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.     

Detection and determination of aflatoxins using affinity chromatography

Aflatoxins B1, B2, G1 and G2 can be easily and rapidly detected in aqueous solutions using an affinity chromatography column coupled to a monoclonal antibody specific for the toxin molecules. Water: methanol extracts of food uncontaminated with aflatoxins were spiked with aflatoxins, diluted with water, and passed through the affinity matrix. The monoclonal antibody is bound to the aflatoxin, which can then be released by using a small volume of methanol. This results in both concentration and separation of the aflatoxin present in solution. As little as 5 ng of aflatoxin can be visualized in the methanol eluate if passed over a small florisil tip under ultraviolet light, while 0.5 ng can be detected in the methanol eluate if analysed by high performance liquid chromatography (HPLC). Thus, this system can be used to test for aflatoxins in contaminated samples by spot testing (> 5 ng) or as a means of HPLC clean-up for quantitative analysis at subnanogram levels. The advantages of this immunological assay in relation to other immunoassays and traditional methods are discussed.     

Aflatoxin determination in commonly consumed foods in Tunisia

BACKGROUND: To investigate natural aflatoxin occurrence, a total of 180 samples of different foods widely consumed in Tunisia were analysed by an in‐house‐validated high‐performance liquid chromatography method including affinity column clean‐up and post‐column bromination techniques. RESULTS: The method used appeared to be rapid, selective and reproducible, and its performances were established. Detection limits were 0.05 ng g^?1 for aflatoxin B1 and 0.025 ng g^?1 for aflatoxins B2, G1 and G2. Aflatoxins were detected in all investigated commodities except rice, with an overall contamination frequency of 34.4% and concentrations ranging from 0.1 to 40.6 ng g^?1. Aflatoxin B1 was found in all contaminated samples. Sorghum, spices and nuts were most contaminated. CONCLUSION: This study has provided an effective analytical method for the reliable determination of aflatoxins in food samples. Over one‐third of the samples investigated were contaminated with aflatoxins. Sorghum, spices and nuts were most contaminated, whereas rice showed no contamination. Copyright © 2010 Society of Chemical Industry     

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。     

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.     

花生油和玉米油中多组分真菌毒素高效液相色谱-串联质谱检测方法的建立

建立花生油和玉米油中黄曲霉毒素、玉米赤霉烯酮、单端孢霉烯族化合物等13种真菌毒素的高效液相色谱-串联质谱检测方法。样品经乙腈-水提取、Multi Sep 226多功能净化柱净化后,分别用电喷雾正离子模式和负离子模式检测。经优化,正离子模式流动相为含2 mmol/L甲酸铵的0.1%甲酸-甲醇溶液,负离子模式流动相为0.1%氨水-乙腈溶液。该方法对花生油和玉米油中13种真菌毒素的检出限为0.05~3.00μg/kg,定量限为0.10~10.00μg/kg,平均加标回收率为66.6%~114.9%。所建方法操作简单、灵敏度高、重复性好,可用于花生油和玉米油中多组分真菌毒素的协同测定。     

Determination of acrylamide in foods by GC/MS using 13C-labeled acrylamide as an internal standard

A method was developed for the determination of trace amounts of acrylamide (AA) in foods. The method includes the addition of 13C-labeled acrylamide-1-13C (AA-1-13C) as an internal standard, extraction with water, bromination, clean-up with a Florisil cartridge column, dehydrobromination and GC/MS analysis in the selected ion monitoring (SIM) mode. Bromination of AA to 2,3-dibromopropionamide (2,3-DBPA) was done using potassium bromide and potassium bromate under an acidic condition. 2,3-DBPA was converted to 2-bromopropenamide (2-BPA) by dehydrobromination with triethylamine before GC/MS analysis. The recoveries of AA from spiked potato chips, corn snack, pretzel and roasted tea were 97-105%, and their relative standard deviations were 0.8-3.9%. The detection limit of AA in foods was 9 ng/g. The method was applied to thirty-one foods purchased from retail markets. AA was found in potato chips at the level of 466-3,340 ng/g, and in other foods at the level of ND-520 ng/g.     

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。     

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.     

The effect of chemical treatment on reduction of aflatoxins and ochratoxin A in black and white pepper during washing

The effect of 18 different chemicals, which included acidic compounds (sulfuric acid, chloridric acid, phosphoric acid, benzoic acid, citric acid, acetic acid), alkaline compounds (ammonia, sodium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide), salts (acetate ammonium, sodium bisulfite, sodium hydrosulfite, sodium chloride, sodium sulfate) and oxidising agents (hydrogen peroxide, sodium hypochlorite), on the reduction of aflatoxins B(1), B(2), G(1) and G(2) and ochratoxin A (OTA) was investigated in black and white pepper. OTA and aflatoxins were determined using HPLC after immunoaffinity column clean-up. Almost all of the applied chemicals showed a significant degree of reduction on mycotoxins (p < 0.05). The lowest and highest reduction of aflatoxin B(1), which is the most dangerous aflatoxin, was 20.5% ± 2.7% using benzoic acid and 54.5% ± 2.7% using sodium hydroxide. There was no significant difference between black and white peppers (p < 0.05).     

高效液相色谱法检测粮油中黄曲霉毒素方法验证

该文报道验证同时检测粮食、植物油中黄曲霉毒素B1、B2、G1和G2的免疫亲和柱净化-柱后光化学衍生-高效液相色谱方法.样品经甲醇-水(体积比为70∶30)提取,通过免疫亲和柱富集和净化,采用Cloversil C18色谱柱(4.6mm×150 mm,粒度5μm),以甲醇:水(50∶50)溶液为流动相,柱后光化学衍生,利...     

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。     

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.     

免疫亲和柱净化-光化学柱后衍生高效液相色谱 荧光法测定粮食中黄曲霉毒素

建立了粮食中黄曲霉毒素B1、B2、G1、G2的免疫亲和柱净化-光化学柱后衍生高效液相色谱荧光检测法。样品经甲醇-水提取,免疫亲和柱净化,高效液相色谱分离,光化学柱后衍生,荧光检测器测定。结果表明,黄曲霉毒素B1、B2、G1、G2的检出限分别为0.50、0.25、0.50、0.25μg/kg,回收率为67.2%～91.7%,RSD小于10%。该方法快速、准确、灵敏度高、重现性好,能满足我国对粮食中黄曲霉毒素限量的检测要求。     

高效液相色谱-串联质谱法测定杂粮豆类中11种真菌毒素

利用高效液相色谱-串联质谱建立杂粮豆类中11种真菌毒素（包括黄曲霉毒素B1、B2、G1、G2、赭曲霉毒素A、T-2毒素、脱氧雪腐镰刀菌烯醇、伏马毒素B1、B2和B3、玉米赤霉烯酮）同时检测的方法。实验对提取溶剂、提取时间和净化方式等进行优化,建立双体系联合提取方法,首先采用甲醇-水（70∶30,V/V）体系提取,再以乙腈-水（84∶16,V/V）体系二次提取,提取时间3 min,在此基础上采用C18吸附剂进行净化处理。结果表明,11种毒素的线性范围良好,相关系数大于0.999,添加回收率为70.0%～108%,相对标准偏差为1.3%～8.6%。所建方法简单、快速、灵敏度高、重复性好,可满足杂粮豆类中11 种真菌毒素的同时检测需要。     

高效液相色谱法同时检测粮谷中的黄曲霉毒素和赭曲霉毒素

建立粮谷类食品中黄曲霉毒素(B1、B2、G1 和G2)和赭曲霉毒素A 的同时检测方法。样品经过甲醇- 水(80:20,V/V)提取,液液萃取净化和富集后,三氟乙酸衍生,采用Agilent ZORBAX SB-C18 色谱柱(4.6mm ×250mm),以乙腈和体积分数2% 冰醋酸溶液为流动相梯度洗脱,在线变换波长荧光检测。根据3 倍信噪比的峰 响应值,确定黄曲霉毒素(B1、B2、G1 和G2)和赭曲霉毒素A 检出限分别为0.06、0.03、0.18、0.05μg/kg 和0.51μg/kg,上述5 种毒素分别在质量浓度0.05～100、0.125～25.00、0.05～100、0.125～25.00μg/L 和0.05～50.00μg/L 范围内呈线性相关,相关系数r 分别为0.9998、0.9998、0.9998、0.9996 和0.9998;在玉米、大米、小麦3 类样品中加标回收率平均为71.73%～115.37%,相对标准偏差为3.00%～9.88%,方法学验证结果表明,黄曲霉毒素和赭曲霉毒素A 5 种毒素同时检测结果与现行国标的单独检测方法检测结果无显著性差异(P ＞ 0.05)。     

Improved sensitive determination method for bromate in bread

An effective clean-up procedure was developed to determine trace levels of bromate in bread by high-performance liquid chromatography with post-column flow reactor detection. Bromate was extracted from bread with deionized pure water. After centrifugation, the supernatant was filtered through a paper filter. The filtrate was filtered through a 0.2 micron nylon filter and chloride ion was removed by an IC-SP M Ag cartridge column or On-Guard Ag cartridge column. The eluate was applied to an Oasis MAX anion exchange cartridge column. The column was washed with 20% acetic acid and water. Bromate was then eluted with 0.5% sodium nitrate solution. The eluate was determined by HPLC with post column flow reactor detection. The method had a quantitation limit of 2 ng/g in bread products. Recoveries of bromate from bread ranged from 68 to 72% at a spiked bromate level of 2-10 ng/g.     

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.     

Sample preparation optimization for the simultaneous determination of mycotoxins in cereals

The efficiency of three extraction solvents and three clean-up procedures was compared for simultaneous extraction and purification of aflatoxins (AFB1, AFB2, AFG1 and AFG2), ochratoxin A (OTA), and zearalenone (ZEA) from spiked cereal samples. The best recovery rates for all mycotoxins were achieved using methanol: water (80:20) as the extraction solvent and AOZ multi-functional immunoaffinity column (IAC), as clean up method with recovery values of 61–89%, while that of Oasis HLB and MycoSep 226 were 37–67% and 44–78%, respectively. Then, five variables in the IAC clean-up conditions, including primary conditioning with phosphate buffer saline (PBS) (0–10 mL) (X1), extract load up volume (10–20 mL) (X2), washing volume with PBS (10–20 mL) (X3), and eluting solution volumes with methanol (1–3 mL) (X4) and acetic acid (0–1.5 mL) (X5), were optimized for the specific purification and enrichment of the mycotoxins. Results showed that primary conditioning and PBS washing did not have a significant effect on the recovery responses of mycotoxins. Optimized conditions were selected as 0, 15, 10, 1.3, and 1.5 mL for X1–X5, respectively. The recovery rates of AFB1, AFB2, AFG1, AFG2, OTA and ZEA were within 93–104% in spiked rice, under optimal conditions. LOD and LOQ were 0.0125 and 0.05 ng/g for AFB1 and AFG1, 0.0037 and 0.015 ng/g for AFB2 and AFG2, 0.05 and 0.2 ng/g for OTA, and 0.5 and 2 ng/g for ZEA, respectively. Extraction of spiked cereal samples with methanol: water (80:20) and clean up using AOZ IAC column in optimal condition provided recovery range of 77–104% for all targeted mycotoxins.     

A Sensitive Immunoaffinity Column-Linked Indirect Competitive ELISA for Ochratoxin A in Cereal and Oil Products Based on a New Monoclonal Antibody

A new sensitive monoclonal antibody (mAb) 1H2 against ochratoxin A (OTA) was reported herein. This mAb belonged to the immunoglobulin G1 (k chain) isotype. In the optimized indirect competitive enzyme-linked immunosorbent assay (icELISA), 1H2 showed a 50 % inhibition concentration (IC₅₀) value of 0.058 ng/mL and a detection limit (IC₁₀) of 0.001 ng/mL. The cross-reactivity of 1H2 with ochratoxin B, aflatoxins, deoxynivalenol, zearalenone, T-2 toxin, or fumonisins was below 0.3 %. Based on this mAb, an immunoaffinity column (IAC)-linked icELISA was developed for OTA detection in the cereal and oil products. The working range of the assay for solid sample was 0.36–16 μg/kg. The recoveries from spiked samples of IAC-linked icELISA ranged from 83 to 101 %. These recoveries were much higher than those of icELISA (21–78 %) and in good agreement with those obtained by using the standard high-performance liquid chromatography method (87–110 %). The results indicated that the mAb 1H2 had the values for studies of OTA in the crude agricultural products.