated on a TLC plate and quantified by densitometry. The analytical method was validated and was adequately reliable and sensitive. The mean recovery rate of ZEA from spiked samples was 92%. The absolute amount of ZEA standard detectable on a TLC plate was 20 ng, giving a limit of detection (LOD) of 100 ng g^-1. In some samples, it was shown that aflatoxins interfere with ZEA. Therefore, to remove this interference, the TLC mobile phase was changed. Data revealed that three of 40 (7.5%) maize samples contained ZEA in the range 100-212 ng g^-1, with a mean of 141 ±51 ng g^-1. This study, which is the first report of ZEA occurrence in Iranian maize, showed that the ZEA level in maize of Mazandaran province was lower than maximum limit for this mycotoxin in Iran.     

Clenbuterol residue analysis by HPLC-HPTLC in urine and animal tissues

A method for clenbuterol residue analysis in urine and animal tissues has been developed. The detection limits are 0.25 micrograms/l and 0.5 micrograms/kg, respectively. The recovery in urine varies from 85% to 90% and in animal tissues from 70% to 74%. The beta 2-agonist was liberated from the tissues by an enzymatic digestion, purified on Chem Elut columns using alkaline conditions and extracted with 0.01 mol/l HCl. Clenbuterol was quantified by high-performance liquid chromatography (HPLC) on a RP-8 column and a post-column reaction procedure. High-performance thin-layer chromatography (HPTLC) was performed on silica gel 60 plates and clenbuterol visualized by means of the modified Ehrlich's TLC spray reagent. Since this method is sensitive, as is HPLC, it was used to obtain a confirmation and to exclude false positive results.     

Validation of a confirmatory analytical method for the determination of aflatoxins B₁, B₂, G₁ and G₂ in foods and feed materials by HPLC with on-line photochemical derivatization and fluorescence detection

A sensitive and selective analytical method for the simultaneous separation and quantitative determination of aflatoxins B1, B2, G1 and G2 in foodstuffs and materials for feed has been validated. The method is based on high performance liquid chromatography with on-line post-column photochemical derivatization and fluorescence detection. The chromatographic separation of aflatoxins was accomplished using a C18 column eluted with an isocratic mobile phase consisting of water, methanol and acetonitrile. The sample preparation required a simple extraction of aflatoxins with MeOH/H2O (80:20, v/v) and a purification step by immunoaffinity column cleanup. The total analysis time, including sample preparation and chromatographic separation, did not exceed 40 min with a run time of 10 min. The on-line photochemical derivatization ensures better results in terms of simplicity, sensitivity and reproducibility with respect to chemical derivatization techniques, and provides an increase of the peak resolution and an extent of automation in comparison with the electrochemical ones. The procedure for the determination of aflatoxins in food samples and cereals for animal consumption was extensively validated following Regulation (EC) No. 882/2004. Detection limits in wheat bran samples of 0.08 μg kg1 for AFB1, 0.02 μg kg1 for AFB2, 0.16 μg kg1 for AFG1 and 0.04 μg kg1 for AFG2 were attained. The method allows high recovery with mean values ranging from 72 to 94% and it satisfies the necessary requirements for sensitivity, linearity, selectivity, precision and ruggedness, demonstrating the conformity of the method with provisions of Regulation (EC) No. 401/2006.     

A Rapid Single-Extraction Method for the Simultaneous Determination of Aflatoxins B1, B2, G1, G2, Fumonisin B1, and Zearalenone in Corn Meal by Ultra Performance Liquid Chromatography Tandem Mass Spectrometry

A single-extraction method to simultaneously determine aflatoxins (B1, B2, G1, G2), fumonisin B1, and zearalenone in corn meal by ultra performance liquid chromatography tandem mass spectrometry, using a triple quadrupole, was optimized, validated, and applied in an occurrence study. Different extraction solutions were tested, with better performance for methanol/acetonitrile/water (60:20:20, v/v/v). Linearity was observed from 0.25 to 1.50 ng/mL for aflatoxins, from 20 to 120 ng/mL for fumonisin, and from 7.00 to 42.00 ng/mL for zearalenone. Significant matrix effects were shown for all groups. Selectivity was demonstrated, as matrix or spectral interferences were not observed at the predicted retention time window of the target analytes. Average recoveries of 87.57, 93.18, 93.35, 94.20, 78.76, and 95.98% were obtained for aflatoxins (B1, B2, G1, and G2) fumonisin and zearalenone, respectively. A z-score of 0.19 was estimated in a corn certified reference material for fumonisin B1. Maximum relative standard deviation values under repeatability and intermediate precision conditions were determined to be 13.6 and 13.6% for aflatoxins, 3.7 and 6.3% for fumonisin, and 3.5 and 4.0% for zearalenone, respectively. In the occurrence study, 50 samples were analyzed and 44% had measurable levels of fumonisin. Zearalenone was detected in 18%. The proposed method showed considerable advantages, considering environmental impacts, efficiency, and reliability.     

Fungi, aflatoxins, and cyclopiazonic acid associated with peanut retailing in Botswana

Peanuts are important food commodities, but they are susceptible to fungal infestation and mycotoxin contamination. Raw peanuts were purchased from retail outlets in Botswana and examined for fungi and mycotoxin (aflatoxins and cyclopiazonic acid) contamination. Zygomycetes were the most common fungi isolated; they accounted for 41% of all the isolates and were found on 98% of the peanut samples. Among the Zygomycetes, Absidia corymbifera and Rhizopus stolonifer were the most common. Aspergillus spp. accounted for 35% of all the isolates, with Aspergillus niger being the most prevalent (20.4%). Aspergillus flavus/parasiticus were also present and accounted for 8.5% of all the isolates, with A. flavus accounting for the majority of the A. flavus/parasiticus identified. Of the 32 isolates of A. flavus screened for mycotoxin production, 11 did not produce detectable aflatoxins, 8 produced only aflatoxins B1 and B2, and 13 produced all four aflatoxins (B1, B2, G1, and G2) in varying amounts. Only 6 of the A. flavus isolates produced cyclopiazonic acid at concentrations ranging from 1 to 55 microg/kg. The one A. parasiticus isolate screened also produced all the four aflatoxins (1,200 microg/kg) but did not produce cyclopiazonic acid. When the raw peanut samples (n = 120) were analyzed for total aflatoxins, 78% contained aflatoxins at concentrations ranging from 12 to 329 microg/kg. Many of the samples (49%) contained total aflatoxins at concentrations above the 20 microg/kg limit set by the World Health Organization. Only 21% (n = 83) of the samples contained cyclopiazonic acid with concentrations ranging from 1 to 10 microg/kg. The results show that mycotoxins and toxigenic fungi are common contaminants of peanuts sold at retail in Botswana.     

液相色谱-串联质谱法同时检测花生油中4种黄曲霉毒素

建立同时测定花生油中4种黄曲霉毒素(包括黄曲霉毒素B1、B2、G1、G2)的液相色谱-串联质谱法(liquid chromatography-tandem mass spectrometry,LC-MS/MS)。该方法经甲醇-水、三氯甲烷依次提取样品,以C18柱分离,流动相为乙腈-0.1%甲酸溶液(梯度洗脱),采用电喷雾正离子多反应监测(multiple reaction monitor,MRM)模式检测。结果表明：标准曲线在0.0～20.0μg/kg进样范围内线性良好;用本法测定4种黄曲霉毒素的回收率在70.8%～108.0%(低加标水平)、82.4%～104.5%(中和高加标水平)之间,相对标准偏差在5.7%～9.7%之间。运用所建立的方法对市售19种不同品牌和批次的花生油中的黄曲霉毒素进行分析,结果显示该方法选择性强、灵敏度高,适用于花生油中4种黄曲霉毒素的定性、定量测定。     

A simple quantitative HPLC method for determination of aflatoxins in cereals and animal feedstuffs using gel permeation chromatography clean-up

A simple and sensitive procedure is described for the analysis of aflatoxins B1, B2, G1 and G2 in cereal and animal feedstuff samples. Aflatoxins are extracted with dichloromethane:water (10:1). Gel permeation chromatography (GPC) using a column packed with Bio-beads S-X3 and dichloromethane:hexane (3:1) as the eluent is used for clean-up of extracts prior to separation and quantification of aflatoxins by HPLC. The eluent fraction containing the aflatoxins is concentrated by evaporation under reduced pressure and the aflatoxins separated by reverse phase HPLC on an ODS column. Quantitative results have been obtained at levels down to 1 microgram/kg, and average recoveries from samples of wheat, maize and compound feed spiked at levels of 10 and 40 micrograms/kg were greater than 80%, 70% and 65% respectively.     

薄层色谱原位富集显微拉曼检测荧光增白剂

利用薄层色谱(TLC)原位富集与显微拉曼光谱(micro-Raman)联用的方法对食品包装材料中荧光增白剂71(C.I.71)和113(C.I.113)进行检测。采用硅胶60F₂₅₄铝板为固定相,甲醇-乙酸乙酯-冰醋酸-水(0.6︰3.4︰0.5︰0.5)混合溶液为展开剂,在UV₂₅₄nm和UV₃₆₅nm下检测定位,用乙醇对薄层板上的目标斑点进行原位富集,在波长532nm条件下进行显微拉曼光谱micro-Raman检测,并对专属性、灵敏度进行考察。结果显示,C.I.71和C.I.113检测限分别为1.25 mg/kg和3.75 mg/kg,该法检测的7批食品包装材料一次性纸杯中,有2批含有C.I.113。所建立的方法专属性强、灵敏度高,可为食品包装材料中荧光增白剂的检测提供备选方法。