Use of high performance liquid chromatography for the identification and estimation of zearalenone, patulin and penicillic acid in food

A method is described for the estimation of 3 u.v.-absorbent mycotoxins in foods by using semi-preparative and analytical h.p.l.c. columns. The foodstuff is extracted using aqueous acetonitrile and fatty matter is removed from the extract by partition into 2,2,4-trimethylpentane. Mycotoxins present in the fat-free extract are then back extracted into chloroform and separated into two fractions on a silica gel column using (a) diethyl ether and (b) chloroform: methanol. Further purification on a semi-preparative alumina column is described prior to measurement of mycotoxin content on analytical h.p.l.c. columns. Recoveries of zearalenone, patulin and penicillic acid added at 2 to 4 times the detection limit to samples of meat, dairy products and cereals ranged from 50 to 100%.     

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).     

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.     

恒温平台石墨炉原子吸收光谱法分别测定食品中总铬及六价铬

三价铬及六价铬是食品中微量元素铬的两种主要存在价态。本文根据其性质及其在食品中的存在形式,研究确定了其分别定量测定的方法。采用10％四甲基氢氧化铵(简称TMAH)于60℃超声波振荡水浴中溶解样品,使铬离子完全溶提出来并不改变其原价态。取一部分样液直接利用石墨炉原子吸收光谱法测定总铬,取另一部分样液,加稀H_2SO_4将溶液pH调至3左右,定容过滤除去大分子沉淀物,然后在pH4.8～5.0下,用EDTA掩蔽三价铬,用DDTC—MIBK螯合萃取分离浓缩六价铬,最后再用石墨炉原子吸收光谱法测定有机相中的六价铬含量,从而达到分别测定六价与三价铬的目的。其中重点探讨研究了样品的前处理方法、六价铬的分离测定条件,干扰因素的消除及标准加入法的应用,并粗略讨论了仪器测定条件。研究结果表明,本方法的准确度、精密度及灵敏度均达到了食品检测的要求。其总铬及六价铬的样品最低检出浓度分别为0.01ppm和0.004ppm;总铬及六价铬的加标平均回收率分别为99.8±5.8％和98.3±6.4％。重复测定红烧赤贝及NBS—SRMl1566标准样品中总铬的结果分别是:0.333±0.017ppm,C.V.5.1％(H_2 SO_4-H_2 O_2法:0.333±0.011,C.V.3.3％);0.593±0.037ppm,C.V.6.2％(H_2SO_4-H_2O_2法:0.557±0.020ppm,C.V.4.0％,保证值为0.69±0.27ppm)。本法     

Development and in-house validation of an LC-MS/MS method for the quantification of the mycotoxins deoxynivalenol,zearalenone, T-2 and HT-2 toxin,ochratoxin A and fumonisin B1 and B2 in vegetable animal feed

Animal feed can be contaminated with various mycotoxins. To ensure animal health and safe food and feed production, the European Commission has recommended increased monitoring of the co-occurrence of deoxynivalenol, zearalenone, ochratoxin A, fumonisin B₁ and B₂, T-2 and HT-2 toxin in feed. Thus, there is a need for an analytical method that enables their simultaneous detection and quantification. This paper describes the development and in-house validation of such a method, in which the mycotoxins were extracted from spiked and naturally contaminated cereal-based compound feed, corn and wheat. The extracts were divided into two aliquots where one was diluted and then analysed directly and the other was cleaned by using MultiSep^®226 and then diluted and analysed. Separation and detection was achieved with LC-ESI-MS/MS by using a triple quadrupole instrument in the SRM mode. The precision (in terms of intra-day repeatability and inter-day reproducibility), accuracy, linearity, apparent recovery and expanded measurement uncertainty in feed, corn and wheat were evaluated. The LODs ranged from 1.0 to 72?μg/kg, and the LOQs ranged from 2.5 to 115?μg/kg. The apparent recovery was higher than 86% for all the mycotoxins, and the precision was better than that defined by the Horwitz equation for all concentrations. Proficiency test materials were analysed to assess the accuracy of the method, and the results were satisfactory for all seven mycotoxins. The method will be used to monitor the occurrence of these mycotoxins in products intended for animal feeding in Sweden.     

磁性固相萃取技术在食品真菌毒素分析中的应用

真菌毒素常见于食品和各种粮食作物中，具有致癌性、诱变性、致畸性等危害，严重威胁人类和动物的健康。由于真菌毒素在食品基质中存在种类多、浓度低、极性范围广等特点，因此建立快速高效的食品样品前处理方法对真菌毒素的痕量分析十分重要。磁性固相萃取（magnetic solid-phase extraction，MSPE）是一种基于磁相互作用的样品前处理技术，凭借简单快速、绿色安全、高效经济等特点，已广泛应用于食品样品真菌毒素分析的前处理中。本文介绍了MSPE的萃取过程和磁性纳米材料的制备和修饰，综述了以无机材料（氧化硅基和碳基）、有机材料（印迹型、有机高分子、表面活性剂和有机小分子）和其他材料（离子液体和单克隆抗体）为代表的MSPE吸附剂在食品样品真菌毒素检测中的应用，并对MSPE技术的发展趋势进行了展望。     

Application of an HPLC–MS/MS method for mycotoxin analysis in commercial baby foods

This article describes the validation of an analytical method for the detection of 21 mycotoxins in baby food. The analytical method is based on the simultaneous extraction of selected mycotoxins by matrix solid-phase dispersion (MSPD) followed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) using a hybrid triple quadrupole-linear ion trap mass spectrometer (QTRAP®). Information Dependent Acquisition (IDA), an extra confirmation tool for samples that contain the selected mycotoxins, was used. The matrix effects were evaluated, and the corrections for the matrix effects were performed using two calibration approaches: external matrix-matched calibration and internal standard calibration. Matrix-matched calibration was ultimately used for accurate quantification, and the recoveries obtained were generally higher than 70%. The analytical method was applied to the analysis of 35 samples of commercial baby foods. No sample exceeded the maximum limit (ML) fixed by the European Union for these mycotoxins in baby food. However, this survey highlighted the occurrence of mycotoxins in cereal-based infant foods.     

Review of current and future analytical methods for the determination of mycotoxins

Chemical methods of analysis for the extraction, clean-up and determination of aflatoxins from animal feedingstuffs and foods are described. The advantages and disadvantages of thin layer chromatography (t.l.c.) and high pressure liquid chromatography (h.p.l.c.) are discussed along with newer techniques such as immunoassay. Methods for other mycotoxins such as ochratoxin, patulin, and the trichothecenes are also included. Problems encountered during collaborative testing of these methods limit the reproducibility that can be achieved.     

Is ergosterol a new microbiological quality parameter in foods or not?

One of the most important spoilage factors of foods is molds. Therefore, it is important to determine the presence of mold in foods quickly because of the deterioration of aroma, flavor, appearance, and textural structure as well as the mycotoxins, which are toxic secondary metabolites of molds. Twenty-five percent of agricultural products worldwide are infected with mycotoxins directly or indirectly. With the global population increasingly rising, the need for access of safe and adequate food in the future has great importance. Quantification of ergosterol, a constituent of the membrane of molds and a precursor of vitamin D₂, is a feasible method for determination of fungal contamination in foods. The aim of this review is to discuss the possibility of using ergosterol as an indicator of mold growth in foods.     

Deoxynivalenol: Rationale for development and application of a urinary biomarker

Mycotoxins are common dietary contaminants in most regions of the world. The frequency of exposure to the various families of mycotoxins is often dependent on geographic location, national wealth and related agricultural and regulatory infrastructure, combined with diversity of diet and degree of food sufficiency. Deoxynivalenol (DON) is a Fusarium mycotoxin that frequently contaminates wheat, corn and barley in temperate regions. A number of acute poisoning incidences have been linked to DON-contaminated foods and chronic exposure to lower levels of DON has been predicted in many regions. DON is a potent animal toxin and exposure in humans may cause gastroenteritis, growth faltering and immune toxicity. An ability to conduct accurate exposure assessment at the individual level is required to fully understand the potential health consequences for humans. To date, such exposure biomarkers have been lacking for many important mycotoxins, including DON. To better assess exposure to DON at the individual level, we have developed a robust urinary assay, incorporating immunoaffinity column (IAC) enrichment and LC–MS detection. Further refinement of this urinary assay, by inclusion of ¹³C-DON as an internal standard, was then undertaken and tested within the UK. DON was frequently observed in urine and was significantly associated with cereal intake. A dietary intervention study demonstrated that avoiding wheat in the diet markedly reduced urinary levels of DON. This biomarker requires further validation but our initial data suggest it may provide a useful tool in epidemiological investigations of the potential health consequences of this common environmental toxin.     

Quantitation of type B-trichothecene mycotoxins in foods and feeds by a multiple stable isotope dilution assay

For quantitation of the trichothecene mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol and 4-acetylnivalenol (Fusarenon X), stable isotope dilution assays (SIDAs) were developed by using analogues labelled with carbon-13 as the internal standards. The SIDAs presented here are the first ones reported for type B-trichothecenes. The synthesis of labelled acetyl derivatives was accomplished by [¹³C₂]-acetylation of unlabelled trichothecenes and subsequent hydrolysis of the products. The mycotoxins were quantified in foods by LC tandem MS after cleanup on multifunctional columns. The method revealed an excellent sensitivity with low detection and quantification limits for all mycotoxins in the low μg/kg range, and good precision in inter-assay stu- dies. The analysis of a certified reference material (maize flour) resulted in a low bias of 2.1% from the certified value for DON and verified excellent trueness of the new method. To prove the suitability of SIDA, a number of cereals and cereal products were analysed and revealed DON contents ranging from 2 to 300 μg/kg. Acetylated derivatives occurred in lower contents and mainly in corn products.     

Occurrence of Aflatoxins and Ochratoxin A in Baby Foods in Portugal

Infants have a more restricted diet and they generally consume more food on a body weight basis than adults. Therefore, the significance and potential health risk of any contaminant in foods consumed by infants is increased and diligent attention must be paid to this particular area. The present study aims to determine the occurrence of aflatoxin M₁ (AFM₁), aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) in processed cereal-based foods (flours) and infant formulae (milk powder) available in the Portuguese market, both sold as conventional and organic origin. Mycotoxin determination was carried out using a method previously applied to duplicate diet samples. This method employed chloroform extraction, liquid–liquid extraction, immunoaffinity column (IAC) cleanup and HPLC analysis with fluorescence detection after post-column derivatisation. Quantification limits were 0.014, 0.004 and 0.028 μg kg⁻¹ for AFM₁, AFB₁ and OTA, respectively. These toxins could only be quantified in 12 of 27 analysed samples (15 positive results): two samples with AFM₁, two samples with AFM₁ and OTA, one sample with AFB₁ and OTA and seven samples with OTA. Positive results concerned four for AFM₁ (26%), one for AFB₁ (7%) and ten for OTA (67%). For these samples, contents ranged between 0.017–0.041 μg AFM₁ kg⁻¹, 0.034–0.212 μg OTA kg⁻¹, and one sample had a value of 0.009 μg AFB₁ kg⁻¹. Considering the presented results, we could provisionally conclude that the presence of these mycotoxins in baby foods does not constitute a public health problem. These are the first results concerning the occurrence of mycotoxins in marketed baby foods in Portugal and this is the first study using the HPLC method, proposed for duplicate diets, in baby food sample analysis.     

Mycotoxins: Current situation

Abstract

Mycotoxins are toxic secondary metabolites of fungi which affect humans and other animals. There are over 300 mycotoxins currently known, and of these only a few have been studied well. Aflatoxin B₁, the major member of aflatoxins and one of the most potent hepatocarcinogens known in nature, is the most well‐studied mycotoxin. In addition to aflatoxins, trichothecenes have been studied from the late 1970s. Natural contamination of various foods and feedstuffs with mycotoxins has been reported from all over the world. Several human and animal intoxications by mycotoxins were reported, such as “turkey's X disease,” alimentary toxic alecukia, and “yellow rain.” Because of concern for human health, many countries have set tolerances for some mycotoxins in foods and feeds, and the number of regulations concerning mycotoxins is increasing.     

Food Safety and Increasing Hazard of Mycotoxin Occurrence in Foods and Feeds

The possible hazard of mycotoxin occurrence in foods and feeds and some food-borne mycotoxicoses is reviewed. Management of the risk of mycotoxin contamination using some useful preventive measures against mycotoxin contamination of foods/feeds during pre- and post-harvesting periods is considered. The physical and chemical methods of mycotoxin decontamination of foods/feeds are briefly described. The use of various feed additives as a method for prevention of the adverse effects of mycotoxins is reviewed. The processing of various foods and feeds is considered in a view to possible mycotoxin decontamination. The necessary hygiene control and risk assessment in regard to mycotoxin contamination of foods and feeds in addition to some useful prophylactic measures are briefly described. A short reference is made concerning the most successful methods of veterinary hygiene control in order to prevent a possible entering of some mycotoxins in commercial channels with a view to human health.     

Simultaneous determination of multi-mycotoxins in palm kernel cake (PKC) using liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Palm kernel cake (PKC) is a useful source of protein and energy for livestock. Recently, it has been used as an ingredient in poultry feed. Mycotoxin contamination of PKC due to inappropriate handling during production and storage has increased public concern about economic losses and health risks for poultry and humans. This concern has accentuated the need for the evaluation of mycotoxins in PKC. Furthermore, a method for quantifying mycotoxins in PKC has so far not been established. The aims of this study were therefore (1) to develop a method for the simultaneous determination of mycotoxins in PKC and (2) to validate and verify the method. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using an electrospray ionisation interface (ESI) in both positive- and negative-ion modes was developed for the simultaneous determination of aflatoxins (AFB₁, AFB₂, AFG₁ and AFG₂), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB₁ and FB₂), T-2 and HT-2 toxin in PKC. An optimum method using a 0.2 ml min^–1 flow rate, 0.2% formic acid in aqueous phase, 10% organic phase at the beginning and 90% organic phase at the end of the gradient was achieved. The extraction of mycotoxins was performed using a solvent mixture of acetonitrile–water–formic acid (79:20:1, v/v) without further clean-up. The mean recoveries of mycotoxins in spiked PKC samples ranged from 81% to 112%. Limits of detection (LODs) and limits of quantification (LOQs) for mycotoxin standards and PKC samples ranged from 0.02 to 17.5 μg kg^?1 and from 0.06 to 58.0 μg kg^?1, respectively. Finally, the newly developed method was successfully applied to PKC samples. The results illustrated the fact that the method is efficient and accurate for the simultaneous multi-mycotoxin determination in PKC, which can be ideal for routine analysis.     

Fate of the fusarium mycotoxins,deoxynivalenol, nivalenol and zearalenone,during extrusion of wholemeal wheat grain

In the European Union, deoxynivalenol in cereals and cereal products is controlled by recent legislation with the objective of minimizing consumer exposure to this mycotoxin. Relatively few studies have examined the loss of Fusarium mycotoxins during processing and whether this is accurately reflected by the processing factors. The behaviour of deoxynivalenol, nivalenol and zearalenone during extrusion of naturally contaminated wholemeal wheat flour has been examined using pilot-scale equipment. Factors examined were temperature and moisture content. Concentrations of the three mycotoxins were little changed by extrusion although the amount of deoxynivalenol decreased at the lowest moisture content. However, this effect did not appear to be temperature-dependent, suggesting that the apparent loss is either due to binding or inability to extract the residue. Under some conditions, concentrations of the mycotoxins, particularly nivalenol, were higher after extrusion.     

Evaluation of enniatins A,A1, B,B1 and beauvericin in Portuguese cereal-based foods

Sixty-one samples of Portuguese cereal-based foods were analysed for the occurrence of emerging mycotoxins called enniatins (A, A1, B and B1) and beauvericin. Samples were extracted with a mixture of acetonitrile/water (85/15, v/v) using an Ultra-Turrax homogeniser, and mycotoxins were detected with liquid chromatography coupled to a mass spectrometer. This method was validated and adequate values of recovery (70–103%) and relative standard deviation (<15%) were obtained. Signal suppression/enhancement was studied and matrix-matched calibration used to minimise this effect, but no additional clean-up step was necessary. The mass spectrometer was operated in selected reaction monitoring (SRM) mode and with selected transitions for each compound to quantify and to qualify them. Fifty-nine per cent of samples were contaminated. The percentages of enniatins were 53%, 49%, 44% and 16% for A1, B, B1, and A, respectively, and for beauvericin it was 1.6%. For the total samples, the mean contamination was 30, 24, 15, 2.1 and 0.1?ng?g^?1 for enniatins A1, B, B1 and A, and beauvericin, respectively. The wheat-based samples showed higher levels and greater prevalence than any other cereals monitored. These results were used to estimate the daily intake of ENs from wheat-based cereal by the Portuguese population. At the same time, the usefulness of this method in the analysis of other important mycotoxins (aflatoxin B₁, ochratoxin A, deoxynivalenol, T-2 toxin, fumonisin B₁ and zearalenone) was evaluated.     

Determination of aflatoxin M1 in breast milk as a biomarker of maternal and infant exposure in Colombia

Chronic exposure to aflatoxins, and especially to aflatoxin B₁ (AFB1), causes hepatocellular carcinoma with prevalence 16–32 times higher in developing compared with developed countries. Aflatoxin M₁ (AFM1) is a monohydroxylated metabolite from AFB1 that is secreted in milk and which can be used as a biomarker of AFB1 exposure. This study aimed to determine AFM1 levels in human breast milk using immunoaffinity column clean-up with HPLC and fluorescence detection. Breast milk samples were obtained from 50 nursing mothers. Volunteers filled in a questionnaire giving their consent to analyse their samples as well as details of their socioeconomic, demographic and clinical data. The possible dietary sources of aflatoxins were assessed using a food frequency questionnaire. A total of 90% of the samples tested positive for AFM1, with a mean of 5.2 ng l^?1 and a range of 0.9–18.5 ng l^?1. The study demonstrated a high frequency of exposure of mothers and neonates to AFB1 and AFM1 in Colombia, and it points out the need to regulate and monitor continuously the presence of aflatoxins in human foods. Further research is needed in order to determine the presence of other mycotoxins in foods and in human samples as well as to devise protection strategies in a country where mycotoxins in human foods are commonly found.     

Organization of the system of control of food contamination with mycotoxins

Mycotoxins are secondary metabolites of microscopic fungi. They are classified with particularly hazardous contaminants of foods and feeds. It has been demonstrated that they are extremely dangerous for the health of man, widely spread and do considerable economic damage. The authors describe in detail the data as to the level of contamination of different foods with aflatoxins, ochertoxins, citrinin, patulin, trichotecenic mycotoxins, and zearalenone. The principal role in the prevention of human mycotoxicoses is played by the control over food contamination with mycotoxins. An organization program of the control has been developed. The principal feature of the program is the organization of the specialized scientific-practical centers responsible for the collection and summarization of the results of monitoring and for the development of prophylactic measures. The authors provide data on the maximum allowable concentrations of aflatoxins in foods validated in different countries. Review the topics concerned with the assessment of the efficacy of the control over food contamination with mycotoxins.     

Natural Occurrence,Analysis, and Prevention of Mycotoxins in Fruits and their Processed Products

Mycotoxins are small toxic chemical products formed as the secondary metabolites by fungi that readily contaminate foods with toxins in the field or after harvest. The presence of mycotoxins, such as aflatoxins, ochratoxin A, and patulin, in fruits and their processed products is of high concern for human health due to their properties to induce severe acute and chronic toxicity at low-dose levels. Currently, a broad range of detection techniques used for practical analysis and detection of a wide spectrum of mycotoxins are available. Many analytical methods have been developed for the determination of each group of these mycotoxins in different food matrices, but new methods are still required to achieve higher sensitivity and address other challenges that are posed by these mycotoxins. Effective technologies are needed to reduce or even eliminate the presence of the mycotoxins in fruits and their processed products. Preventive measures aimed at the inhibition of mycotoxin formation in fruits and their processed products are the most effective approach. Detoxification of mycotoxins by different physical, chemical, and biological methods are less effective and sometimes restricted because of concerns of safety, possible losses in nutritional quality of the treated commodities and cost implications. This article reviewed the available information on the major mycotoxins found in foods and feeds, with an emphasis of fruits and their processed products, and the analytical methods used for their determination. Based on the current knowledge, the major strategies to prevent or even eliminate the presence of the mycotoxins in fruits and their processed products were proposed.