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。     

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。     

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%.     

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。     

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

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 methylene dichloride solution is cleaned up on a polypropylene column, filled with 0.5 g silica gel 60. The aflatoxins are eluted with chloroform-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 micrograms/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.     

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.     

Aflatoxins in spices marketed in Portugal

Seventy-nine prepackaged samples of 12 different types of spice powders (five cardamom, five cayenne pepper, eight chilli, five cloves, seven cumin, five curry powder, five ginger, five mustard, 10 nutmeg, 12 paprika, five saffron and seven white pepper) were selected from supermarkets and ethnic shops in Lisbon (Portugal) for estimation of aflatoxins by immmunoaffinity column clean-up followed by HPLC. Aflatoxin B (AFB) was detected in 34 samples of prepackaged spices (43.0%). All of the cayenne pepper samples were contaminated with levels ranging from 2 to 32 µ g AFB / kg. Three nutmeg samples contained levels ranging from 1 to 5 µ g/kg, three samples had levels ranging from 6 to 20 µ g/kg, and there were two with 54 µ g/kg and 58 µ g/ kg. Paprika contained levels of aflatoxin B₁ ranging from 1 to 20 µ g/kg. Chilli, cumin, curry powder, saffron and white pepper samples had levels ranging from 1 to 5 µ g/kg. Aflatoxins were not detected in cardamom, cloves, ginger and mustard. None of the samples analysed contained aflatoxins B₂, G₁ and G₂.     

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.     

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.     

Occurrence of certain mycotoxins in corn and corn-based products and thermostability of fumonisin B1 during processing

A total of 57 samples of corn and corn-based products collected from various districts of Egypt were analyzed for Fusarium mycotoxins (T-2, diacetoxyscripenol (DAS), deoxynivalenol (DON) and fumonisin B1 (FB1) and aflatoxins. FB1 was detected in about 80%, 53.85%, 33.3%, and 28.57% of yellow corn, corn meal, white corn, and popcorn samples, respectively. The levels of FB1 ranged from 10 to 780 microg/kg. T-2 and DAS were detected in 5% and 10% of yellow corn samples, respectively, and DON was detected in white corn and popcorn samples at levels of 28.8 and 10.1 microg/kg, respectively. Starch samples were found to be free from Fusarium mycotoxins. Baking balady bread at 450 degrees C/min reduced FB1 to 72.4% while baking Franco bread at 250 degrees C/20 min reduced FB1 to 57.4%. Boiling of macaroni and corn in water completely removed FB1 from contaminated samples. On the other side, corn flakes samples were found to be contaminated with aflatoxins B1 and G1 at levels ranging from 6 to 10 ppm, whereas 2.9% of samples were contaminated with aflatoxin B1 > 35 ppm and G1 > 16 ppm.     

A survey of fumonisins, deoxynivalenol, zearalenone and aflatoxins contamination in corn-based food products in Argentina.

The presence of mycotoxins in corn-based foods available in Argentina was determined in order to make a preliminary exposure assessment. Thirty-eight samples [corn meal ('polenta') and corn flakes] of different local brands were analysed for zearalenone, deoxynivalenol and aflatoxins by TLC and fumonisins (FB1, FB2 and FB3) by HPLC. None of the 38 samples contained any detectable amount of aflatoxins (< 2 micrograms/kg), zearalenone (< 50 micrograms/kg) and deoxynivalenol (< 50 micrograms/kg). By contrast fumonisin contamination was found in 95% of the samples. The highest fumonisin levels were found in corn meal: FB1 (range positives: 60-2860 micrograms/kg; mean positive value: 556 micrograms/kg), FB2 (61-1090 micrograms/kg; 232 micrograms/kg) and FB3 (18-1015 micrograms/kg; 150 micrograms/kg). Low levels of fumonisin B1 were detected in 16/17 corn flakes samples (2-38 micrograms/kg). Total fumonisin levels in corn meal were more than 1000 micrograms/kg in 24% (5/21) of the samples. Although it is not the staple food in Argentina, maize consumption is very important, especially among children. A daily fumonisin intake of 11.3 micrograms/kg of body weight was estimated for child consumers (1-5 years old) based on an average consumption of 200 g of corn meal/day. Calculated at an average rate for all children (consumers or not) the intake estimate was 0.9 microgram/kg of body weight.     

Aflatoxins contamination in spices and processed spice products commercialized in Korea

A survey for total aflatoxins (aflatoxins B₁, B₂, G₁, and G₂) was conducted on 88 spices and processed spice products commercialized in Korea. The presence of aflatoxins was determined by high-performance liquid chromatography (HPLC) with fluorescence detector using immunoaffinity column clean-up. Total aflatoxins (AFs) are detected in 12 samples (13.6% of incidence) including seven red pepper powder, two red pepper pastes (Kochujang), two curry and one ginger product. The contamination levels are 0.08–4.45 μg/kg as aflatoxin B₁ and 0.08–4.66 μg/kg as AFs. The liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis on contaminated samples was conducted for the confirmation of detected aflatoxins. The 12 samples which showed aflatoxins by HPLC/FLD were confirmed as aflatoxins by LC–MS/MS.     

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.     

Aflatoxins in Turkish dried figs intended for export to the European Union

Dried figs for export from Turkey from crop years 2003 through 2006 were tested for aflatoxin B1 and total aflatoxins. For export to the European Union, consignments of 0.5 to 10 tonnes of dried figs were sampled according to European Commission regulations, and high-pressure liquid chromatography (HPLC) was used to determine concentrations of aflatoxins Bl, B2, G1, and G2. For each consignment of dried figs, a 30-kg sample (comprising 100 subsamples) was divided into three 10-kg subsamples, which were separately blended and analyzed with HPLC. This monitoring effort was conducted for figs from 2003, 2004, 2005, and up to June 2006, for a total of 10,396 30-kg samples (28,489 analyses). The incidence of contamination with aflatoxin B1 at higher than 2 ng/g was on average 0.6, 2.0, 4.0, and 2.4% for 2003, 2004, 2005, and up to June 2006, respectively, whereas contamination with total aflatoxins at higher than 4 ng/g was 2.6, 3.0, 5.1, and 2.7%. There was significant variability in contamination between replicate 1-kg samples, indicating small numbers of individual contaminated figs were probably responsible. There were also substantial differences in the relative proportions of aflatoxins B1, B2, G1, and G2 among samples, suggesting different contributing fungal sources.     

A sensitive analytical method for six aflatoxins in rainbow trout muscle and liver

A highly sensitive analysis method for six aflatoxins (aflatoxin B?, B?, G?, G?, M? and aflatoxicol) in rainbow trout muscle and liver was developed. Aflatoxins (AFs) were extracted with acetonitrile-water (9 : 1), purified on an immunoaffinity column, and subjected to HPLC with fluorescence detection after post-column photochemical derivatization. The recoveries of AFs at 0.05 μg/kg spiking levels were 71.4-82.4% in muscle and 80.1-93.0% in liver, and the repeatability relative standard deviations (RSDr) were 0.87-4.6% in muscle and 2.0-6.2% in liver. Limits of quantitation (LOQs) and limits of detection(LODs)of AFs were estimated to be 0.004-0.029 μg/kg, and 0.002-0.012 μg/kg, respectively.     

OCCURRENCE OF AFLATOXINS IN VARIOUS NUTS COMMERCIALIZED IN TURKEY

Nuts are important agricultural commodities in Turkey as they are exported and largely consumed domestically. Two hundred and seventeen samples of hazelnuts, pistachio nuts and peanuts were randomly collected from public markets, bazaars, supermarkets and retail stores in several regions of Turkey and analyzed for the incidence of aflatoxin (B1, B2, G1, G2) contamination by high-performance liquid chromatography. The levels of aflatoxin B1 (AFB1) and the total aflatoxins in the majority of samples analyzed (87.09%) were so low that they were not quantifiable. Thirty-one samples (14.28%) were found to have low levels of aflatoxins, below the Turkish National regulatory limits of 5 µg/kg for AFB1 and 10 µg/kg for total aflatoxins. However, four samples (1.84%) showed a level of contamination that exceeded the maximum tolerated levels set in the Turkish regulations. The highest value of AFB1 was 36.81 µg/kg in pistachio nuts. This article reports the data of the first survey on the presence of aflatoxins in nuts sampled in three distinct regions of Turkey.

PRACTICAL APPLICATIONS

The formation of aflatoxins depends not only on the genetic potential of mold strains but also on environmental factors, especially during post-harvest transportation and storage. Although further national surveys must be performed on a regular base, the results of the present study indicate a reduced level of aflatoxin contamination of nut-based products compared to earlier observations. The results conclude that implementation of stricter quality control measures, technical assistance to private sector actors and regulatory initiatives to support employment of these strategies undertaken in recent years by the National Authorities have paid back.     

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)。     

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.     

Co-occurrence of aflatoxins B 1 , B 2 , G 1 , G 2 , zearalenone and fumonisin B 1 in Brazilian corn

Two hundred and fourteen unprocessed corn samples (1997-98 harvest), collected at wholesale markets in different regions in Brazil, were surveyed for the occurrence of mycotoxins. The samples were analysed for aflatoxins B 1 , B 2 , G 1 , G 2 , zearalenone and fumoni1sin B 1 using in-house validated methods. The occurrence of aflatoxin B 1 , zearalenone and fumonisin B 1 was found in 38.3, 30.4 and 99.1% of the samples, respectively. Aflatoxin B 1 , zearalenone and fumonisin B 1 contamination levels varied from 0.2 to 129, 36.8 to 719, and 200 to 6100 μg/kg, respectively. The cooccurrence of the two carcinogenic mycotoxins aflatoxin B 1 and fumonisin B 1 was observed in 100% of the aflatoxin-contaminated samples (82 samples). Cooccurrences of aflatoxin B 1 : zearalenone: fumonisin B 1 and aflatoxin B 1 : aflatoxin B 2 : fumonisin B 1 were found in 18 and 43 samples, respectively.     

Determination of aflatoxins in animal feeds by HPLC with multifunctional column clean-up

A reversed-phase HPLC method with fluorescence detection for the determination of the aflatoxins B1, B2, G1 and G2 in 42 animal feeds, comprising corn (16), soya bean meal (8), mixed meal (13), sunflower, wheat, canola, palm kernel, copra meals (1 each) was carried out. The samples were first extracted using acetonitrile:water (9:1), and was further cleaned-up using a multifunctional column. Optimum conditions for the extraction and chromatographic separation were investigated. By adopting an isocratic chromatographic system using a mobile phase comprising acetonitrile:methanol:water (8:27:65, v/v/v), the separation of the four aflatoxins was possible within 30 min. Recoveries for aflatoxins B1, B2, G1 and G2 were 98 ± 0.7%, 95 ± 1.0%, 94 ± 3.6% and 97 ± 4.3%, respectively. The results show that eight samples (19%) were contaminated with aflatoxins, ranging from 6.5 to 101.9 ng g^?1. Total aflatoxin levels in three samples exceed the legal limits of many countries of 20 ng g^?1.