SAPIENZA UNIVERSITÀ DI ROMA

Transcription

1 SAPIENZA UNIVERSITÀ DI ROMA Facoltà di Scienze Matematiche, Fisiche e Naturali Dipartimento di Chimica LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRIC METHODOLOGIES FOR THE DETERMINATION OF AFLATOXINS AS SENSITIVE MARKERS OF FOOD SAFETY AND QUALITY Supervisor: Prof. Aldo Laganà Ph.D thesis of: dr. Patrizia Foglia Chimica Analitica dei Sistemi Reali - XIX ciclo

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3 CONTENTS Foreword...1 INTRODUCTION AND GENERAL REMARKS...5 Mycoses and Mycotoxicoses...5 Mycotoxin...6 Toxicology and Health...15 Mycotoxin control strategies...21 Mycotoxin analysis: an Outline...25 Legislative regulations...33 AIM OF PRESENT WORK...36 Chapter 1 AFLATOXINS Introduction General information Chemistry and Biotransformation Absorption and Distribution Effects of aflatoxins on animal and human health: Aflatoxicosis Possible intervention strategies Current legislation on aflatoxins Analytical methodologies Experimental Reagents and Chemicals Cautions and Safety considerations Instrumentation...85 Extraction and Clean-up apparatus...85 LC-MS/MS apparatus Quantitation and Statistical evaluation...88 i

4 1.3. Results and Discussion Extraction and Clean-up Liquid chromatography-mass spectrometry Conclusions...95 Chapter 2 AFLATOXINS IN MAIZE Background Experimental Samples Sample preparation Recovery experiments LC-MS/MS analysis Results and Discussion Extraction and Clean-up Liquid chromatography-mass spectrometry Recovery and precision Method performances Effect of subsampling Real sample analysis Conclusions Chapter 3 AFLATOXINS IN MILK Background Experimental Samples Sample preparation Recovery experiments LC-MS/MS analysis Results and Discussion ii

5 Extraction and Clean-up Liquid chromatography-mass spectrometry Recovery and precision Method performance comparison Real sample analysis Comparison between aflatoxins M1 and B Enhanced Product Ion (EPI) scan acquisition Conclusions Chapter 4 AFLATOXINS IN CHEESE Background Experimental Samples Sample preparation Recovery experiments LC-MS/MS analysis Quantitation and statistical evaluation Results and Discussion Extraction and Clean-up Method comparison Matrix effect Recovery and precision Quantification limit Real sample analysis Conclusions APPENDIX 1: Bioterrorism APPENDIX 2: Environmental factors affecting mycotoxin production APPENDIX 3: Using aflatoxin-contaminated corn iii

6 APPENDIX 4: Liquid chromatography-mass spectrometry References List of Abbreviations List of Tables List of Figures Supporting Information iv

7 Foreword The identification of some particular components that are in food allows the recognition of reliable marks useful to assess the safety and quality level of a product. The need to verify the level of substances with suspected and/or probable dangerousness in the foodstuff creates a continuous demand of suitable analytical procedures able to determine and quantify them. As a result of several food crises in the last decade, food safety is nowadays an integral aspect of food protection. Therefore, there is a clear need for food safety experts at industrial, scientific and governmental level. The guiding principle of EU-food safety policy is based on comprehensive, integrated approach. Quality assurance and food safety are of paramount importance to all companies and organizations involved in the food production chain. This means to ensure elimination of food hazard, tracking and tracing of food products and a high level of human health and consumer protection, throughout the food production chain ( farm to table ) across all food sectors. In this way the farm to table policy covers all sectors of the food chain, including feed production, primary production, food processing, storage, transport and retail sale. A successful food policy demands the traceability of feed and food. This includes the obligation to ensure that adequate procedures to facilitate such traceability must be introduced to check feed and food and to withdraw them from market where there is a risk to consumer s health. 1

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11 Introduction INTRODUCTION AND GENERAL REMARKS Mycoses and Mycotoxicoses Fungi are major plant and insect pathogens, but they are not nearly as important as agents of disease in vertebrates, i.e., the number of medically important fungi is relatively low. Frank growth of fungi on animal hosts produces the diseases collectively called mycoses, while dietary, respiratory, dermal, and other exposures to toxic fungal metabolites produce the diseases collectively called mycotoxicoses. Mycoses are frequently acquired via inhalation of spores from an environmental reservoir or by unusual growth of a commensal species that is normally resident on human skin or the gastrointestinal tract. For many mycoses, the ordinary portal of entry is through the pulmonary tract, but direct inoculation through skin contact is not uncommon. In contrast to mycoses, mycotoxicoses are examples of poisoning by natural means and thus are analogous to the pathologies caused by exposure to pesticides or heavy metal residues. The majority of mycotoxicoses, on the other hand, results from eating contaminated foods. Skin contact with mould-infested substrates and inhalation of spore-borne toxins are also important sources of exposure [Bennett 2003]. The distinction between a mycotoxin and a mushroom poison is based not only on the size of the producing fungus, but also on human intention. Mycotoxin exposure is almost always accidental. In contrast, with the exception of the victims of a few mycologically accomplished murderers, mushroom poisons are usually ingested by amateur mushroom hunters 5

12 Introduction who have collected, cooked, and eaten what was misidentified as a delectable species [Moss 1996]. The Dictionary of the Fungi gives the widest definition, stating that a toxin is a non enzymatic metabolite of one organism which is injurious to another, and a mycotoxin is a toxin produced by a fungus, especially one affecting humans or animals [Kirk 2001]. The symptoms of a mycotoxicosis depend on the type of mycotoxin; the amount and duration of the exposure; the age, health, and sex of the exposed individual; and many poorly understood synergistic effects involving genetics, dietary status, and interactions with other toxic insults. Although the total number affected is believed to be smaller than the number afflicted with bacterial and viral infections, fungal diseases are nevertheless a serious international health problem. One of the characteristics shared by mycoses and mycotoxicoses is that neither category of illness is generally communicable from person to person [Bennett 2003]. Mycotoxin The name mycotoxin combines the Greek word for fungus mykes and the Latin word toxicum meaning poison. The definition of mycotoxins is somehow vague and it is difficult to define mycotoxin in a few words. All mycotoxins are produced as secondary metabolites by filamentous and microscopic fungi, especially by saprophytic moulds growing on foodstuffs or animal feeds, in the field and during storage [Krogh 1987, Miller 1997]. 6

13 Introduction Representing a variety of chemical families, the chemical structures of mycotoxins produced by these fungi are very diverse, as are the characteristics of the mycotoxicoses they can cause [ICMSF 1996]. In terms of structural complexity, mycotoxins vary from simple C4-compounds, to complex substances [Culvenor 1989, Steyn 1985] and range in mostly low molecular weight from about 200 to 500 Da. Modern mycotoxicology began with the discovery of the aflatoxins in the early 1960s. The term mycotoxin was coined in 1962 in the aftermath of an unusual veterinary crisis near London, England, during which approximately 100,000 turkey poults died from an acute necrosis of the liver. When this mysterious turkey X disease was linked to a peanut (groundnut) meal contaminated with secondary metabolites from Aspergillus flavus (aflatoxins), it sensitized scientists to the possibility that other occult mould metabolites might be deadly [Bennett 2003]. The following researches led to the discovery of additional mycotoxins produced by other fungi. The period between 1960 and 1975 has been termed the mycotoxin gold rush because so many scientists joined the well-funded search for these toxigenic agents. In those years it became clear that mycotoxins have been the cause of human illness and death as well. It is now well established that mycotoxicoses have been responsible for major epidemics in man and animals at least during recent historic times. The most important have been ergotism (Saint Anthony s fire), caused by the toxic sclerotia of Claviceps sp. which, contaminating rye flour, killed thousands of people in Europe in the last thousand years; alimentary toxic aleukia (ATA), caused by the consumption of Fusarium-contaminated grain, which was responsible for the death of many thousands of people in 7

14 Introduction the Russia in the 1940s; stachybotryotoxicosis, which killed tens of thousands of horses and cattle in the Russia in the 1930s; aflatoxicosis which has caused death and disease in many animals, and perhaps man as well, in England, in South and East Asia, and human primary liver cancer (PLC) in Africa and South East Asia also correlated with the ingestion of aflatoxins. Ochratoxin A (OTA) is suspected to play a role in Balkan endemic nephropathy (BEN) in ex-yugoslavia and chronic interstitial nephritis (CIN) in North Africa [Piva 2005]. Each of these diseases is now known to have been caused by growth of specific moulds which produced one or more potent toxins, usually in one specific kind of commodity or feed. Depending on the definition used, and recognizing that most fungal toxins occur in families of chemically related metabolites, some 300 to 400 compounds are now recognized as mycotoxins, of which approximately a dozen groups regularly receive attention as threats to human and animal health [Cole 1981]. Some fungi produce a single toxin only, while others may produce many toxic compounds, which may be shared across fungal genera. Nevertheless, there are mycotoxins related to a specific genus. These toxins are mainly produced by five genera of fungi: Aspergillus, Penicillium, Fusarium, Alternaria, and Claviceps. These fungi produce mycotoxins belonging to eight groups that are of relevance in food industry: aflatoxins, citrinin, fumonisins, ochratoxins, patulin and other small lactones, trichothecenes, zearalenone and ergot alkaloids (Table I.1). 8

15 Introduction Table I.1. Mycotoxins of relevance to human health [Geisen 1998]. Genus Mycotoxins Aspergillus Aflatoxins, ochratoxin A, sterigmatocystin, cyclopiazonic acid. Penicillium Patulin, ochratoxin A, citrinin, penitrems, cyclopiazonic acid. Fusarium Alternaria Claviceps Trichothecenes (T-2 toxin, deoxynivalenol, nivalenol, diacetoxyscirpenol), zearalenone (F2 toxin), fumonisins, moniliformin. Tenuazonic acid, alternariol, alternariol methylether, altertoxins. Ergot alkaloids. The major mycotoxin-producing fungi are not aggressive pathogens in plants. However, mycotoxins are produced by several genera in plants during the growing season when portals of entry are provided and environmental conditions are appropriate [CAST 2003]. 9

16 Introduction Table I.2. Mycotoxins found in foods and foodstuffs. Commodity Situation Potential mycotoxins Cereals Maize and peanuts pre-harvest fungal infection pre-harvest fungal infection deoxynivalenol, T-2 toxin, nivalenol, zearalenone, alternariol, alternariol monomethyl ether, tenuazoic acid, fumonisins aflatoxins Maize and sorghum pre-harvest fungal infection fumonisins Stored cereals, nuts, spices dump storage conditions (storage abuse) aflatoxins and ochratoxin Fruit juice mould growth on fruit patulin Dairy products Meat and eggs Oilseeds animal consumption of mould contaminated feeds animal consumption of mould contaminated feeds pre-harvest fungal infection aflatoxin M1, cyclopiazonic acid, ochratoxin, compactin, cyclonaldic acid patulin, citrinin, ochratoxin, cyclopiazonic acid, cyclopaldic acid, citromycetin, roquefortine, fumonisins tenuazonic acid, alternariol Taken from Sweeney While all mycotoxins are of fungal origin, not all toxic compounds produced by fungi are called mycotoxins. The target and the concentration of the metabolite are both important. Fungal products that are mainly toxic to bacteria (such as penicillin) are usually called antibiotics. Fungal 10

17 Introduction products that are toxic to plants are called phytotoxins by plant pathologists (confusingly, the term phytotoxin can also refer to toxins made by plants; see Graniti 1972 for a cogent discussion of the etymology of phytotoxin and its use in plant pathology). Mycotoxins are made by fungi and are toxic to vertebrates and other animal groups in low concentrations. Other low-molecular weight fungal metabolites such as ethanol that are toxic only in high concentrations are not considered mycotoxins [Bennet 1987]. Finally, although mushroom poisons are definitely fungal metabolites that can cause disease and death in humans and other us, they are rather arbitrarily excluded from discussions of mycotoxicology. Moulds (i.e., microfungi) make mycotoxins; mushrooms and other macroscopic fungi make mushroom poisons (see above: mycoses and mycotoxicoses section). It is difficult to prove that a disease is a mycotoxicosis. Moulds may be present without producing any toxin. Thus, the demonstration of mould contamination is not the same thing as the demonstration of mycotoxin contamination. In other words, the presence of a recognized toxinproducing fungus does not automatically imply the presence of the associated toxin as many factors are involved in its formation. Conversely, the absence of any visible mould does not guarantee freedom from toxins as the mould may have already died out while leaving the toxin intact. Moreover, even when mycotoxins are detected, it is not easy to show that they are the etiological agents in a given veterinary or human health problem. Toxigenic fungi are ubiquitous in nature and mycotoxin-producing mould can grow on a wide range of substrates. For agricultural commodities, the severity of crop contamination tends to vary from year to year based on 11

18 Introduction weather and other environmental factors that affect mycotoxin presence in raw and stored commodities [Bennett 2003]. Data on optimal temperature and water activity for toxin production by Aspergillus, Penicillium, and Fusarium spp. in culture are provided in Table I.3 and their interaction is shown in Figure I.1 (for major information see also Appendix 2). Table I.3. Examples of optimal conditions for mycotoxin production. Microorganism (mycotoxin) Temp ( C) Aw a Reference Aspergillus flavus, A. parasiticus (aflatoxin) Hill and others 1985 Aspergillus ochraceus (ochratoxin) Penicillium verrucosum (ochratoxin) Aspergillus carbonarius (ochratoxin) Fusarium verticillioides, F. proliferatum (fumonisin) Fusarium verticillioides, F. proliferatum (deoxynivalenol) Fusarium graminearum (zearalenone) Ramos and others Cairns and others Mitchell and others Marin and others Hope and Magan Sanchis 2004 Penicillium expansum (patulin) Sanchis 2004 a Water activity. The majority of data generated on environmental optima for mycotoxin production was obtained from cultures rather than actual field or storage environments. Taken from Murphy

19 Introduction Atmosphere Fungicides Microbial competition. ph Temperature FUNGUS MYCOTOXINS Time Substrate composition Water content Plant pathogens Mechanical damage Figure I.1. Interaction of environmental factors influencing the mycotoxin production. The presence of mycotoxins in food and feed commodities is related to climatic and other growth-related factors that influence the production of these secondary metabolites by the fungi. Thus, there are considerable differences between various regions of the world as well as year-to-year fluctuation within countries. There are also considerable differences between countries and even within countries with regard to the intake of food commodities, thus making exposure assessments and therefore risk assessments country specific [Kuiper-Goodman 1995]. In favourable environmental condition toxigenic fungi occur regularly in worldwide food supplies due to mould infestation of susceptible agricultural products, such as grains, spices, nuts, dried fruit, coffee, oil seed, vegetable and fruits [Krogh 1987, Miller 1997] in the field, during storage, or at later points. The mycotoxins are formed during growth of moulds on foods. Some mycotoxins are only present in the mould, while 13

20 Introduction most of them are excreted in foods. In liquid foods and in fruit, the diffusion of mycotoxins can be very fast, leaving no part of the product uncontaminated. In solid food the diffusion is slow living the major part of the product uncontaminated [Filtenborg 1996]. Mycotoxins may also be found in beer and wine resulting from the use of contaminated barley, other cereals and grapes in their production [Bacaloni 2005]. The estimate usually given is that one quarter of the world s crops are contaminated to some extent with mycotoxins. Their occurrence in food, beverages and feed can be caused by direct contamination of plant materials or products thereof, or by carry-over of mycotoxins and their metabolites into animal tissues, milk and eggs as the result of livestock eating contaminated feed entering in this way the human food chain. The end result is that mycotoxins are commonly found in foods and because mycotoxins are generally lipophilic (except for fumonisins), they tend to accumulate in the fat fraction of plants and animals [Bennett 2003, Hussein 2001, Zöllner 2006]. The economic consequences of mycotoxin contamination are profound. These toxins account for millions of dollars annually in losses worldwide in human health, animal health, and condemned agricultural products. Crops with large amounts of mycotoxins often have to be destroyed. Alternatively, contaminated crops are sometimes diverted into animal feed. Giving contaminated feeds to susceptible animals can lead to reduced growth rates, alteration in nutrient absorption and metabolism, effects on the endocrine and exocrine systems, suppression of the immune system, various illness, and death. Moreover, animals consuming mycotoxin-contaminated feeds can produce meat and milk that contain toxic residues and biotransformation products. Thus, aflatoxins in cattle 14

21 Introduction feed can be metabolized by cows into aflatoxin M1, which is then secreted in milk (see chapter 3). Ochratoxin in pig feed can accumulate in porcine tissues. Court actions between grain farmers, livestock owners, and feed companies can involve considerable amounts of money. Therefore, the ability to diagnose and verify mycotoxicoses is an important aspect of the mycotoxin problem [Bennett 2003]. Diagnosis of mycotoxicoses in animals is difficult as they may be similar to diseases with other causations. Some of the symptoms observed with a mycotoxicosis may be secondary, resulting from an opportunistic disease that is present because of immune suppression due to exposure to mycotoxins. Diagnosis is further complicated by a lack of research and feed analyses, nonspecific symptoms and interactions with other stress factors [Whitlow 2002]. This is even more difficult in cases where more than one mycotoxin is involved because the toxins can produce additive, and sometimes synergistic, effects in animals [CAST 2003]. Toxicology and Health Mycotoxicoses, like all toxicological syndromes, can be categorized as acute or chronic [Hussein 2001]. Acute toxicity generally has a rapid onset and an obvious toxic response, while chronic toxicity is characterized by low-dose exposure over a long time period (many toxins are present in low amounts in daily intaken food [Sforza 2006]), resulting in cancers and other generally irreversible effects [James 1985]. Accepting that it is often difficult to distinguish between acute and chronic effects, many papers on mycotoxicoses blur this basic dichotomy entirely, and it is not always easy to interpret the published data on purported health effects. Almost 15

22 Introduction certainly, the main human and veterinary health burden of mycotoxin exposure is related to chronic exposure (e.g., cancer induction, kidney toxicity, immune suppression). However, the best-known mycotoxin episodes are manifestations of acute effects (e.g., turkey X syndrome, human ergotism, stachybotryotoxicosis) [Bennett 2003]. In animals, acute diseases include liver and kidney damage, attack on the central nervous system, skin disorders and hormonal effects. Nerve toxins may cause trembling or even death. Skin disorders may be necrotic lesions or photosensitivity, while hormonal effects include abortions in cattle, swollen genitals in pigs and a variety of poorly defined disorders including vomiting in pigs, feed refusal and failure to thrive. Metabolism and defense mechanisms are important factors in understanding mycotoxin toxicity in specific species or individual animals. Specificity of such mechanisms is well demonstrated in the significant difference between ruminants and non-ruminants in handling mycotoxins. Ruminants have generally been more resistant to the adverse effects of mycotoxins. In vitro studies have shown the ability of the rumen microbiota to degrade mycotoxins. Understanding the metabolic pathways of mycotoxins in ruminants and non-ruminants could enable researchers and public health officials to gain insight on how to assess the associated risks of mycotoxin exposure in various species [Hussein 2001]. Besides causing toxicological syndromes, mycotoxins induce powerful and dissimilar biological effects. Some are carcinogenic (aflatoxins, ochratoxins, fumonisins and patulin), mutagenic (aflatoxins and sterigmatocystin), teratogenic (ochratoxins), estrogenic (zearalenone), hemorrhagic (trichothecenes), immunotoxic (aflatoxins and ochratoxins), nephrotoxic (ochratoxins), hepatotoxic (aflatoxins and phomopsins), 16

23 Introduction dermotoxic (trichothecenes) and neurotoxic (ergotoxins, penitrems, lolitrems and paxilline), whereas others display antitumoral, cytotoxic, and antimicrobial properties [Steyn 1995]. Toxins which act on the liver and kidney are especially difficult to detect and levels much lower than those producing acute effects are often carcinogenic. When eaten in minute quantities in the daily diet, they can cause cancers in experimental animals long after the time of eating. It is probable that humans can be affected the same way [ICMSF 1996]. In order to demonstrate that a disease is a mycotoxicosis, it is necessary to show a dose-response relationship between the mycotoxin and the disease. For human populations, this correlation requires epidemiological studies. Supportive evidence is provided when the characteristic symptoms of a suspected human mycotoxicosis are evoked reproducibly in animal models by exposure to the mycotoxin in question [Bennett 2003]. Exposure assessments are quite sketchy at best, given that there is no publicly accessible ongoing systematic surveillance for human mycotoxin exposures [Murphy 2006]. Advances in the statistical estimation of uncertainty make extrapolations increasingly relevant. For the risk assessment, the results of the exposure assessment (estimated probable daily intake, or PDI) are compared with the hazard assessment (estimated tolerable daily intake, or TDI) to indicate the degree of concern. The hazard assessment is usually based on the determination of a noobserved-effect-level (NOEL) in long-term toxicological studies, and the application of a safety factor. The exposure assessment is evaluated with data on the occurrence of mycotoxins in various commodities and food intake data. There are many uncertainties in both the exposure assessment and the hazard assessment, which can therefore greatly influence the 17

24 Introduction overall risk assessment, and thus the actual health risks are suggested to be somewhat less critical than estimated [Kuiper-Goodman 1995]. Due to the variation in mycotoxin content of human foods across world regions and seasons, and the continually improving toxicological data sets for mycotoxins, increasingly sophisticated models will be developed to assess human health risk from these food borne toxins. Progress in the science of risk assessment is allowing a greater level of certainty regarding risk, but toxin interactions and emerging human epidemics of various chronic and infectious diseases will continue to pose major challenges in this field [Murphy 2006]. Acute effects are generally produced by high amounts of toxins present in food or feed, so that fatal incidents are usually restricted to the less developed areas of the world [Piva 2005], or to livestock. In general, mycotoxin exposure is more likely to occur in parts of the world where poor methods of food handling and storage are common, where malnutrition is a problem, and where few regulations exist to protect exposed populations. People who have enough to eat normally avoid foods that are heavily contaminated by moulds, so it is believed that dietary exposure to acute levels of mycotoxins is rare in developed countries. However, even in developed countries, specific subgroups may be vulnerable to mycotoxin exposure: many mycotoxins survive processing into flours and meals [Bennett 2003]. In addition to the complex nature of mycotoxin contamination, there is also the possibility for multiple toxins, either from the same or from different fungal species, to be simultaneously present in the same plant. Additive effects may occur when more than one mycotoxin responsible for the same toxicity through the same mechanism of action are found 18

25 Introduction together. In addition, synergistic effects may also occur; in this case the final toxic effect observed is greater than the sum of the toxic effects of each mycotoxin [D Mello 1999, Speijers 2004]. This happens when one chemical increases the target site concentration of another, e.g., by increasing the absorption or decreasing the metabolic degradation, or if the compounds act at different stages of the same toxicity pathway. Really very little is known about the effects of long-term low-level exposure, especially with regard to co-contamination with multiple mycotoxins. This lack of data shows that the issue of combined toxicity is very complicated. Various combinations of the above compounds have been identified, indicating that their behaviour in such cases is altered. Some of these combinations are summarized in Table I.4. Table I.4. Frequently occurring combinations of mycotoxins in different plant products. Mycotoxins References Ochratoxin and Citrinin Pohland et al., 1992; Vrabcheva et al., 2000 Ochratoxin and Zearalenone Halabi et al., 1998 Ochratoxin and Penicillic acid Stoev et al., 2001 Ochratoxin and Aflatoxin B1 Sedmikova et al., 2001 Patulin and Citrinin Martins et al., 2002 Fumonisin B1 and Moniliformin Gutema et al., 2000 Aflatoxin B1, Fumosin B1, Zearalenone, Deoxynivalenol, Nivalenol Deoxynivalenol, Nivalenol, Diacetoxyscirpenol, T-2, HT-2, and other trichothecenes Sardjono et al., 1998; Eskola et al., 2001; Gonzalez et al., 1999 Eskola et al., 2001; Pronk et al., 2002 Taken from Speijers

26 Introduction In general, a lot of such studies are difficult to interpret. Probably the best approach to start with is to try to understand how mycotoxins can interfere at cellular level and thus interact with the toxicity of another mycotoxins. It is also very important to understand how mycotoxins interfere with the cellular machinery, and where in the cascade of actions, a mycotoxin can interfere with cellular functions and how exposure to multiple toxins can lead to extremely complicated biological responses within a relative simple system like a single cell. Mycotoxins with similar mode of action would be expected to have at least additive effects. Conversely, some interactions could have subtractive effects (for example, the ability of cyclopiazonic acid to prevent the lipid peroxidation induced by patulin or the ability of fungal serine palmitoyl transferase inhibitors, such as sphingofungins, to prevent the fumonisin-induced accumulation of free sphingoid bases). The theoretical consideration based on the cellular mode of action what interaction between mycotoxins can be predicted is not a final answer. The toxicokinetic behaviour, metabolism and the toxicodynamic aspects are of influence on the final outcome when man or experimental animals are exposed to a mixture of some mycotoxins. It is unknown what happens if different types of cells/tissue are involved or more organ systems are affected by the mycotoxins [Speijers 2004]. In practice, the outcome of combined exposure to mycotoxins might either quantitatively or even qualitatively be different from what would be predicted. The result can also depend on the species or the type of endpoint studied [Pohland 1992]. Therefore, further specific experiments, which are complicated and include a lot of effort, are needed. This 20

27 Introduction information on co-contamination is limited, thereby elevating the challenge faced throughout the farm-to-fork continuum. However, the impact of mycotoxins on health depends on the amount of mycotoxin consumed, the toxicity of the compound, e.g. acute or chronic effects, the body weight of the individual, the presence of other mycotoxins (synergistic effects) and other dietary effects [Kuiper- Goodman 1991]. All of the following criteria have to be satisfied to link a mycotoxin to a specific human disease: occurrence of the mycotoxin in food supplies; human exposure to the mycotoxin; correlation between exposure and incidence; reproducibility of the characteristic symptoms in experimental animals; similar mode of action in human and animal models [Hsieh 1990]. Mycotoxin control strategies Most mycotoxins are chemically stable. This makes it important to avoid the conditions that lead to mycotoxin formation as far as possible. This is difficult to achieve for the growing crop that is subject to the prevailing climate and conditions. In food manufacturing, destruction of mycotoxins by conventional food processing is difficult because they are typically highly resistant to physical and chemical treatments so they tend to survive storage and processing even when cooked to quite high temperatures [Scott 1991]. In the marketplace, mycotoxins can be a hurdle to international trade, leading to increased regulation of foods and feeds that may contain them and removal from the market of commodities not meeting regulatory limits. 21

28 Introduction A number of decontamination procedures, broadly based on physical, chemical and biochemical principles, have been investigated. However, the general consensus now prevailing is that preventive measures offer greater potential than remedial procedures [D Mello 1999]. The first line of defence against the introduction of mycotoxins is at the farm level and starts with implementation of good agricultural practices to prevent infection. Preventive strategies should be implemented from prethrough post-harvest. Pre-harvest strategies include maintenance of proper planting/growing conditions (for example, soil testing, field conditioning, crop rotation, irrigation), antifungal chemical treatments (for example, proprionic and acetic acids), and adequate insect and weed prevention. Harvesting strategies include use of functional harvesting equipment, clean and dry collection/transportation equipment, and appropriate harvesting conditions (low moisture and full maturity). Postharvest measures include use of drying as dictated by moisture content of the harvested grain, appropriate storage conditions, and use of transport vehicles that are dry and free of visible fungal growth [CAC 2003, Quillien 2002]. Unfortunately, even commodities dried to a satisfactory degree before storage can develop local pockets favourable to mycotoxin growth as a result of moisture generated by insect respiration and local condensation [Williams 2004]. Various physical means, such as aeration, cooling, hermetic storage and modified atmospheres, have been used effectively to reduce insect and fungal growth in stored grains in some countries, thereby controlling mycotoxin formation. Irradiation with gamma-rays, which is used for insect control, is unsuitable for fungal control since the doses required are 22

29 Introduction greater than those permitted for use in grains. Addition of natural products extracted from medicinal plants has been used successfully on a laboratory scale against a variety of fungi. Addition of biological control agents such as bacteria and yeasts has shown some promise. Use of an integrated approach, combining low levels of more than one control agent, may contribute to fungal control and to reducing contamination by mycotoxins [WHO 2002]. While implementation of these precautions go a long way toward reducing mycotoxin contamination of foods, they alone do not solve the problem and should be an integral part of an integrated Hazard Analysis and Critical Control Point (HACCP)-based management system [Lopez- Garcia 1999]. Inclusion of mycotoxin control in HACCP plans, an important aspect of an overall management approach, should include strategies for prevention, control, and quality from farm-to-fork. In the food industry, post-harvest control of mycotoxins has been addressed via HACCP plans, which include use of approved supplier schemes. Implementation at pre-harvest stages of the food system needs more attention. Such action provides a critical front-line defense to prevent introduction of contaminants into the food and feed supplies [FAO/IAEA 2001]. There is considerable on-going research on methods to prevent preharvest contamination of crops with emphasis on mechanisms by which the affected plants may inhibit growth of moulds or destroy mycotoxins that they produce. These approaches include developing host resistance through plant breeding and through enhancement of antifungal genes by genetic engineering, use of bio-control agents, and targeting regulatory 23

30 Introduction genes in mycotoxin development [Brown 1998, D Mello 1999, Duvick 2001, Karlovsky 1999, Munkvold 2003]. There are hybrids currently in use that limit mycotoxin production; however, the potential to reach unacceptable levels remains [Murphy 2006]. Another avenue for reducing mycotoxin levels would be to reduce insect injury to plant kernels. Insects play an important role in the proliferation of mould growth in the field and in storage. Resistance developed through the use of several Bacillus thermophilus genes in corn, wheat, and other cereal grains to minimize insect damage has led to effective reduction in Fusarium ear rot (F. verticillioides and F. proliferatum) mycotoxin levels in grain [Munkvold 2003]. As of now, none of these methods has solved the problem. Because mycotoxins are natural contaminants of foods, their formation is often unavoidable. Many efforts to address the mycotoxin problem simply involve the diversion of mycotoxin-contaminated commodities from the food supply through government screening and regulation programs [Bennett 2003]. Since it is normally impracticable to prevent the formation of mycotoxins, the food industry has established internal monitoring methods. Similarly, complete elimination of any natural toxicant from foods is an unattainable objective. Therefore, government regulatory agencies survey for the occurrence of mycotoxins in foods and feeds and establish regulatory limits. Guidelines for establishing these limits are based on epidemiological data and extrapolations from animal models, taking into account the inherent uncertainties associated with both types of analysis. 24

31 Introduction Estimations of an appropriate safe dose are usually stated as a tolerable daily intake [Kuiper-Goodman 1998, Smith 1995]. In this context the development and application of analytical methodologies play a fundamental role in assessing nature and level of mycotoxin contamination in food and feed. Mycotoxin analysis: an Outline Any analytical scheme for mycotoxins falls into five discrete stages: Sampling essential. Sample preparation essential. Extraction essential. Clean-up can be eliminated. Separation can be eliminated. Determination can be simple yes/no. The sampling stage is one of the most critical steps in any analysis and this is particularly the case with mycotoxins, where the contamination is known to be extremely heterogeneous. Fungi tend to develop in isolated pockets in commodities. This results in a very uneven distribution within a consignment of the mould as it grows and any associated mycotoxin. Hence, it is vital that a strategy is developed to ensure that a sample taken for analysis is truly representative of the whole consignment. There has been considerable work on sampling particularly for aflatoxins, and the general rule of large sample sizes made up of multiple subsamples is well recognized, although not always widely practised. There does need to be a balance between the rigour of statistical considerations 25

32 Introduction being applied to sampling and the practical constraints which often govern what can be carried out in practice [Gilbert 2000]. However, it is carried out, sampling is inevitably difficult and slow to execute, as is the subsequent grinding and mixing of the bulk sample prior to sub-sampling for analysis. The sampling stage is not optional, and is an inevitable time constraint in carrying out analysis irrespective of how fast the subsequent stages can be made. Most mycotoxins are toxic in very low concentrations so this requires sensitive and reliable methods for their detection. Sampling and analysis taken together represent an extremely demanding challenge for the analyst. Failure to achieve a satisfactory performance can lead to unacceptable consignments being accepted or satisfactory loads being unnecessarily rejected. All methods for the determination of mycotoxins require preliminary extraction of the mycotoxin from the commodity into a suitable solvent. This extraction is usually carried out in a high-speed blender or by mechanical shaking over a period of time. The extraction has to be as much as possible a selective process. Since frequently food matrices are rather complex, a non-selective extraction technique can lead to analytical interferences caused by coextracted components present in the matrix. Regardless of the extraction option which is chosen, it is likely that the extract may require at least two additional treatments prior to analysis. First, if the extract volume is large, it may require concentration in order to insure adequate mass of analyte to be present in a small volume, increasing in this way the sensitivity. Second, this primary extract obtained will certainly require some additional purification. Without this, both the quality of the chromatographic separation and sensitivity of the 26

33 Introduction instrumental response will suffer: interferences can produce large and tailed peaks overlapping those for the analytes, and even using specific detectors, the presence of great amounts of coextractives can saturate the detector or create false-positive peaks. The clean-up stage of the analysis essentially involves, therefore, preliminary separation of the mycotoxin from other coextracted substances, and an initial concentration step. The specificity and sensitivity of the end-measurement for the analysis will determine the extent of subsequent clean-up that is required. The development of any analytical procedure is thus a matter of balancing the relative performances of each stage in the assay. Thus, the less specific the measurement step, the more clean-up and the more chromatographic separation is required [Gilbert 2000]. In taking an overview of mycotoxin methods, it is helpful to identify three broad analytical categories: screening methods, validated official methods and research methods. Screening methods Screening methods are typified as being rapid test methods. In general they tend to be only qualitative frequently giving a yes/no answer above a predetermined threshold limit although they may be quantitative or semiquantitative. By definition, screening methods have an identified and accepted failure rate. Low false-positive rates are usually acceptable on the basis that positive results will need to be confirmed by more rigorous analytical methods. The use of screening methods which may be prone to false-negatives are however more difficult to justify. 27

34 Introduction Screening methods are relatively quick to introduce into the market-place as they do not necessarily require validation or official endorsement. In the mycotoxin area, screening methods have abounded, ranging from the low cost to more recent innovations based on antibody technology varying from various cups, test cards and dip-stick format to conventional ELISA (enzyme-linked immunosorbent assay) strips. ELISA-based kits are simple to use and allow analysis of many samples per day. However, they are generally expensive, and may suffer from cross-reactivity phenomena giving rise to false-positive results that must be confirmed by GC or LC coupled with mass spectrometry [Gilbert 2000]. Tremendous advances have been made in terms of the availability of mycotoxin screening methods, with test kits now offering the possibilities of obtaining results on a sample extract in a matter of a few minutes. There is no doubt that these kits are simple to use and analysis can be undertaken in the field without the need for recourse to a laboratory environment. The disadvantages of mycotoxin test kits can be the cost (particularly for developing countries which probably have the greatest need for this technology), the lack of attention with regard to sample extraction conditions and a tendency to disregard sampling requirements. Many test kits require use of aqueous-based extraction systems which may not be optimum for maximum extraction of the mycotoxin from the matrix of interest. There is also a potential conflict between the need to take large samples made up of a number of sub-samples for mycotoxin analysis to get representative results, which is very time consuming, and the main selling-point of test kits which is the speed of analysis. The area of largest impact of immunological based test kits has been the introduction of affinity columns. Not only can affinity columns be used in a format for 28

35 Introduction rapid testing but they can be used to replace conventional sample clean-up for mycotoxins in combination with an instrumental endpoint. The relatively high cost of affinity columns makes the prospect of multiple use attractive. There is a need for multiple use columns which will have a higher antibody loading and ease of regeneration without subsequent loss of recovery [Gilbert 2000]. The need for rapid yes/no decisions has led to a number of new screening methods. In particular, rapid and easy-to-use test kits, based on immunoanalytical principles or the generation of artificial macromolecular receptors employed in molecularly imprinted polymers (MIPs), have made good progress [Krska 2005]. Further research in mycotoxin analysis is pursued in the field of biosensors and electronic nose [Tognon 2005] and also the potential of near-infrared reflectance spectroscopic (NIRS) techniques as screening method was demonstrated [Berardo 2005]. The availability of sensitive and fast methods of analysis that can be used in situ or for decentralized tests is highly desirable. In this perspective, electrochemical methods, e. g. based on immunosensors and screenprinted electrodes, have shown important advantages compared to traditional methods currently in use, because of cost effectiveness, ease of handling and sensitivity [Alarcón 2006]. Official methods Official methods are those which have been validated by interlaboratory collaborative trial, for which performance characteristics such as repeatability (r), reproducibility (R) and limits of detection/quantification have been established [IUPAC 1995]. 29

36 Introduction A number of international organizations are involved in the validation of analytical methods, including AOAC International, the International Organization for Standardization and its European equivalent, the European Committee for Standardization, and the International Union of Pure and Applied Chemistry. Methods of analysis are accepted by these organizations only after they have been validated within their harmonized protocol for the conduct of collaborative studies [WHO 2002]. Such methods will be used as referee or reference methods particularly in the event of dispute between two parties. Official methods may originate as research methods initially, and with refinement gain the robustness to justify validation. It may not always be possible to use an official method, either because it is not suitable for a particular toxin matrix combination, because some reagents and instruments are not available, or because it is not costeffective or practical [WHO 2002]. Official methods are normally based on conventional laboratory equipment and instrumentation, although instrumentation once regarded as sophisticated (such as combined gas chromatography-mass spectrometry) is now considered as being normal for most laboratories and is beginning to feature in official methods. While many of these methods use high performance liquid chromatography (HPLC), a number are still thin layer chromatography-based and only a few fully utilize recent developments in antibody technology. A recent European project has tackled the need for official methods validated at new low European regulatory limits [Gilbert 2000]. 30

37 Introduction There is a future need for increased validation of more mycotoxin methods to cover a wider range of matrices and at the demanding regulatory limits now being set in many countries. Research Methods Research methods are those at the cutting edge of analytical science. Research methods are not limited in accessibility to instrumentation and thus sophisticated equipment may be employed. In this category, combined liquid chromatography-mass spectrometry (LC-MS) has been increasingly featured as an important tool for identification, and is also the most useful research approach for unequivocal confirmation of identification. Research methods are not formally validated and only require internal quality assurance measures to be undertaken [Gilbert 2000]. For analysing organic compounds in complex matrices, even more when they are present in a slight quantity, very sensitive and selective analytical methods are needed. These methods include thin layer chromatography (TLC), enzymatic assays, capillary gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis-mass spectrometry (CE-MS), and highperformance liquid chromatography (LC) with UV, fluorescence detection, combined with derivatization where necessary, or with MS detection [Hines 1995, Krska 2001a, Krska 2001b, Manetta 2005, Martins 2001, Sforza 2006, Stroka 2000, Trucksess 1995, Van Egmond 1991]. In the area of mycotoxin analysis, the most promising development was observed in mass spectrometry. In recent years, the initial, enthusiastic idea that atmospheric pressure ionization followed by tandem MS is a 31

38 Introduction panacea for complex analytical problems has been revised. More and more experimental evidence [Bogialli 2003a, Kebarle 1993, Zöllner 2000] proves that, especially for multicomponent analysis in complex samples, the matrix effect can weaken the ionic signal to a large, unforeseeable extent. The yield of protonation (or deprotonation) of the analytes during electrospray ionization can be decreased by competition effects due to the co-presence of matrix components. As a consequence, sensitivity decreases and, unless ideal internal standards are available, time-consuming internal calibrations are required to achieve accuracy [Bogialli 2003b]. Selective extraction methods [Matuszewski 1998], suitable chromatography [Bogialli 2003a,b], or both of them [Matuszewski 1998] could minimize analyte ion suppression. Triple-quadrupole (QqQ) MS/MS is recognized as a high sensitivity and selectivity technique for confirmation and quantitative purposes in analysis of complex matrices. The availability of sensitive detection, using ionization sources such as electrospray (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo ionization (APPI), drastically improved the possibilities of employing LC-MS in the analysis of mycotoxins. In addition, multiple reaction monitoring (MRM) with tandem mass spectrometry (MS/MS) results in enhanced performance, providing additional selectivity and increased sensitivity (based on S/N) enabling selective and accurate analyses over a wide linear range [Zöllner 1999]. For these reasons, more recently methods based on LC-MS and LC-MS/MS are rapidly spreading in routine analysis. 32

39 Introduction Legislative regulations In recent years, the general concern about the potential effects of mycotoxins on the health of humans and animals is increasing. Measures have been set up by authorities in many countries to monitor and control mycotoxin levels, especially for agricultural import products from third world countries. Various factors play a role in decision-making processes focused on setting limits for mycotoxins. These include scientific factors to assess risk (such as the availability of toxicological data), food consumption data, knowledge about the level and distribution of mycotoxins in commodities, and analytical methodology. Economic factors, such as commercial and trade interests and food security issues, also have an impact. Weighing the various factors that play a role in the decision making process to establish mycotoxin tolerances is therefore of crucial importance. Despite the difficulties, mycotoxin regulations have been established in many countries during the past decades, and newer regulations are still being issued [FAO 2004]. Maximum tolerable levels and guideline levels have been established in different food and feed products, often down to the ppb or ppt level [Hussein 2001, Van Egmond 1997, 2001/466/EC, 2002/32/EC, 2004/683/EC, 2003/2174/EC]. Such regulation have been fixed or are going to be fixed for a number of mycotoxins like aflatoxins; the trichothecenes deoxynivalenol, diacetoxyscirpenol, T-2 toxin and HT-2 toxin; the fumonisins B1, B2 and B3; agaric acid; the ergot alkaloids; ochratoxin A; patulin, phomopsins; sterigmatocystin and zearalenone [FAO 2004]. Respective levels are under debate for other mycotoxins. 33

40 Introduction Mycotoxins are regulated in more than 99 countries worldwide [FAO 2004]. The U.S. Food and Drug Administration, the European Union, the Institute of Public Health in Japan, and many other governmental agencies around the world test products for aflatoxins and other mycotoxins and have established guidelines for safe doses. However, regulations vary from country to country on the type of mycotoxin, matrix (type of food or feed) as well as the maximum allowed level [Anklam 2002]. The United States use one set of guidelines, the European Union uses another, and Japan yet another, and many other guidelines have also been developed, so there is a need for worldwide harmonization of mycotoxin regulations. Unfortunately, sometimes the regulatory community seems to be setting limits based more on current analytical capabilities than on realistic health factors [Bennett 2003]. The Food and Agriculture Organization of the United Nations has published a series of compendia summarizing worldwide regulations for mycotoxins [FAO 1997]. International legislation on foods and feeds is established by Codex Alimentarius (CAC) created in 1963 by FAO and WHO. The Codex Alimentarius system for development of legislation concerning contaminants, including mycotoxins in foods and feeds, is laid down in considerable detail [Codex Alimentarius Commission 2000]. The Codex Committee on Food Additives (CCFAC) serves as the body responsible for the risk management component of the Codex Alimentarius risk analysis process in relation to contaminants in general and mycotoxins in foods and feeds in particular. The body responsible for the risk assessment component of the Codex Alimentarius risk analysis process is JECFA. It is the role and privilege of JECFA to provide Codex Alimentarius with scientifically based assessment of the toxicity of food additives and 34

41 Introduction contaminants, such as mycotoxins, and to establish safe levels for human consumption. Moreover, the Codex General Standards for Contaminants and Toxins in Food (GSCTF) covers also feeds and raw commodities. The GSCTF contains the most important principles for laying down Codex Maximum Limits (MLs) for contaminants and toxins in foods and feeds [FAO 2000]. The General Standard, however, does not yet contain figures pertaining to the MLs for all contaminants and toxins in all food groups. Development of MLs as well as sampling plans and Codes of Practice to reduce the contamination of food by certain mycotoxins are presently in progress in the CCFAC [Codex Alimentarius Commission 2001]. In the European Union (EU), a similar process takes place. The legal basis for European Commission regulations concerning specific contaminants such as mycotoxins became available with the framework Council Regulation (EEC) No. 315/93 laying down Community procedures for contaminants in foods [(EEC) 315/93]. The EU regulations and proposals are roughly similar to the worldwide Codex legislation, but contain more details [2001/466/EC]. In EU, risk assessment is assigned to the Scientific Committee on Food (SCF) that examines the indications from Member States, and then recommends tolerable limits for food safety consume. As a result of the establishment of the European Union and the aim for harmonization of the common market within the EU, regulations concerning certain contaminants (e.g. Regulation 466/2001) were drafted. 35

42 Introduction AIM OF PRESENT WORK Throughout the world, contamination of food commodities and animal feedstuffs by certain mycotoxins is considered a serious food safety problem, so mycotoxin occurrence has become of great concern worldwide. Presumably, 25% of worldwide produced foods are contaminated by mycotoxins. This study will be focused on the major and best known mycotoxin class well-known as the cause of human disease: the aflatoxins (AFs). Many agricultural commodities are vulnerable to aflatoxins that have assumed significance due to their deleterious effects on human beings, poultry and livestock. For all these reasons, it is important to devise accurate, specific and sensitive methods for determining AFs in food and feedstuff. However, the analysis of these compounds in complex matrices at low concentration levels is not easy. The aim of this study was to develop a sensitive, accurate and reliable confirmatory procedure, based on liquid chromatography tandem mass spectrometry, for analysing aflatoxins in food samples. Indeed, though the Commission Decision 2002/657/EC [2002/657/EC] provides that Methods based only on chromatographic analysis without the use of molecular spectrometric detection are not suitable for use as confirmatory methods, an official method based on LC-MS for the aflatoxin analysis does not exist at the moment. Particular attention was paid to optimize the extraction and clean-up steps (without using still expensive immunoaffinity columns) as well as chromatographic conditions in order to enable sensitive and accurate analysis over a wide linear range. 36

43 Introduction In particular, the research had the purpose to study the aflatoxin contamination throughout the maize-milk-milk product food chain and specifically of AFB1, AFB2, AFG1 and AFG2 in maize and AFM1 in vaccine milk and in milk products. The first part of this work was focused on the development of an able and suitable multiresidual analytical methodology for the determination of aflatoxins in maize since it is a widely used animal feedstuff, for cattle especially. [ eef_cattle.pdf]. The analysis of this kind of matrix made possible to understand if and in what amount there was maize contamination by aflatoxins that may be metabolized and therefore could be found in milk and in milk derivatives. Subsequently, the AFM1 in milk have been examined. Then cheese, as a matrix strictly related, was studied. By studying these matrices, we can have a comprehensive situation of the aflatoxins risk throughout maize-milk-milk product food chain. Once the methodology was developed it was utilized to make a survey about the presence of each compound in the real samples. To make the comprehension of this research more immediate, I chose to handle the individual matrices in different chapters. However, the common parts inherent to the materials and methods, the instrumentation which has been used, the notions, the considerations, the results and discussion of general nature, are discussed in a general chapter. 37

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45 Chapter 1 AFLATOXINS 39

46

47 Chapter 1 Aflatoxins Chapter 1 AFLATOXINS 1.1. Introduction General information Aflatoxins (AFs) are a class of structurally related mycotoxins, isolated and characterized in 1960 after the turkey X disease caused the death of more than 100,000 turkey poults in Great Britain. The cause was found to be a feed containing Brazilian peanuts, which was infested heavily with Aspergillus flavus. The toxic material derived from the fungus A. flavus was given the name "aflatoxin" (A+fla+toxin) by virtue of its origin. Since then, the aflatoxins have been the most widely studied mycotoxins. Aflatoxins are a group of naturally occurring toxic metabolites produced by a polyketide pathway by many strains of Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius. Aspergillus bombycis, Aspergillus ochraceoroseus, Aspergillus nomius, and Aspergillus pseudotamari are also aflatoxin-producing species, but they are encountered less frequently [Goto 1996, Klich 2000, Peterson 2001]. A. flavus and A. parasiticus are common in most soils and are usually involved in decay of plant materials. From the mycological perspective, there are great qualitative and quantitative differences in the toxigenic abilities displayed by different strains within each aflatoxigenic species. For example, only about half of A. flavus strains produce aflatoxins [Klich 1988], while those that do may produce more than 10 6 µg/kg [Cotty 1994]. It should be mentioned that Aspergillus oryzae and Aspergillus sojae, species that are widely used in Asian food fermentations such as soy sauce, miso, and sake, are closely related to the aflatoxigenic species A. flavus and A. parasiticus. Although 41

48 Chapter 1 Aflatoxins these food fungi have never been shown to produce aflatoxin [Wei 1986], they contain homologues of several aflatoxin biosynthesis pathway genes [Klich 1995]. Deletions and other genetic defects have led to silencing of the aflatoxin pathway in both A. oryzae and A. sojae [Takahashi 2002, Weidenbörner 2001a]. There are approximately 20 aflatoxins identified, with the aflatoxins B1, B2, G1, G2, M1 and M2 as most common. Toxigenic A. flavus generally produces only aflatoxins B1 and B2, whereas A. parasiticus generally produces aflatoxins B1, B2, G1 and G2 [Davis 1983]. From these, the M aflatoxins were isolated from urine and milk and identified as mammalian metabolites of AFB1 and AFB2 [Patterson 1978]. Aflatoxins with the index number 1 show greater toxic property compared to aflatoxins with the index number 2. For aflatoxins with the index 1, there is no threshold dose below that no tumor formation would occur. Only a zero level of exposure will result in no risk. AFs are biologically active compounds that, even in small amount, have powerful hepatotoxic, immunosuppressive, mutagenic, carcinogenic, and teratogenic effects [Groopman 1988, Hussein 2001, Peraica 1999] in human and animal populations [Eaton 1994, Newberne 1969, Peers 1973, Shank 1972]. Among them, AFB1 is usually the major aflatoxin produced by toxigenic strains and is the most commonly found in food and also the best studied. It is highly toxic, in terms of both acute and chronic toxicity [Moss O.M. 2002, Sweeney 1998], and is considered as the most potent natural hepatic carcinogen known [Cullen 1993, Squire 1981]. Many substrates support growth and aflatoxin production by aflatoxigenic moulds. These compounds may contaminate a wide variety of agricultural 42

49 Chapter 1 Aflatoxins commodities and foodstuff, especially if having high carbohydrate and/or fat contents [Agag 2004, Nilüfer 2002]. Besides cereals (maize in particular), foods most commonly contaminated by aflatoxins include peanuts, pecans, almonds, hazelnuts, Brazil nuts, pistachio nuts, and walnuts [Creppy 2002, Weidenbörner 2001a and 2002], sorghum, barley, rye, wheat, peanuts, soya, rice, cottonseed and various derivative products made from these primary feedstuffs [Agag 2004], figs, oilseeds, tobacco, and a long list of other commodities is a common occurrence [Bennett 2003]. The formation of aflatoxins is influenced by physical, chemical and biological factors. The physical factors include temperature and moisture. The chemical factors include the composition of the air and the nature of the substrate. Biological factors are those associated with the host species [Agag 2004]. Like the genetic ability to make aflatoxin, contamination is highly variable. Sometimes crops become contaminated with aflatoxin in the field before or during harvest; infection is most common after the kernels have been damaged by insects, birds, mites, hail, early frost, heat and drought stress, windstorms and other unfavourable weather [Bennett 2003]. Moreover, factors that stress plants often influence aflatoxin production. Greatest stress factors include soil moisture content, temperature range and nutrient-deficient soils [Payne 1992] (see also Appendix 3). Even more problematic is the fate of crops stored under conditions that favour mould growth. In storage, usually the most important variables are the moisture content of the substrate and the relative humidity of the surroundings [Bennett 2003]. AFs are widespread in many countries, especially in tropical and subtropical regions where temperature and humidity conditions are optimal for mould growth and for production of toxins [IARC 2002, Moss 43

50 Chapter 1 Aflatoxins 2002, Rustom 1997]. Today, the climate tropicalization makes aflatoxicosis a worldwide problem of current concern and has received great attention during the last three decades. Several products, used in animal feeding, are frequently contaminated by aflatoxins. The frequent incidence of these toxins in agricultural commodities as well as in farm animals has a potentially negative impact on the health and economies of the affected regions [Rustom 1997]. The main source of human exposure to aflatoxins is contaminated food. Two pathways of the dietary exposure have been identified: (a) direct ingestion of aflatoxins (mainly AFB) in contaminated foods of plant origin such as maize and nuts and their products, (b) ingestion of aflatoxins carried over from feed into milk and milk products including cheese and powdered milk, where they appear mainly as aflatoxin M1 [Concon 1988, Moss 2002, WHO 1979]. In addition to the carry-over into milk, residues of aflatoxins may be present in the tissues of animals that consume contaminated feed [WHO 1979]. Aflatoxin residues have been found in animal tissues, eggs and poultry following the experimental ingestion of aflatoxin-contaminated feed [Agag 2004]. In developed countries, sufficient amounts of food combined with regulations that monitor aflatoxin levels in these foods protect human populations from significant aflatoxin ingestion. However, the methods used to ensure minimal contamination in developed countries cannot realistically be used in developing countries, because of the characteristics of the food systems and the technological infrastructure in those countries; therefore, aflatoxins are uncontrolled in these situations. The result is a 44

51 Chapter 1 Aflatoxins divide in the prevalence of aflatoxicosis exposure between people living in developed and developing countries [Williams 2004]. Therefore, in countries where populations are facing starvation or where regulations are either not enforced or non-existent, routine ingestion of aflatoxin may occur [Cotty 1994]. Worldwide, liver cancer incidence rates are 2 to 10 times higher in developing countries than in developed ones [Henry 1999]. Unfortunately, strict limitation of aflatoxin-contaminated food is not always an option Chemistry and Biotransformation When discovered, only two toxic components of aflatoxin were identified on thin layer chromatography plates and were named AFB and AFG due to their blue or green fluorescence under ultraviolet light, respectively. In 1963, the chemical and physical nature of the aflatoxins B1, B2, G1 and G2 were characterized. Chemically, aflatoxins are polycyclic, unsaturated and highly substituted coumarins (difuranocoumarin derivates). Their structure (Figure 1.1) consists of a bifuran ring fused to a coumarin nucleus with a pentenone ring (in B and M aflatoxins), or a six-membered lactone ring in G aflatoxins [Agag 2004]. 45

52 Chapter 1 Aflatoxins O O O O H O H O O O AFB1 H O OCH 3 H O OCH 3 AFB2 Molecular formula: C17H12O6 Formula Weight: Molecular formula: C17H14O6 Formula Weight: O O O O OH O OH O AFM1 O O AFM2 H O OCH 3 H O OCH 3 Molecular formula: C17H12O7 Formula Weight: Molecular formula: C17H14O7 Formula Weight: O O O O H O O H O O AFG1 O O AFG2 H O OCH 3 H O OCH 3 Molecular formula: C17H12O7 Formula Weight: Molecular formula: C17H14O7 Formula Weight: Figure 1.1. Structures and molecular weights of the major aflatoxins. 46

53 Chapter 1 Aflatoxins Also, an 8,9 double bond is found in the form of a vinyl ether at the terminal furan ring in AFB1 and AFG1, but not AFB2 and AFG2. However, this small difference in structure is associated with a very significant change in activity; whereby AFB1 and AFG1 are considerably more carcinogenic and toxic than AFB2 and AFG2 [Jaimez 2000]. Structurally the dihydrofuran moiety, containing double bond, and the constituents liked to the coumarin moiety are of importance both in producing biological effects and high response in fluorescence spectrometry. From a chemical point of view, the highly conjugated and rigid aflatoxin moieties give rise to native fluorescence characteristics of this kind of compounds. Moreover, it should be noticed that the small structural variations distinguishing the aflatoxins have a drastic influence on the cited fluorescence properties i.e.: G2 and B2 derivatives are far more fluorescent than their unsaturated homologues: B1 and G1. The four compounds were separated by the colour of their fluorescence under long wave ultraviolet illumination: the B aflatoxins fluoresce blue (B=blue), whereas the G aflatoxins show green fluorescence (G=green) under UV light [Agag 2004]. Two other aflatoxins M1 and M2 are distinguished as results of metabolic processes in the digestive tract of mammals fed aflatoxinated preparation and were found in milk the first time (hence the M designation) [Patterson 1978]. Other metabolites, aflatoxins B2a, G2a, aflatoxicol, aflatoxicol H1 and aflatoxins P1 and Q1 have been identified [FDA 1979]. Of the aflatoxins present in food AFB1, AFG1 and AFM1 are of primary importance and, together with aflatoxicol, present possible health concerns. Although aflatoxins B1, B2 and G1 are common in the same food sample, AFB1 predominates (60-80% of the total aflatoxin content). Generally AFB2, 47

54 Chapter 1 Aflatoxins AFG1 and AFG2 do not occur in the absence of AFB1. In most cases AFG1 is found in higher concentrations than AFB2 and AFG2 [Weidenbörner 2001a]. Aflatoxins are inhibitors of nucleic acid synthesis because they have a high affinity for nucleic acids and polynucleotides. The carcinogenicity and mutagenicity of aflatoxins B1, G1 and M1 are considered to arise as the result of the formation of a reactive epoxide at the 8,9-position of the terminal furan ring [Agag 2004]. Cytochrome P450 enzymes convert aflatoxins to the reactive 8,9-epoxide form (also referred to as aflatoxin-2,3 epoxide in the older literature), which is capable of binding to both DNA and proteins [Eaton 1994]. Aflatoxins have also been shown to decrease protein synthesis, lipid metabolism, and mitochondrial respiration. They also cause an accumulation of lipids in the liver, causing a fatty liver. This is due to impaired transport of lipids out of the liver after they are synthesized. This leads to high fecal fat content. Aflatoxins act, after bioactivation in the liver by binding of biological molecules such as essential enzymes, blockage of RNA polymerase and ribosomal translocase (inhibiting protein synthesis) and formation of DNA adducts [Angsubhakorn 1981, Hsieh 1990]. It is the formation of DNA-adducts that leads to gene mutations and cancer. One such mutation is suspected to occur in the human p53 tumor suppression gene at codon 249 [Eaton 2004]. AFB1 is metabolized by the liver through the cytochrome P450 enzyme system to the major carcinogenic metabolite AFB1-8,9- epoxide (AFBO), or to less mutagenic forms such as AFM1, Q1, or P1 [Crespi 1991, Shimada 1989]. As shown in Figure 1.2 there are several pathways that AFBO can take, one resulting in cancer, another in toxicity, and others in AFBO excretion. 48

55 Chapter 1 Aflatoxins Figure 1.2. Overview of biotransformation pathways for aflatoxin B1 (Bammler and others 2000). Mechanistically, it is known that the reactive aflatoxin epoxide binds to the N 7 position of guanines. Studies have shown that AFBO induces conversions from G (guanine) to T (thymine) at the 3rd nucleotide of the codon, making it a mutational hotspot [Bressac 1991, Hsu 1991]. This mutation has been found with greater frequency among patients with hepatocellular carcinomas [IARC 1993a,b]. A reactive glutathione S- transferase (GST) system found in the cytosol and microsomes catalyzes the conjugation of activated aflatoxins with reduced glutathione, leading 49

56 Chapter 1 Aflatoxins to the excretion of aflatoxins. Variations in the level of the glutathione transferase system as well as variations in the cytochrome P450 system are thought to contribute to the differences observed in interspecies aflatoxin susceptibility [Bennett 2003] Absorption and Distribution Because aflatoxins are very liposoluble compounds, they are readily absorbed from the site of exposure (usually the gastrointestinal tract) into blood stream. Absorption of AFs from the respiratory system has been reported in workers at feed mills, although there have been no studies to determine the quantitative importance of this route of absorption of AFs in poultry. Taking as example AFB1, when AF is ingested by animals, it is readily absorbed via the gastrointestinal tract into the portal blood and is carried to the liver where it is metabolized. In the liver cells AFB1 is converted to classes of metabolites (hydroxylated derivatives) that enter systemic circulation and are afterward eliminated through urine and bile or milk, or may be transmitted to edible animal products. There are free or unconjugated primary metabolites of AFB1, water-soluble conjugates of these metabolites, metabolites that are covalently bound to cellular macromolecules and degradation products of these AFB1 adducts [Agag 2004]. A portion of AFB1 is activated and bound to liver tissues. Some water-soluble conjugates of AFB1 metabolites are excreted into the bile and subsequently the faeces. Other water-soluble conjugates and degradation products of AFB1 macromolecule adducts and the uncojugated AFB1 50

57 Chapter 1 Aflatoxins metabolites are excreted into the general circulatory blood for systemic distribution into milk or eggs and edible tissues [Eaton 1994]. In the liver cells, AFB1 is altered by cytoplasmic reductase to form aflatoxicol and by microsomal mixed-function oxidase system to form aflatoxins M1, Q1, P1 and B1 -epoxide (the most toxic and carcinogenic derivative). All of which are less toxic than B1 and are subject to conjugation with other molecules and rapid elimination from the body [Agag 2004] Effects of aflatoxins on animal and human health: Aflatoxicosis The diseases caused by aflatoxin consumption are loosely called aflatoxicosis. Many aflatoxins exhibit acute and chronic toxicity including mutagenic, carcinogenic and teratogenic effects in a wide range of organisms [Jaimez 2000]. In animals, the aflatoxins cause liver damage, decreased milk and egg production, reduced reproductively and suppressed immunity in animals consuming low dietary concentrations. The principal target organ for aflatoxins is the liver. After the invasion of aflatoxins into the liver, lipids infiltrate hepatocytes and lead to necrosis or liver cell death. The main reason for this is that aflatoxin metabolites react negatively with different cell proteins, which lead to inhibition of carbohydrate and lipid metabolism and protein synthesis. In correlation with the decrease in liver function, there is a derangement of the blood clotting mechanism, icterus (jaundice), and a decrease in essential serum proteins synthesized by the liver. In acute toxicity the clinical signs include gastrointestinal 51

58 Chapter 1 Aflatoxins dysfunctions, decreased feed intake and efficiency, weight loss, jaundice, drop in milk production, nervous signs, bleeding, ascitis, pulmonary edema and death. All species of animals are susceptible to aflatoxicosis, but outbreaks occur mostly in pigs, sheep and cattle. Bovine species are generally less sensitive compared to non-ruminants because aflatoxins are partly degraded by the forestomach flora. Beef and dairy cattle are more susceptible to aflatoxicosis than sheep or horses. Young animals of all species are more susceptible than mature animals to the effects of aflatoxin. In poultry, beside inappetance, weight loss, decreased egg production, leg and bone problems, poor pigmentation, fatty liver, kidney dysfunction, bruising and death, suppression to natural immunity and susceptibility to parasitic, bacterial and viral infections can occur [Agag 2004]. The susceptibility of individual animals to aflatoxicosis varies considerably depending on dose, duration of exposure, species, age, sex weight, diet, exposure to infectious agents, and the presence of other mycotoxins and pharmacologically active substances [FDA 1979]. Carcinogenesis has been observed on some of above-mentioned animals; trout are the most susceptible. Animals which consume sub-lethal quantities of aflatoxin for several days or weeks develop a sub-acute (chronic) toxicity syndrome which commonly includes moderate to severe liver damage. Even with low levels of aflatoxins in the diet, there will be a decrease in growth rate, lowered milk or egg production, and immunosuppression. There is some observed carcinogenicity, mainly related to aflatoxin B1. Liver damage is apparently due to the yellow colour that is characteristic of jaundice, and the gall bladder will become swollen. Immunosuppression is due to the 52

59 Chapter 1 Aflatoxins reactivity of aflatoxins with T-cells, decrease in Vitamin K activities, and a decrease in phagocytic activity in macrophages [Agag 2004]. Thousands of studies on aflatoxin toxicity have been conducted, mostly concerning laboratory models or agriculturally important species [Cullen 1993, Eaton 1994, Newberne 1969]. Literature across all species provides clear evidence that the dose and duration of exposure to aflatoxin clearly have a major effect on the toxicology and may cause a range of consequences: 1) large doses lead to acute illness and death, usually through liver cirrhosis; 2) chronic sub-lethal doses have nutritional and immunologic consequences; and 3) all doses have a cumulative effect on the risk of cancer [Williams 2004]. Because of the differences in aflatoxin susceptibility in test animals, it has been difficult to extrapolate the possible effects of aflatoxin to humans. Aflatoxins are well recognized as a cause of liver cancer, although lung cancer is also a risk among workers handling contaminated grain; besides they have additional important toxic effects. In farm and laboratory animals, chronic exposure to aflatoxins compromises immunity and interferes with protein metabolism and multiple micronutrients that are critical to health. These effects have not been widely studied in humans, but the available information indicates that at least some of the effects observed in animals also occur in humans [Williams 2004]. Although susceptibility of humans to aflatoxins is not well known, results of epidemiological studies carried out in Africa and Asia, where there is a high incidence of hepatoma, have revealed an association between cancer incidence and the aflatoxin content of the diet [Jaimez 2000]. Clinically, the main features of acute human aflatoxicosis are edema of the legs and feet, abdominal pain and vomiting as well as liver dysfunction, 53

60 Chapter 1 Aflatoxins convulsions gastrointestinal haemorrhage, hematemesis, fever, diarrhea, and coma. Hypertension, hypoglycaemia and elevated serum transaminases are the most constant biochemical abnormalities. Fatty degeneration in the liver and kidneys, and cerebral edema are the major findings in autopsy [Agag 2004]. Acute aflatoxicosis results in death. The Reye s syndrome, with encephalopathy and fatty degeneration of the viscera is a common cause of death among children at rural areas, with the incidence increasing during the later part of the rainy season. Chronic aflatoxicosis results in cancer, immune suppression, gastrointestinal and hepatic neoplasms and other slow pathological conditions [Bennet 2003]. Also the illness known as cirrhosis of Indian childhood is partly due to an aflatoxic poisoning. The expression of aflatoxin related diseases in humans may be influenced by factors such as age (susceptibility to aflatoxin is greatest in the young, sex (according to the concentration of testosterone), nutritional status, and/or concurrent exposure to other causative agents such as viral hepatitis (HBV) or parasite infestation. In some areas where aflatoxin contamination and HBV occur together, hepatomas are the predominant cancer and they may be a predominant cause of death. Moreover, ingestion of aflatoxin, viral diseases, and hereditary factors have been suggested as possible aetiological agents of childhood cirrhosis [Rogers 1993]. Exposure to aflatoxins in the diet is considered an important risk factor for the development of primary hepatocellular carcinoma (HCC), particularly in individuals already exposed to hepatitis B. Simultaneous hepatitis B 54

61 Chapter 1 Aflatoxins and AFB1 infections commonly occur in regions with high rates of hepatocellular carcinoma (HCC)[Murphy 2006]. In classical epidemiology, several studies have linked liver cancer incidence to estimated aflatoxin consumption in the diet [Li 2001, Peers 1973, Van Rensburg 1985]. The results of these studies have not been entirely consistent, and quantification of lifetime individual exposure to aflatoxin is extremely difficult. The incidence of liver cancer varies widely from country to country, but it is one of the most common cancers in poor countries [Bennett 2003]. The presence of hepatitis B virus infection, an important risk factor for primary liver cancer, complicates many of the epidemiological studies. In molecular epidemiology, it is possible to demonstrate with more certainty the association between putative carcinogens and specific cancers. Biomonitoring of aflatoxins can be done by analysing for the presence of aflatoxin metabolites in blood, milk, and urine. Moreover, excreted DNA adducts and blood protein adducts can also be monitored. The aflatoxin B1-N 7 -guanine adduct represents the most reliable urinary biomarker for aflatoxin exposure but reflects only recent exposure. Numerous studies have shown that carcinogenic potency is highly correlated with the extent of total DNA adducts formed in vivo [Bennett 2003, Eaton 1994]. Inactivation of the p53 tumor suppressor gene may be important in the development of primary hepatocellular carcinoma. There is also considerable evidence associating aflatoxin with neoplasms in extrahepatic tissues, particularly the lungs. To recapitulate, there is no other natural product for which the data on human carcinogenicity are so compelling. Based on epidemiological evidence, the WHO-International 55

62 Chapter 1 Aflatoxins Agency for Research on Cancer (IARC) has classified AFs as potential carcinogenic agent to humans (Group 1) [IARC 2002]. Biomarkers of exposure The currently favoured method of measuring human exposure consists of the analysis of body fluids for the presence of aflatoxin derivatives. Urinary excretion not only serves as evidence that humans have the necessary biochemical pathways for carcinogenesis [IARC 1993a,b], but also provides a reliable biomarker for exposure to AFB1 [Groopman 2005]. Each biochemical process results in derivatives that have a characteristic half-life within the body, and thus the exposure can be assessed over a period of days, weeks, or months. Recent exposure to aflatoxin is reflected in the urine as directly excreted AFM1 and other detoxification products, but only a small fraction of the dose is excreted in this way. Measurements of aflatoxin and its by-products in urine have been found to be highly variable from day to day. This reflects the wide variability in the contamination of food samples, and, for this reason, the measurement of AFM1 on a single day may not be a reliable indicator of a person s chronic exposure [Groopman 1993, Makarananda 1998, Wild 1998]. The aflatoxin-albumin adduct is measured in peripheral blood and has a half-life in the body of days. Therefore, it is a measure that integrates the exposure over a longer period and hence is a more reliable indicator of a person s chronic exposure. However, it should be remembered that the fraction of the ingested aflatoxin processed into any particular metabolite is variable. A given concentration of any particular biomarker cannot be used to make assumptions about the total dose or the amounts directed into any other 56

63 Chapter 1 Aflatoxins competing pathway. The evidence of contamination in market and food samples and the human biomarker data show that, regardless of food preparation practices, the human populations of developing countries are widely and significantly exposed to aflatoxins, but usually at a level less than that needed for direct acute illness and death [Williams 2004]. The data on the temperature conditions needed for aflatoxin synthesis; the vulnerability of staple commodities to contamination; the systems for food production, storage, and marketing; and the regulation enforcement failures all indicate that there is high risk of chronic aflatoxin exposure in developing countries. Population data from the FAO database indicate that ~4.5 billion people live in this zone Possible intervention strategies The traditional approach to preventing exposure to aflatoxins has been to ensure that foods consumed have the lowest practical aflatoxin concentrations. In developed countries, this has been achieved for humans largely by regulations that have required low concentrations of the toxins in traded foods (see later). However, this approach has certain limitations and clearly has failed as a control measure for developing countries. The effective enforcement of regulations defining the concentrations of aflatoxins permitted in various foods in North America and Europe has turned aflatoxin into a problem with significant economic but minor human health consequences. To prevent the economic loss associated with failure to meet the regulations, there are three main points of leverage: production, storage, and processing [Williams 2004]. 57

64 Chapter 1 Aflatoxins Production Around 1970 it was established that contamination, or at least invasion by the causal fungi, could start in the field before harvest. Environmental conditions such as drought during the grain growth stage, insect damage in the field, variety, and soil characteristics have proven to be determining factors in pre-harvest contamination. These conditions are now sufficiently well understood to describe the risk of contamination of major crops. The result is that management can be used to minimize contamination, and the practice of inoculating the fields with nonaflatoxigenic strains of fungi may shortly be a new tool in the battle to prevent economic loss. Cultural practices are mainly preventive in nature and to be effective, cultural control of aflatoxins must take into consideration all the environmental and agronomic factors that influence pod and seed infection by A. flavus and aflatoxin production. These factors may vary considerably from one location to another, and between seasons in the same location. Growing same crop continuously on the same land may lead to build up of high populations of A. flavus. Hot and arid environments also favour the growth of A. flavus in the soil; because of the importance of drought as a factor predisposing crops to contamination, irrigation is a very important means of ensuring food quality. Moreover optimum plant populations should be established, since too high population may lead to severe drought stress where rain fall is sub optimal in a growing season. To prevent the contamination of the crop it is very important to select the cultivar, which should fit a particular growing season and mature at the end of the rainy season so that post harvest field drying can be done under 58

65 Chapter 1 Aflatoxins favourable conditions [Williams 2004]. Harvesting the crop at optimum maturity is also very important. Harvest should begin soon after the crop matures if adequate dryer capacity is available. Delaying harvest usually increases losses from field-borne diseases, insects, birds, and weather [Vincelli 1995]. More recent developments have made use of biotechnology to introduce genes that either prevent the formation of aflatoxin as a result of fungal metabolism or prevent or decrease fungal action. Several breeding lines with resistance to A. flavus colonization of seeds comparable to that of resistance sources and with greater yield potential have been bred. Use of resistant varieties could be considered as part of an integrated aflatoxin management program incorporating cultural and crop handling procedures appropriate to different agro ecological situations. Microorganisms have often been suggested as agents of control for aflatoxin contamination. The best bio competitive agent to control A. flavus in the field would be atoxigenic strains of A. flavus (spores of non-toxic A.flavus), because these strains as compared to other potential microbial bio competitive agents, would be adaptable to environmental conditions identical to the toxigenic strains and would be biologically active at same time as well. The aflatoxin contamination process is however so complex that in most cases a combination of approaches will be required to eliminate or even control the toxin problem. These approaches offer considerable long-term promise, but time and sizeable investment are still needed before the research can affect human health. In developing countries, many of these pre-harvest opportunities to minimize contamination are not exploited by producers. 59

66 Chapter 1 Aflatoxins Insect damage in the field is not controlled by pesticides or by cultural practices; drought is a common phenomenon, and most crops are produced without irrigation as an option. Harvesting is usually done without machinery, and drying is usually carried out very inefficiently and is dependent on the weather. Adverse weather at harvest results in slow and inadequate drying and brings attendant risks of contamination [Williams 2004]. Storage It is well understood that much of the contamination of commodities with aflatoxins occurs during storage. To preserve quality in storage, it is necessary to prevent biological activity through adequate drying (<10% moisture), elimination of insect activity that can increase moisture content through condensation of moisture resulting from respiration, low temperatures, and inert atmospheres. In other words, the conditions needed to prevent the development of contamination are known, but it is not always easy to produce them in storage systems in developing countries. One fact that makes storage such an important issue for these countries is the subsistence nature of most farming there. Most people in rural areas grow and store their own food. Consequently, most food is stored in small, traditional granaries, and there is little investment in the management of the conditions [Williams 2004]. Studies of grain quality in such storage structures show a steady increase in the aflatoxin content over time, which reflects the failure to maintain appropriate conditions [Hell 2000]. There are some small cautions that can be very important to prevent the contamination of the crop. For example, it is very important to clean out all harvesting, handling, and drying equipment and storage bins prior to harvest. Before storage it is necessary 60

67 Chapter 1 Aflatoxins to remove all broken kernel and foreign material, which can provide a source of contamination. Also mow around storage bins to discourage insect/rodent activity is very useful [Vincelli 1995]. Achieving and preserving the conditions that prevent contamination is likely to prove a significant challenge for small-scale (household and farm level) storage and to be beyond the resources of most, even if they could be convinced of the value of making the effort. Processing Processing of commodities can be used to reduce the aflatoxin content and thereby prevent economic loss. Three main approaches exist: dilution, decontamination, and separation. With regard to dilution, where regulations are enforced, the easiest means of satisfying the requirement is (unfortunately) to mix grain low in aflatoxin with grain exceeding the regulated limits. Thus, although the concentration is reduced, consumers are still exposed to the same overall aflatoxin burden. This approach fails when there is not enough clean grain to allow adequate dilution of the contaminated stock or when the infrastructure to hold stocks and achieve the desired mixing is lacking. With regard to decontamination, considerable effort has been expended to develop methods by which contaminated commodities may be treated to denature aflatoxins. Treatment with ammonia, alkaline substances [Phillips 1993], and ozone can denature aflatoxins, but whether this change is permanent is not clear. For instance, the processing of corn with caustic soda, as is used in traditional Mesoamerican cooking, has been shown to reduce the aflatoxin content, but there is some evidence both that the chemical change may be reversible and that, after consumption, the aflatoxins may be reformed in the acid conditions in the stomach. 61

68 Chapter 1 Aflatoxins With regard to separation, considerable success in reducing aflatoxin contamination can be achieved by separating contaminated grain from the bulk. This approach depends on the heavy contamination of only a small fraction of the seeds, so that removing those leaves a much lower overall contamination. Study of the distribution of aflatoxin in peanuts shows that a major portion (80%) of the toxins is often associated with the smaller and shrivelled seed, and thus screening can lower the overall concentration in the bulk. Further removal of aflatoxin contaminated seeds may be achieved by color sorting, which, in the case of peanuts, is most effective when the seeds are blanched. A consequence of this sorting approach to aflatoxins is the fate of the now highly concentrated aflatoxins in the grain removed from the bulk. The poorest producers and laborers often consume those nuts, which should have been discarded, or they feed them to their animals [Williams 2004]. Whereas it is highly desirable that feed is not contaminated, the reality is that much of the grain fed to animals is contaminated, even in developed countries, and this condition results in substantial losses to producers if the feed is not treated. It is also clear that, in areas where regulations are not enforced, humans are commonly exposed to aflatoxins. In developed countries where regulations allow higher aflatoxin concentrations in animals, the agricultural industries have developed alternative approaches (chemoprotection and enterosorption) to limit biologically effective exposure without the high cost of preventing contamination. Also physical treatments, such as heat, microwaves, gamma-rays, X-rays, ultra violet light, are sometimes used [Creppy 2002]. 62

69 Chapter 1 Aflatoxins Chemoprotection Chemoprotection is based on manipulating the biochemical processing of aflatoxins to ensure detoxification rather than preventing biological exposure. Chemoprotection against aflatoxins has been demonstrated with the use of a number of compounds that either increase an animal s detoxification processes or prevent the production of the epoxide that leads to chromosomal damage. One technical solution is drug therapy, because several compounds are able to decrease the biologically effective dose [Williams 2004]. Inhibition of AFBO formation (through disruption of the cytochrome P450 system) and/or adduct formation are important strategies for prevention of these damaging mutations. In animal models, metabolic detoxification of AFBO is facilitated by induction of glutathione S-transferase (GST). This enzyme catalyzes the reaction that binds glutathione to AFBO and renders it non carcinogenic. It is interesting to note that mice, which are resistant to aflatoxin carcinogenesis, have GST activity levels 3 to 5 times higher than rats, which are susceptible, and humans have lower GST activity than rats. Synthetic dithiolthione compounds, such as oltipraz (an FDA approved antischistosomal drug), and antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and ethoxyquin, are among the most effective against aflatoxin carcinogenesis [Bammler 2000, IARC 1993a,b, Kwak 2001). Chlorophyllin has also been shown to protect against human AFB1 toxicity [Kensler 2004]. It remains to be seen if long-term administration of oltipraz could lessen human risk for AFB1 63

70 Chapter 1 Aflatoxins carcinogenesis; reduction of aflatoxin intake, however, would result in reduced liver cancer rates. However, sustained long-term therapy is expensive, may have side effects, and is not likely, given the health budgets of developing countries and their other pressing health problems. For the animal feed industries, a major focus has been on developing food additives that provide protection from the toxins. One approach has been the use of esterified glucomanoses and other yeast extracts that provide chemoprotection by increasing the detoxification of aflatoxins [Williams 2004]. Enterosorption Another approach has followed the discovery that certain clay minerals can selectively adsorb aflatoxin. Enterosorption is based on the mind of adding a binding agent to food to prevent the absorption of the toxin while the food is in the digestive tract; the combined toxin-sorbent is then excreted in the faeces [Williams 2004]. While many toxins are adsorbed to surface-active compounds, such as activated charcoal, the bonding is not often effective in preventing uptake from the digestive system. Various sorbents have different affinities for aflatoxins and therefore differ in preventing the biological exposure of the animals consuming contaminated foods. There have been several claims for different adsorption agents, but their efficiency in preventing aflatoxicosis varies with the adsorbent [Phillips 1993]. With enterosorption, there is also a risk that non-specific adsorbing agents may prevent the uptake of micronutrients from the food. In vitro tests of hydrated sodium calcium aluminosilicates (HSCAS) suggest that there is little other adsorption of micronutrients. The use of HSCAS additives in 64

71 Chapter 1 Aflatoxins contaminated feeds has proven effective in preventing aflatoxicosis in turkeys, chickens, lambs, cattle, pigs, goats, rats, and mice. The use of radiolabeled aflatoxin shows that the addition of clay in a proportion of 0.5% of the volume to a contaminated feed reduced exposure in chicks by ~95%. Selected calcium montorillonites have proven to be the most highly selective and effective of these enterosorbents. This approach is now widely used in animal production industries worldwide, and HSCAS is estimated by one manufacturer to be added to 10% of all animal feeds [Phillips 1993, Williams 2004] Current legislation on aflatoxins The International Agency for Research on Cancer (IARC) identifies aflatoxins as a Group 1 human carcinogen [IARC 2002]. In order to reduce aflatoxicosis risk, most of the developed countries have regulated the maximum residue levels (MRLs) for AFs in food and control measures. The limits are highly variable, depending on the degree of development and economic involvement of the countries in setting regulatory limits. The aflatoxin regulations are often detailed and specific for various foodstuffs, for dairy products and for feedstuffs. Different countries have different legislative regulations for AFs. The European Commission has set the MRLs for AFB1 and total AFs (AFB1, AFB2, AFG1 and AFG2) in cereals, grains, groundnuts, dried fruit and other corn products ready for retail sale to 2 µg/kg and 4 µg/kg, respectively; [2003/2174/EC] whereas the current MRLs established by US Food and Drug Administration (FDA) are higher (20 µg/kg) [FDA 2004a]. In milk, 65

72 Chapter 1 Aflatoxins AFM1 maximum allowable level was set to 0.05 µg/kg by European Union [2003/2174/EC] and 0.5 µg/kg by U.S. Food and Drug Administration [FDA 2004b]; (see Table 1.1). 66

73 Table 1.1. Guidance and regulations on aflatoxins in food and feed. Food/Feed Maximum level aflatoxin (μg/kg) (B1 + B2 + B1 G1 + G2) M1 Source United States lrd/fdaact.html FOOD Foods 20 Brazil nuts, pistachio nuts, peanuts, and peanut products 20 Milk 0.5 FEED Corn and peanut products intended for: Finishing beef cattle 300 Finishing swine 100 lb ( 45 kg) 200 Breeding beef cattle, breeding swine, or mature poultry Corn, peanut products, and other animal feeds and feed ingredients but excluding cottonseed meal, intended for immature animals Corn, peanut products, cottonseed meal, and other animal feed ingredients intended for dairy animals, for animal species, or when the intended use is unknown Cottonseed meal for beef cattle, swine, poultry 300

74 European Union Food/Feed FOOD Groundnuts, nuts and dried fruit: Groundnuts, nuts and dried fruit and processed products thereof, intended for direct human consumption or use as an ingredient in foodstuffs Groundnuts to be subjected to sorting, or other physical treatment, before human consumption or use as an ingredient in foodstuffs Nuts and dried fruit to be subjected to sorting, or other physical treatment, before human consumption or use as an ingredient in foodstuffs Maximum level aflatoxin (μg/kg) (B1 + B2 + B1 G1 + G2) 2 a 4 a 8 a 15 a 5 a 10 a M1 Source COMMISSION REGULATION (EC) No 2174/2003 of 12 December 2003 Cereals (including buckwheat, Fagopyrum sp) Cereals (including buckwheat, Fagopyrum sp) and processed products thereof intended for direct human consumption or use as an ingredient in foodstuffs Cereals (including buckwheat, Fagopyrum sp), with the exception of maize, to be subjected to sorting, or other physical treatment, before human consumption or use as an ingredient in foodstuffs Maize to be subjected to sorting, or other physical treatment, before human consumption or use as an ingredient in foodstuffs

75 Food/Feed Maximum level aflatoxin (μg/kg) (B1 + B2 + B1 G1 + G2) M1 Source Milk (raw milk, milk for the manufacture of milk based products and heat-treated milk Spices: Capsicum spp. (dried fruits thereof, whole or ground, including chillies, chilli powder, cayenne and paprika) Piper spp. (fruits thereof, including white and black pepper) Myristica fragrans (nutmeg) Zingiber officinale (ginger) Curcuma longa (turmeric) FEED b All feed materials 20 Complete feedingstuffs for cattle, sheep and 20 goats with the exception of: complete feedingstuffs for dairy animals 5 complete feedingstuffs for calves and lambs 10 Complete feedingstuffs for pigs and poultry (except young animals) Other complete feedingstuffs 10 Complementary feedingstuffs for cattle, sheep and goats (except complementary feedingstuffs for dairy animals, calves and lambs) Complementary feedingstuffs for pigs and poultry (except young animals) Other complementary feedingstuffs COMMISSION DIRECTIVE 2003/100/EC of 31 October 2003

76 Food/Feed Maximum level aflatoxin (μg/kg) (B1 + B2 + B1 G1 + G2) M1 Source Foods for infants and young children Baby foods and processed cerealbased foods for infants and young children c Infant formulae and follow-on formulae, including infant milk and follow-on milk d Dietary foods for special medical purposes e intended specifically for infants COMMISSION REGULATION (EC) No 683/2004 of 13 April 2004 (a) The maximum levels apply to the edible part of groundnuts, nuts and dried fruits. If nuts in shell are analysed, it is assumed when calculating the aflatoxin content all the contamination is on the edible part. (b) Maximum content relative to a feedingstuff with a moisture content of 12 %. (c) The maximum level for baby foods and processed cereal-based foods for infants and young children refer to the dry matter. The dry matter is determined in accordance with the provisions of Commission Directives 98/53/EC and 2002/26/EC. (d) The maximum level for infant formulae and follow-on formulae refer to the product ready to use (marketed as such or after reconstitution as instructed by the manufacturer). (e) The maximum level for dietary foods for special medical purposes intended specifically for infants refer in the case of milk and milk products, to the products ready for use (marketed as such or reconstituted as instructed by the manufacturer), in the case of products other than milk and milk products, to the dry matter. The dry matter is determined in accordance with the provisions of Commission Directives 98/53/EC and 2002/26/EC.

77 Chapter 1 Aflatoxins Considering the carry-over into milk and the established adverse effects on animal health, approximately 45 countries have set specific levels for aflatoxin B1 in feed for dairy animals [FAO 2004, Van Egmond 2003]. To support compliance with the maximum levels in milk intended for human consumption, stringent maximum levels were also set in the EU for feedstuffs which might be consumed by dairy cows [2002/32/EC]. A limit of 5 µg/kg feed for dairy cattle is applied in the EU countries and in the new member states, but only in few countries outside Europe. In contrast, grains for animal feed in the United States are allowed till 300 ppb aflatoxin (see Table 1.1). Moreover, Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed replaced since 1 August 2003 Council Directive 1999/29/EC of 22 April 1999 on the undesirable substances and products in animal nutrition. The main modifications can be summarised as follows: - extension of the scope of the Directive to include the possibility of establishing maximum limits for undesirable substances in feed additives. - deletion of the existing possibility to dilute contaminated feed materials instead of decontamination or destruction (introduction of the principle of non-dilution). - deletion of the possibility for derogation of the maximum limits for particular local reasons. - introduction the possibility of the establishment of an action threshold triggering an investigation to identify the source of contamination ( early warning system ) and to take measures to reduce or eliminate the contamination ( pro-active approach ). 71

78 Chapter 1 Aflatoxins In particular the introduction of the principle of non-dilution is an important and far-reaching measure. In order to protect public and animal health, it is important that the overall contamination of the food and feed chain is reduced to a level as low as reasonably achievable providing a high level of public health and animal health protection. The deletion of the possibility of dilution is a powerful mean to stimulate all operators throughout the chain to apply the necessary prevention measures to avoid contamination as much as possible. The prohibition of dilution accompanied with the necessary control measures will effectively contribute to safer feed. Thus with the entry into force of Directive 2002/32/EC on 1 August 2003 feed materials such as groundnut, copra, palm-kernel, cotton seed, maize and products derived from the processing thereof, have to comply with the level of 20 µg/kg. It can be observed that some feed materials regularly exceed the 20 µg/kg aflatoxin B1 level which will result that significant amounts of some of these feed materials can only be used for animal feed after an effective detoxification treatment. On the other hand, it can be observed that current feeding practices include the more frequent use of these feed materials directly on the farm, which could imply additional risks for elevated levels of aflatoxin M1 in the case of dairy animals. Although it is generally acknowledged that the current legislation is sufficient to guarantee that the aflatoxin M1 level in the overall milk supply is below 0.05 µg/kg, some experts are of the opinion that the current levels in EU legislation for aflatoxin B1 in complete feeds and complementary feeds for dairy animals/for animals in lactation do not provide sufficient guarantees, particularly in the case of high yielding dairy cattle, that the 72

79 Chapter 1 Aflatoxins produced milk will comply with the EU legislation on aflatoxin M1 in milk at farm level. According to these experts, the current maximum levels for aflatoxin B1 in complementary feed and complete feed should be lowered [EFSA 2004] Analytical methodologies Due to the widespread occurrence and toxicity of the aflatoxins, reliable methods are required for their identification and quantitation in different matrices to ensure safety and compliance with current legislation. Thus, the availability of sound analytical methods for monitoring the presence of aflatoxins along the food chain is of the utmost importance for keeping contamination under control. Several analytical methods have been developed for determining aflatoxins in various foodstuff (cereal-based foods, milk, spices, beer etc), feeds, animal tissue and biological fluids. As seen in the introduction section, the sampling stage is one of the most critical steps in aflatoxin analysis (and mycotoxins in general), where the contamination is extremely heterogeneous [Gilbert 2000]. There is a great deal of statistical variability in aflatoxin analyses for several agricultural commodities: contaminated kernels are usually not uniformly distributed in a load, so one sample might contain high toxin levels from a hotspot while the next sample does not. So often the results on a particular sample will depend on how many contaminated kernels were present in the sample, and on how high the level of contamination was in those kernels [Vincelli 1995]. 73

80 Chapter 1 Aflatoxins It is thus crucial that sampling is carried out in a way that ensures that the analytical sample is truly representative of the consignment. Failure to do this may invalidate the subsequent analysis [Eman]. One cannot escape some variability in testing for aflatoxins. So it is important to recognize that great sampling variability exists, and to use methods that minimize this variability. As the distribution of aflatoxin M1 in liquid milk can be expected to be reasonably homogeneous, sampling of liquid milk for aflatoxin M1 will be more accurate than sampling of granular feed products. Most of the uncertainty in estimates of aflatoxin M1 in milk is probably associated with the analytical procedure [WHO 2002]. Sampling and sample preparation remain a considerable source of error in the analytical identification of aflatoxins. Thus, systematic approaches to sampling, sample preparation, and analysis are absolutely necessary to determine aflatoxins at the parts-per-billion level. All analytical procedures for aflatoxins usually follow the general pattern for mycotoxin assays: extraction, purification (or clean-up), concentration, separation, and determination. For sample preparation, various techniques have been used for the extraction of aflatoxins from different matrices and for the subsequent purification of the extracts. Extraction depends to a great extent on the physicochemical properties of the commodities contaminated with aflatoxins [Ikins 1991]. Due to the diverse nature of products that can be contaminated, there is not a single adequate method of extraction for all products. Commodities with high lipid and pigment content require a more selective treatment followed by extensive purification methods than those with a low content of these 74

81 Chapter 1 Aflatoxins components. As aflatoxins are weakly polar components and are soluble in slightly polar solvents and insoluble in completely non-polar solvents, normally they are extracted using mixtures of organic solvents such as acetone, chloroform, or methanol, or combinations of these solvents with water [Jaimez 2000]. On the other hand, the use of small amounts of water in combination with the cited solvents humidifies the substrate increasing the penetration of the organic solvent in the sample and enhancing aflatoxin extraction [Ikins 1991]. The addition of non-polar solvents, like hexane, for fat partitioning is also used [Moss 1979]. Test extracts are cleaned up before instrumental analysis to remove coextracted materials that often interfere with the determination of target analytes. In practice, the choice of solvent depends on the following clean-up and separation procedure. When purification and clean-up are required, liquid liquid partitioning or, more recently, solid-phase (SPE) extraction cartridges and inmunoaffinity columns (IAC) are used [EFSA 2004]. Since Eppley in 1968 developed silica-gel purification columns for aflatoxin analysis, a large variety of solid-phase extraction columns have been developed for this purpose. The bonded phase may either be polar (silica cartridges) or non-polar (C18, C8, C2, cyclohexyl or phenyl cartridges). Other types of solid-phase extraction columns are the multifunctional Mycosep columns that, contrary to the others, allow the passage of mycotoxins and retain the interfering substances [Jaimez 2000 and references therein]. The most modern clean-up tool for aflatoxin analysis is the use of immunoaffinity cartridges (IAC) that have been shown to have a great potential to increase method specificity and sensitivity by selective enrichment and isolation of the target aflatoxins 75

82 Chapter 1 Aflatoxins [Papp 2002]. These columns are composed of monoclonal antibodies specific for aflatoxins, which are immobilized on Sepharose and packed into small cartridges. A sample containing aflatoxins is loaded onto the affinity gel column, and the antigen target aflatoxin is selectively complexed by the specific antibodies on the solid support into an antibody-antigen complex. The column is then washed with water to remove all other matrix components of the sample. Target aflatoxin is eluted from the column with a small volume of pure acetonitrile, and the eluate is concentrated and analysed. The combination of immunoaffinity clean-up and liquid chromatography coupled with fluorescence detection (see later) offers the best means for efficient purification and precise determination of low concentrations of aflatoxins. The method may, however, be too expensive for routine use in developing countries [JECFA 2001]. Many methods of analysis have become available for the determination of aflatoxins, both for screening and for quantitative estimates. Screening methods are particularly useful since they can be carried out quickly, easily, and economically. Theoretically, screening methods should never give false-negative results. The screening tests used for aflatoxins are usually immunochemical, both radioimmunoassays and enzyme immunoassays, although enzyme immunoassays are used more often [WHO 2002]. Most enzyme immunoassays for aflatoxins are heterogeneous, with separation of the immunocomplex and the unreacted material. One of the commonest heterogeneous enzyme immunoassays, the ELISA (enzymelinked immunosorbent assays), is generally used to determine aflatoxins. Several direct competitive ELISAs for aflatoxins are available 76

83 Chapter 1 Aflatoxins commercially and are usually designed for rapid screening [WHO 2002]; the 96-well microtitre plate assay is most commonly used for quantitative measurements. ELISA detection limits are comparable with those of HPLC methods. However, a disadvantage is the possibility of false-positive results due to cross-reactions and more important the possibility of falsenegative results [Anklam 2002]. The immunological methods have been used since the 1990s and are nowadays accepted as official methods for aflatoxin determination in some food commodities; however, it is still necessary to evaluate their efficiency in different foodstuffs and to correlate their results with other accepted analytical methods [Candlish 1991]. ELISA methods can be used for screening purposes only, because they are not reliable when used as a quantitative method [Azer 1991, Gilbert 1993]. For legal purposes, positive results in an ELISA method require confirmation by an accepted reference method [WHO 2002]. Techniques other than immunochemical procedures can, in principle, be used for rapid analysis for aflatoxins. One such technique involves electrochemical flow injection monitoring on filter-supported bilayer lipid membranes or a portable field test involving a patented, membrane-based flow-through enzyme immunoassay, which can be carried out on farms. The method has not been formally validated [JECFA 2001]. New approaches for rapid methods are currently made on the basis of biosensors, dip-stick like kits as well as other immunochemistry-based techniques such as surface plasmon resonance (SPR) ( or the ORIGEN Technology ( which are based on flowcells and can be used for a high throughput analysis of samples and consequently successful screening of a large sample throughput in a small 77

84 Chapter 1 Aflatoxins time interval, still maintaining reliability. All these different immunochemistry-based applications clearly show the direction of innovation that can be expected in the near future for screening tests as one of the two main pillars of a reliable and cost-effective measurement and prevention strategy. Other promising approaches are based on non-destructive techniques, such as near- or mid-infrared spectroscopic methods [Pettersson 2001], Fourier transform infrared photo-acoustic spectroscopy (FTIR-PAS) [Greene 1999] as well as other methods which do not measure the toxin itself, but indicate that with a certain probability the material is contaminated [Stroka 2002]. For quantitative analytical methods, once purified extracts are obtained, the concentration of aflatoxins can be determined in one of several ways. Most quantitative methods involve thin-layer chromatography [Goto 1988, Stroka 2000, Trucksess 1984, Van Egmond 1991] or/and high performance liquid chromatography [Akiyama 2001, Dragacci 2001, Manetta 2005, Martins 2001, Reddy 2001, Srivastava 2001, Van Egmond 1991]. TLC was the first method principle used for the determination of aflatoxins. Although TLC techniques were extensively used for aflatoxin analysis, afterwards an increase in the use of high-performance thin-layer chromatography (HPTLC) has been noted. The accuracy of TLC is less than that of HPLC [Ellis 1991], but the results obtained using HPTLC are similar to that of HPLC and more consistent than ELISA data [Jaimez 2000]. However, the present trend is the use of HPLC as an election technique for aflatoxin analysis. With advances in HPLC methods in the 1980s, laboratories moved away from TLC to HPLC determination since these techniques offer several 78

85 Chapter 1 Aflatoxins advantages compared to TLC such as automation, high performance separation and generally lower detection limits, while on the other hand the instrumental requirements increased as well. Aflatoxins can be separated and detected using either normal- or reversed-phase HPLC methods mainly with UV or fluorescence detection. In the first research studies with HPLC, the normal stationary phase (NP HPLC) was used coupled with a detection system for UV absorption which was insufficient to determine the aflatoxins at sub-nanogram levels. Since aflatoxins have fluorescent properties, it was soon demonstrated that systems for determination with fluorescence detection were more sensitive. One of the most important problems is the main aflatoxins (B1, B2, G1 and G2) fluorescence dependence towards the composition of the solvent. For example, mobile phases used for NP-HPLC, contained chloroform or dichloromethane. Under these conditions, the fluorescence detection at nanogram levels was only possible for AFG1 and AFG2 because the fluorescence emission of AFB1 and AFB2 was markedly quenched. This made it necessary to simultaneously use an UV detector for AFB1 and AFB2 and a fluorescence one for AFG1 and AFG2. Manabe et al. [Manabe 1978] found that the addition of an organic acid to the mobile phase enhanced the fluorescence intensities of AFB1 and AFB2. This method, with slight modifications, was used for aflatoxins determination in diverse agricultural products, mixed feed and for the determination of AFM1 and AFM2 in milk. Aflatoxins can be resolved by RP-HPLC columns with methanol water or acetonitrile water, but in these aqueous solvents, the fluorescence of AFB1 and AFG1 is rather weak. In general, RP-HPLC systems are used more frequently than those of normal-phase due to the easier manipulation as 79

86 Chapter 1 Aflatoxins well as the smaller toxicity of watery mobile phases. Because in these types of solutions, AFB1 and AFG1 fluorescence diminishes, different derivatization procedures have been tested including the use of strong acids such trifluoroacetic acid (TFA) and oxidants such as chloramine T, iodine and bromine. Furthermore, with automated HPLC systems, postcolumn derivatization appears preferable in order to reduce the manipulations required on each sample, the relative merits of the TFA methods are counter- balanced since TFA is not the most suitable reagent for post-derivatization because of its toxicity and its corrosive properties on the pumping devices. Recently, different methods of post-column derivatization have been developed especially with iodine, bromine or pyridinium bromide perbromide [Jaimez 2000 and references therein]. LC with fluorescence detection (FD) in combination with pre-column derivatization [Akiyama 2001, Goda 2001] and post-column derivatization [Barmark 1994, Joshua 1993] is often used due to the high selectivity and sensitivity of these methods. With the availability of IACs for aflatoxins, recent analytical methods combining the analytical advantages of both methodologies (immunoselectivity and chromatographic separation) have been developed and validated successfully for many applications [Anklam 2002, Jaimez 2000 and references therein them; Abbas 2002, Aycicek 2005, Kussak 1995a,b, Medina-Martìnez 2000, Nilŭfer 2002, Stroka 2000, Tekinşen 2005]. Even though these validated methods apply HPLC as a separation technique, it was shown that the use of TLC in combination with IACs is a promising application that can compare with HPLC concerning the performance requirements [Stroka 2002]. 80

87 Chapter 1 Aflatoxins With the advent of API interfaces in the late 1980s, the coupling of LC to MS became accessible (see Appendix 4). The high selectivity and sensitivity of MS detection methods associated with the resolution of LC provide decisive advantages to perform qualitative as well as quantitative analysis of a wide range of molecules at trace levels. LC-MS has been recently used for structural elucidation in aflatoxin studies. Just a limited number of papers describing different MS methods for AF analysis in various matrices have been published [Cappiello 1995, Cavaliere 2006a,b, Kussak 1995c, Schatzki 2002, Takino 2004, Tuomi 2001, Vahl 1998, Ventura 2004], however, only few papers report LC coupled to mass spectrometry (MS) or tandem MS (MS/MS) for analysing AFs in food of our interest (maize, milk and cheese) and none reaches suitable quantifying level that satisfy the limits in force (for the matrices concerning this work, see the specific references in the following chapters). In this field, LC-MS seems to be just a minor alternative to the already well established, reliable and robust LC-FD methodology. Really it is the only confirmation technique. In many countries, public health agencies rely on mass spectrometry (MS) for unambiguous confirmation of xenobiotics in foodstuff. The EU Commission Decision 2002/657/EC requires rigorous confirmation which provides the use of mass spectrometry. This means also that analytical methodologies capable of high performance are now required. LC-MS provides decisive advantages in performing identification as well as determination of analytes at trace levels. However, the coupling of both techniques is really efficacious if a suitable combination of sample preparation, chromatographic conditions and interface is selected. Both, atmospheric pressure chemical ionization (APCI) and electrospray 81

88 Chapter 1 Aflatoxins ionization (ESI) techniques are commonly used in LC-MS. Common problems for ionization methods such as ESI include the suppression of the analyte signal by compounds with higher ionization efficiency than the target compounds which are present in excess. In addition, adduct formation with background ions are sometimes prevalent in ESI, so it is often necessary to select suitable additives in the LC mobile phase. Atmospheric pressure photoionization (APPI) is the latest interface introduced in the field of soft ionization techniques for coupling MS to liquid-phase separation systems (for more details see Appendix 4). According to recent investigations, APPI seems to be a more reliable alternative to ESI [Zöllner 2006]. In particular some studies have shown that APPI can provide higher signal-to-noise ratios respect to APCI [Hanold 2004, Meng 2002]. However, the mechanism of the APPI process is still not completely understood and it is not easy to establish which compounds are amenable to APPI instead of APCI or ESI. In order to obtain reliable results, and hence give consumers and producers confidence in testing methods, there is an urgent need for internationally validated methods, which could serve as confirmatory methods and form the other main pillar in a reliable and cost-effective measurement and prevention strategy. Future analytical work for aflatoxin with LC-MS should, therefore, provide fully validated assays that are in agreement with recent EU regulations about aflatoxin analysis. 82

89 Chapter 1 Aflatoxins 1.2. Experimental Reagents and Chemicals Standards of AFM1 (Cyclopenta[c]furo[3',2':4,5]furo[2,3-h][1]benzopyran- 1,11-dione, 2,3,6a,9a-tetrahydro-9a-hydroxy-4-methoxy-, (6aR,9aR)- (9CI)); AFB1 (Cyclopenta[c]furo[3',2':4,5]furo[2,3-h][1]benzopyran-1,11-dione, 2,3,6a,9a-tetrahydro-4-methoxy-, (6aR,9aS)- (9CI)); AFB2 (Cyclopenta[c]furo[3',2':4,5]furo[2,3-h][1]benzopyran-1,11-dione, 2,3,6a,8,9,9a-hexahydro-4-methoxy-, (6aR,9aS)- (9CI)); AFG1 (1H,12H- Furo[3',2':4,5]furo[2,3-h]pyrano[3,4-c][1]benzopyran-1,12-dione, 3,4,7a,10atetrahydro-5-methoxy-, (7aR,10aS)- (9CI)) and AFG2 (1H,12Hfuro[3',2':4,5]furo[2,3-h]pyrano[3,4-c][1]benzopyran-1,12-dione, 3,4,7a,9,10,10a-hexahydro-5-methoxy-, (7aR,10aS)- (9CI)) -from Aspergillus Flavus- and formononetin (FORM) (7-hydroxy-3-(4-methoxyphenyl)-4 benzopyrone) were purchased from Sigma-Aldrich (St. Luis, MO, USA). Individual stock solutions were prepared from pure aflatoxins dissolved in acetonitrile at 50 µg/ml level for AFM1 and at 1 mg/ml for the other aflatoxins, stored at -20 C in amber glass vials and kept in the dark at room temperature (22-25 C) until use. Also FORM was prepared in acetonitrile at 1 mg/ml, but in this case storage not required amber vials and dark conditions. Working standard solutions were prepared by suitable dilution of stocks. These solutions were kept at 4 C and renewed weekly. Acetonitrile, acetone, methanol, and dichloromethane were HPLC grade from Carlo Erba (Milan, Italy) while toluene was for residue analysis from Fluka (Fluka Chemie GmbH, Buchs, Switzerland). All organic solvents were used as received. Concentrated ammonia, formic and acetic acids were RPE grade from Carlo Erba. Ultra-pure water was 83

90 Chapter 1 Aflatoxins produced from distilled water by a Milli-Q system (Millipore Corporation, Billerica, MA, USA). AFM1, not present in vegetables since it is an AFB1 hepatic metabolite, was used as internal standard (IS) for maize analysis. AFG1 was used as internal standard (IS-1) in reversed phase (RP)-chromatography (for milk and cheese analysis), whereas FORM was used as internal standard (IS-2) in normal phase (NP)-chromatography (for milk analysis) Cautions and Safety considerations Aflatoxins are carcinogenic compounds, consequently, solutions and extracts should be handled with extreme care. Gloves and other protective clothing have to be worn as safety precaution during compound handling. Crystalline aflatoxin standards have to be handled in a glove box. Aflatoxins are subject to light degradation, so it is necessary to protect analytical work from daylight and keep aflatoxin standard solutions in amber vials or aluminium foil. Special attention should be taken with glassware. The use of non acid-washed glassware (e.g., vials, tubes, flasks) for aflatoxin aqueous solutions may cause a loss of aflatoxin. Thus, prior to use, soak new glassware in dilute acid (e.g., 10% sulfuric acid) for several hours to remove possible active adsorption sites for AFs; then, rinse extensively with distilled water to remove all acid traces (check with ph paper) [Dragacci 2001]. Aflatoxin residues can be destroyed using 3% sodium hypochlorite. Glassware used for standard or sample should be soaked in 3% aqueous sodium hypochlorite to destroy AF residue before cleaning and re-use. Then, clean with detergent, rinse thoroughly with 84

91 Chapter 1 Aflatoxins distilled water and wash with methanol and methylene chloride. These steps were necessary to minimize the glass adsorption losses Instrumentation Extraction and Clean-up apparatus C18 (Discovery DSC-18), Envi-Chrom P, polypropylene tubes, polyethylene frits, and the vacuum manifold were from Supelco (Bellefonte, PA, USA); sand (Crystobalite, mash size) was supplied by Fluka AG (Fluka Chemie GmbH, Buchs, Switzerland); Lichrolut (R) EN ( µm) by Merck (Darmstadt, Germany), Oasis HLB cartridges (200 mg) from Waters (Milford, USA) and Carbograph-4 was purchased by LARA (Rome, Italy). Carbograph-4 is a graphitized carbon black (GCB) with a surface area of 210 m 2 /g and particle size range of mesh, similar to Carboprep 200 (Restek, Bellefonte, PA, USA) and Envicarb X (Supelco, Bellefonte, PA, USA). PTFE syringe filters (0.45 and 0.20 µm; 13 mm diameter) were from Alltech (Deerfield, IL, USA). A Polytron homogenizer was purchased from Kinematica (Luzern, CH). An ALC (Milan, Italy) multispeed refrigerated centrifuge PK131R was used. A homemade apparatus was used for hot water extraction (HWE): briefly, it consisted of an LC pump to supply solvent through 1/16-in.-O.D. (1/30-in.-i.d.) stainless steel tubing (including a 2-m preheating coil) to the extraction cell. Both the preheating coil and the extraction cell were placed inside a GC oven. A 10 cm x 8.3 mm-i.d. stainless steel column matched with stainless steel frits (2 µm pore size) was used as extraction cell. 85

92 Chapter 1 Aflatoxins LC-MS/MS apparatus Liquid chromatography was performed by using a Perkin-Elmer series 200 micropumps (Norwalk, CT, USA) including a vacuum solvent degassing unit, and coupled with a Perkin-Elmer autosampler equipped with a 20 µl loop. The purified samples were chromatographed on a suitable column (see specific chapters) with a Securityguard ODS, 4 x 2.1 mm i.d. precolumn supplied by Phenomenex (Torrance, CA, USA). The column was maintained in an oven (Timberline Instruments, Inc., Boulder, CO, USA) at 45 C. A Q-TRAP linear ion trap mass spectrometer or API 2000 triplequadrupole mass spectrometer (Applied Biosystems/MDS Sciex Concord, Ontario, Canada) were used. They were coupled with a TurboIonSpray (TISP), a heated nebulizer-atmospheric Pressure Chemical Ionization (HN-APCI) or a PhotoSpray sources (APPI). Applied Biosystems/MDS Sciex Analyst software version or was employed for data acquisition and processing. Mass calibrations and resolution adjustments on the resolving quadrupoles were automatically performed by using a 10 5 mol/l solution of poly(propylene glycol) introduced via a model 11 Harvard infusion pump (Harvard apparatus, Hollison, MA, USA). Compound spectra were preliminarily recorded by connecting the Harvard infusion pump to the interface. In order to optimize the tuning parameters for each compound, 1-5 ng/µl standard solutions in a suitable solvent (depending on the ion source and the chromatography used) were infused at 10 µl/min. Interfaces were operated in the positive ionization mode. The [M + H] + ions were selected by the first quadrupole and fragmented in the collision cell (Q2) operating at medium pressure (arbitrary scale). From the MS/MS 86

93 Chapter 1 Aflatoxins full-scan spectra, two suitable transitions were selected for acquisition in multiple reaction monitoring (MRM) mode, used for compound identification and quantification. The ESI needle voltage was set at 5500 V, the curtain gas was set to 20 (arbitrary units), nebulizer gas and auxiliary gas were set, respectively, to 30 and 45 and probe temperature was 400 C (see some little differences for milk in section). Nitrogen was used both as turbo and collision gas. The optimized tuning parameters selected for detection of each compound are reported Table 1.2. Table 1.2. Liquid chromatography-tandem mass spectrometry conditions and precursor ion/product ion pairs for aflatoxin analysis. Analyte DP a (V) EP b (V) RCE c (%) Precursor ion (m/z) Product ions (m/z) AFM ; 259 AFG ; 257 AFG ; 283 AFB ; 287 AFB ; 241 FORM ; 197 a Declastering potential. b Entrance potential. c Relative collision energy, expressed as % respect to the maximum voltage difference value between the high pressure entrance quadrupole (Q0) and collisional cell quadrupole (RO2) (+/- 130 V) permitted by the instrument. 87

94 Chapter 1 Aflatoxins Quantitation and Statistical evaluation Two set of calibration lines, named standard and matrix-matched respectively, were constructed. Standard solutions were prepared by diluting appropriate volumes of the working standard and IS solutions, with the starting chromatographic mobile phase; matrix matched solutions were prepared by spiking analyte-free samples after extraction and cleanup with known and appropriate volumes of the working standard and IS solutions, and following the remaining specific procedure for each matrix. Both standard and matrix matched solutions were fortified at six concentration levels, and 20 µl were injected. As to cheese, AFM1 was quantified using external calibration, matrix matched, or standard addition procedures (see 4.3 section). Standard additions procedure was done at three levels of spiking after evaluating the original contamination level. For each analyte the combined ion current profile for both transitions was extracted from the LC-MRM dataset, and the peak area plot versus injected amount or concentration was obtained by measuring the resulting peak area and relating this area to that for the IS. Unweighted regression lines for standard and matrix matched calibration were calculated and compared for evaluating the matrix effect on ionization efficiency and therefore signal intensity. For aflatoxin quantitation, the matrix-matched calibration plot was used and corrections for recoveries were done. Statistical evaluations were performed by ANOVA (p = 0.05). Linearity was evaluated in separate experiments in which calibration lines were constructed in a concentration range wider than that used for quantification of samples. In all cases R 2 > was found. 88

95 Chapter 1 Aflatoxins Analyte instrumental limits of quantification (LOQs) were estimated by the LC-MS/MS MRM chromatogram resulting by analysing each aflatoxin in standard solution at a low known concentration. The sum ion currents of both precursor-fragment ion transitions relative to each analyte were extracted; the resulting trace was smoothed twice by applying the smoothing method (Analyst software). Thereafter, the peak height-toaveraged background noise ratio (S/N) was measured. The background noise (N) estimate was based on 3σ of the peak-to-peak baseline signal near the analyte peak and was directly provided by the instrumentation software. A definition of LOQ as the amount giving S/N=10 of each analyte was adopted (for LOD this value is S/N=3). Method quantification limits (MQLs) were then calculated in the same way by analysing a sample extract fortified at known level. The method identification limit (MIL) was defined [Cavaliere 2006b], in compliance with the Commission Decision 2002/657/EC, as the analyte concentration in the sample giving a S/N=3 for the less abundant of the two selected transitions for MRM mode. This parameter with respect to MDL (method detection limit) for which the value S/N=3 is calculated on the sum ion currents of both transitions is totally devoid of ambiguity. Total recovery was assessed by spiking analyte-free samples, performing the extraction and clean-up procedures, measuring the peak areas, calculating the peak area ratios relative to the opportune IS added after clean-up, and comparing these data with those obtained by spiking the extracts of the same sample after clean-up. 89

96 Chapter 1 Aflatoxins 1.3. Results and Discussion Extraction and Clean-up As reported in the introduction, one of goals of this work was to perform sample preparation without using the very expensive immunosorbent cartridges. Low sensitivity and poor precision were the main problems arising when a simple solid-phase extraction (SPE) was tried. For clean-up purposes, various adsorbents suitable for the extraction of compounds, such as Envi-Chrom, Lichrolut, Oasis HLB (poli(divinylbenzene-co-n-vinylpyrrolidone)) and graphitized carbon black (GCB, Carbograph-4), were tested. The choice of a GCB material for cleaning-up the extracts was due to the good results in terms of selectivity and accuracy achieved with respect to the low recovery and/or strong matrix effect achieved with the other adsorbents probably correlated to coextracted and coeluted compounds [Bogialli 2003b]. As formerly reported, GCBs can behave as sorbents with reversed-phase, polar interaction and ion-exchanger properties. Moreover, they possess a particular affinity for aromatic compounds with respect to aliphatic ones [Altenbach 1995, Andreolini 1987, Crescenzi 1996]. The remarkable similarity between its surface and the difurancoumarin structure of AFs [Altenbach 1995] (Figure 1.3) may facilitate mutual interactions. 90

97 Chapter 1 Aflatoxins Figure 1.3. Sketch showing the probable coplanar interactions between the graphitized carbon black surface and the aflatoxin B1 molecule. For all these reasons, this material proved to be suitable for this application in terms of recovery and efficiency in removing interfering substances, even though the well-known loadability limitations of graphitized carbon blacks must be accounted [Andreolini 1987, Crescenzi 1996, Laganà 2003]. In order to avoid breakthrough for the less retained compounds, the extract, or a fraction of it, added to 500 ml water, was submitted to the clean-up procedure and passed through a Carbograph-4 cartridge at a flow rate of ml/min. Dilution of the sample with an appropriate volume of water resulted in both lower matrix effect and higher recovery for the less retained compounds. This behaviour has already been recognized [Andreolini 1987, Laganà 2003] and might be due to some kind of coextracted molecules that can play a double role, namely, displacement from active adsorption sites during loading on Carbograph cartridge and competition for ion desorption from charged droplets 91

98 Chapter 1 Aflatoxins during the electrospray ionization process. The volume of 500 ml water was selected as the best compromise to maximize recovery and minimize signal suppression, and to obtain reasonable loading time. An aliquot of methanol (2 or 5 ml depending on matrix - see specific chapters) was passed through the Carbograph-4 cartridge before elution to remove traces of water from the cartridge. Dichloromethane/methanol mixtures 80:20 or 90:10 (v/v) are usually effective in recovering neutral compounds strongly retained from GCBs while the same acidified mixtures are selective eluents for acidic compounds [Cavaliere 2005, Faberi 2005]. In table 1.3 the elution curves for AFs adsorbed on 250 mg of Carbograph- 4 from 500 ml of water by using different organic mixtures are reported. As can be seen, although the compounds are non-acidic by nature, only the addition of acetic acid to a mixture rich in dichloromethane allowed the total recovery of AFs. Probably acetic acid acts as competitor for dipolar interaction between groups of AFs and the chemical heterogeneities on the Carbograph-4 surface. The phenomenon is not correlated with acidity so that CH2Cl2:CH3OH:CH3COOH (88:10:2, v/v/v) mixture gives a better result than CH2Cl2:CH3OH:CH3COOH (85:10:5, v/v/v) one. 92

99 Chapter 1 Aflatoxins Table 1.3. Recoveries of aflatoxins adsorbed on 6 ml cartridge filled with 250 mg of Carbograph-4 by using 20 ml of different organic mixtures. Recovery % Elution mixtures CH2Cl2:CH3OH (80:20, v/v) CH2Cl2:CH3OH:CH3COOH (85:10:5, v/v/v) CH2Cl2:CH3OH:CH3COOH (88:10:2, v/v/v) CH2Cl2:CH3COOH (98:2, v/v) AFB1 AFB2 AFG1 AFG2 AFM The analytes did not show degradation or insolubility phenomena during the concentration step and the reconstitution of extracts before LC-MS/MS analysis Liquid chromatography-mass spectrometry During electrospay AFs can give rise to both negative [M-H] - and positive [M+H] + and [M+Na] + ions. In water/methanol and water/acetonitrile solutions, operating in positive mode, sodiated adduct by far prevails on proton adduct formation (clearly visible in the Q1 spectra). In order to find the best conditions in terms of sensitivity, different mobile phase modifier - such as ammonium acetate, ammonium formate and formic acid - were tested. Then, the target analytes were chromatographed under the suitable conditions reported in experimental sections, and MS spectra were acquired in both Q1 and MRM modes. Effective results were obtained 93

100 Chapter 1 Aflatoxins with ammonium formate. Several concentrations of ammonium formate (0, 1, 2, 5, 10, 25 mmol/l) were prepared. As can be seen in Figure 1.4 (were AFB1 is taken for example), the addition of ammonium formate reversed the AFs behaviour in-source, and at the optimum concentration of 2 mmol/l, the signal for [M+H] + increased by a factor 6.5 (probably ammoniated fragment is not a preferential adduct compared with protonated one or it lead to the loss of ammonia leaving a [M+H] + adduct and therefore a significant enhancement of the [M+H] + ions). Surprisingly, also in negative mode the use of 2 mmol/l ammonium formate doubled the signal intensity which became about 70% of that in positive mode. More surprisingly, the MS/MS noise in negative mode was about 5-10 times higher than that in positive mode, depending on the transition. As a consequence, LC with water/acetonitrile containing 2 mmol/l ammonium formate and positive ESI was chosen for lower detection limit. 94

101 Chapter 1 Aflatoxins [M+H]+ [M+Na]+ Intensity (cps) Ammonium formate concentration (mmol/l) Figure 1.4. The effect of ammonium formate addition to the acetonitrile/water mobile phase on the H + and Na + adduct formation by aflatoxin B1 molecule during positive electrospray ionization. Different kinds of reversed phase-columns, such as low and high coverage C18, high-purity silica based C18, and polar reversed phase, were tested. Among the columns tested, the best compromise in separation was obtained with the Alltima C Conclusions The common parts showed in this chapter will be utilized for the development of new LC-MS/MS confirmatory methods studied to identify and quantify the major aflatoxins, at present known, that can arise from fungal species A. flavus and A. parasiticus potentially colonizing food throughout the maize-milk-milk product food chain. An important advantage of the methods that will be proposed is the cleanup on Carbograph-4. In suitable conditions it allows both simultaneous 95

102 Chapter 1 Aflatoxins multiresidue and selective analyses for target aflatoxin/s without using the very expensive immunoaffinity columns. The methods developed in this work have been specifically studied for confirmatory analysis purpose. It is important to remember the need of methodology based on mass spectrometry in compliance with the Commission Decision 2002/657/EC. The work for the determination of aflatoxins described in the next chapters meets these requirements: the limits of quantitation reached in the confirmatory methods developed, were always below current or proposed regulation for food studied. The foodstuff and commodity contamination caused by aflatoxins, and mycotoxins in general, is very frequent. It is very important to consider with great attention that the contemporary presence of different toxins in the same product is possible. In this way the toxins can have a toxic synergistic action not included from single low limits. Then, it is crucial to monitor simultaneously their presence where they can be found. The research carried out has brought the possibility to have validated methods that can be used as guardianship the consumers and the producers, like reliable confirmation methods. These latter are an important instrument which clarifies the role that the substances assumed with the diet may play in the various aspects of health and pathologies. 96

103 Chapter 2 AFLATOXINS IN MAIZE 97

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105 Chapter 2 Aflatoxins in maize Chapter 2 AFLATOXINS IN MAIZE 2.1. Background Aflatoxins can be produced when Aspergillus flavus and Aspergillus parasiticus attack and grow on certain plants and plant products. As mentioned above, aflatoxins may contaminate a wide variety of agricultural commodities and foodstuff, especially if they have high carbohydrate and/or fat contents [Nilüfer 2002]. In particular, maize (corn, Zea mays) is frequently infected by Aspergilli that can produce aflatoxins [Abdullah 1998]. It has been demonstrated that maize is among the commodities with the highest risk of AF contamination [Jelinek 1989] whilst barley, wheat, soybeans, beans, sorghum, oats, pulses, cassava, millet and rice are resistant or only moderately susceptible to aflatoxin contamination in the field [CAST 1989]. Maize is probably the commodity of greatest worldwide concern, because it is grown in climates that are likely to have contamination with aflatoxins and corn is the staple food of many countries. Therefore, maize is source of direct and indirect exposure, since nowadays it is a widely used food in the breeding, especially for cattle [ Initially, it was believed that A. flavus was only a problem on corn in storage. But it has since been shown that it can also attack corn in the field. Field infection of corn by A. flavus can result in aflatoxin production in the corn prior to harvest [Wrather 2006]. During hot, humid conditions, microscopic spores are produced on corn residue and in survival bodies at the soil surface. These spores are carried by air movement, and some of them land on the silks. The spores 99

106 Chapter 2 Aflatoxins in maize germinate, and the fungus colonizes the silks if hot conditions continue. The fungus can then grow down the silk channel and around the developing ear. Yellow-brown silks that are still moist are most susceptible to colonization and invasion down the silk channel. Fresh, unpollinated silks are relatively resistant, and brown, dry silks can be colonized, but growth of the fungus down the silk channel is limited. Once fungal growth is present under the husk, the fungus may infect uninjured kernels if the plant is stressed once the dough stage is reached [Vincelli 1995]. On corn in the field A. flavus is evident as a greenish-yellow to yellowish-brown, felt-like or powdery mould growth on or between the corn kernels. 100

107 Chapter 2 Aflatoxins in maize Figure 2.1. Adapted from Payne, G. A. (1998). Process of contamination by aflatoxin producing fungi and their impact on crops. Pgs in: Mycotoxins in Agriculture and Food Safety. K. K. Sinha and D. Bhatnagar, eds. Marcel Dekker, New York. 101

108 Chapter 2 Aflatoxins in maize However, the growth of the mould itself does not always indicate toxin presence since the AF yield depends on particular growth conditions as well as genetic predisposition [Buchi 1969]. High temperatures (26-43 C) and a relative humidity of 85% are optimum for A. flavus growth and aflatoxin production. Drought conditions and moisture stress are always more characterizing climate. Under these conditions A. flavus and, thus, aflatoxins have the potential to be a considerable problem on corn. Drought and heat during the growing season are by far the most common stress factors associated with preharvest aflatoxin contamination, especially during pollination and as kernels mature. High night time temperatures range may be particularly important. Other factors that can enhance the risk of aflatoxin contamination include nitrogen deficiency, excessive plant populations, and poor root development. Another means of infection is through wounds caused by birds, insects or weather factors such as hail, early frost that cracks the pericarp and wind storms. Kernel injury by insects or birds provides infection sites that are easily colonized by the fungus. Certain insects can carry spores of A. flavus and introduce them onto senescing silks and into wounded kernels [Vincelli 1995]. A. flavus can also develop or continue to develop on corn in storage. The extent and severity of both invasion by A. flavus and the production of aflatoxin in the stored grain are influenced by several factors including moisture content and temperature of stored grain, condition of grain going into storage and length of storage. A. flavus grows best on corn at C and % moisture. Moisture content below 13% and temperature of the corn within -15 and -12 C of the average monthly air temperature prevent invasion by A. flavus. Then 102

109 Chapter 2 Aflatoxins in maize as the fungus grows, respiration occurs releasing heat and moisture into the surrounding environment in the grain mass. This results in an increase in the moisture content and temperature of the surrounding corn, causing a hot spot. If moisture content and temperature continue to rise, the environment for A. flavus becomes more favourable [Vincelli 1995, Wrather 2006]. Shelled corn should be dried to 15.5% or below within hours of harvest to minimize the risk of mould growth and aflatoxin contamination. Clean corn that is dried to 15 to 15.5% moisture should store well for up to 6 months if it is cooled quickly and held at the recommended temperatures. Corn that will be held for 9 months should be dried to 14%. A 12-month storage period requires a 13% storage moisture to reduce the risks of mould development and aflatoxin production). Natural air and low-temperature drying systems do not always achieve this when corn moisture exceeds 18% so most grain drying systems are used [Vincelli 1995]. Aflatoxin contamination of corn is typically associated with previous seen stresses in all stages. Thus, any practice to minimize these factors will reduce the risk of AF contamination (see also section and Appendix 3). Due to AF hazardous nature for humans and animals, national and international authorities established maximum residue levels (MRLs) for aflatoxins in maize. The European Commission has set the MRLs for AFB1 and total AFs allowed in corn products ready for retail sale to 2 µg/kg and 4 µg/kg, respectively [2003/2174/EC]; whereas the current MRLs established by US Food and Drug Administration (FDA) are 20 µg/kg [FDA 2004a]. 103

110 Chapter 2 Aflatoxins in maize In order to ensure safety and compliance with current legislation, many methods are presently available for determining aflatoxins in maize. Among these methods, thin layer chromatography and gas chromatography have found wide applicability [Honma 2004, Majerus 1992, Park 1994, Shotwell 1981, Solovey 1999], but liquid chromatography is nowadays the most preferred separation technique [Park 1990, Pazzi 2005, Roos 1997, Sobolev ASAP, Yazdanpanah 2001] commonly in combination with fluorescence detection. Several studies describing different MS methods for aflatoxin analysis in various foods have been published [Cappiello 1995, Cavaliere 2006a,b, Kussak 1995c, Schatzki 2002, Takino 2004, Tuomi 2001, Vahl 1998, Ventura 2004]; however, only two papers report LC coupled to mass spectrometry or tandem mass spectrometry, both using APCI source, for analysing aflatoxins in maize [Abbas 2002, Pazzi 2005]. In this chapter it will be described the development of a sensitive, accurate and reliable confirmatory procedure, based on chromatography/ electrospray- tandem mass spectrometry (LC/ESI-MS/MS), for the simultaneous analysis of AFB1, AFB2, AFG1 and AFG2 in corn meal samples. Particular attention was paid to optimizing the extraction and clean-up steps (without using the still expensive immunoaffinity column) as well as chromatographic and mass-spectrometric conditions in order to provide sensitive and accurate analysis over a wide linear range. The confirmatory method was evaluated in terms of accuracy, precision and method detection limits (MDLs). Finally, this study also includes application of the LC-MS/MS developed methods to analyse maize meal brands commonly found in stores and maize samples grown in experimental field in

111 Chapter 2 Aflatoxins in maize 2.2. Experimental Samples Two kinds of samples were analysed: 8 maize meal brands commonly found in stores, and 40 maize samples grown in five field trials in In the first case, samples were purchased from Italian retail markets, while the Experimental Institute of Cereal Research (Rome, Italy) kindly provided maize samples from field trials near Caleppio (Lodi), Luignano (Cremona), Vigone (Turin), Rottaia (Pisa), and Palazzolo (Udine). The selected field locations covered different pedo-climatic areas in north Italy, where maize is extensively farmed. Four hybrids (Matea, Tevere, Cotos, and Eleonora, belonging to FAO 300, 500, 600 and 700 classes, respectively), widely used for cultivation in Northern Italy and representing different maturity groups (from short season to full season), were used in this study. Samples of every hybrid type were harvested in the five locations beginning about 28 day after black layer maturity [Ritchie 1982] and, in a second step, after 15 days. The harvested ears were immediately dried, machine-shelled, grounded and conserved in sealed plastic bags at 0 C until analysis Sample preparation Extraction One gram of maize meal was homogenized for 15 s with 10 ml of acetonitrile/water (80:20, v/v), using a Polytron homogenizer. Doubling extraction time did not increase analyte recoveries. The homogenized 105

112 Chapter 2 Aflatoxins in maize sample was then placed on the top of a C18 cartridge positioned in the vacuum manifold. C18 cartridges were prepared by filling 6 ml polypropylene tubes with 250 mg of the adsorbent placed between two polyethylene frits. Immediately before using these cartridges were activated with 5 ml of acetonitrile/water (80:20, v/v). The vacuum was adjusted to the maximum and the extract was collected into a 25 ml volumetric flask. The extraction vessel was washed twice with 7 ml of the extracting solvent mixture, and these washings were also passed through the C18 cartridge and collected in the volumetric flask; the volume was then adjusted to 25 ml. Clean-up For this step, Carbograph-4 cartridges, prepared by placing 100 mg of the adsorbent inside 3 ml polypropylene tubes between two polyethylene frits, was employed. Before processing samples, Carbograph-4 cartridges were attached to a vacuum manifold apparatus and washed sequentially with 5 ml of dichloromethane/methanol/acetic acid (88:10:2, v/v/v), 3 ml of methanol and 5 ml of Milli-Q water. A 10 ml aliquot of the extract was diluted to 500 ml with water and passed through the pre-conditioned Carbograph-4 cartridge at a flow rate of about 20 ml/min. If the water-diluted sample resulted opalescent sampling flow rate may decrease. Locating a small quartz wool flock above the upper frit ensured a constant flow rate was maintained during extraction. The cartridge was washed with 25 ml of water followed by 2 ml of methanol for decreasing the residual water content. The vacuum was adjusted to provide a flow rate of about 5 ml/min, and aflatoxins 106

113 Chapter 2 Aflatoxins in maize were eluted with 15 ml of dichloromethane/methanol/acetic acid (88:10:2, v/v/v). The eluate was collected into a 1.4 cm i.d. round-bottomed glass vial, spiked with IS solution and evaporated to dryness at 40 C under a gentle nitrogen flow. The residue was reconstituted with 1 ml of the starting mobile phase for LC, and the obtained solution was forced through a PTFE syringe filter (0.45 µm; 13 mm diameter). A 20 µl aliquot of the final solution was analysed by LC/ESI-MS/MS Recovery experiments For recovery studies, aflatoxin-free samples were fortified as follows: 1 g of sample was placed in a flat amber glass vessel and soaked in 1 ml of acetone solution containing different volumes (10, 20, 40, 200 µl) of a composite working standard solution at 0.05 ng/µl level of each analyte, taking care to uniformly spread it on the sample. An intimate contact between analytes and sample was obtained by mixing with a spatula for five minutes. The samples were allowed to air drying at 25 C in a ventilate oven for about one hour, to eliminate the organic solvent. The spiked sample was extracted as described above and analysed LC-MS/MS analysis The purified samples were chromatographed on an Alltima C18 column (250 x 2.1 mm i.d., 5 µm particle size) from Alltech (Deerfield, IL, USA) with a Securityguard ODS, 4 x 2.1 mm i.d. precolumn supplied by Phenomenex (Torrance, CA, USA). The column was maintained in an oven (Timberline Instruments, Inc., Boulder, CO, USA) at 45 C. 107

114 Chapter 2 Aflatoxins in maize A gradient elution with acetonitrile/water (95:5, v/v) as mobile phase A, and water as mobile phase B, both containing 2 mmol/l ammonium formate was used. After an isocratic step at 20% A for 2 min, A was linearly increased to 70% in 16 min, then brought to 100% and held constant for 10 min. The flow rate was 200 µl/min. An API 2000 triple-quadrupole mass spectrometer, coupled with a TurboIonSpray (TISP) source was used and an Analyst software version was employed for data acquisition and processing. Mass calibrations and resolution adjustments on the resolving quadrupoles were automatically performed as described in section where also the tuning parameters selected for detection of each compounds were reported (and also showed after) Results and Discussion Extraction and Clean-up A sub-sample of 1 g could be selected for extraction provided that the laboratory sample was finely grinded and thoroughly mixed. Aflatoxin extraction from cereal matrices was usually performed by an organic solvent, such is methanol, acetonitrile, acetone or their aqueous solutions [Abbas 2002, Pazzi 2005, Takino 2004]. The extraction procedure, as described in section, was optimized after evaluating the performance of different techniques, such as pressurized liquid extraction (PLE), matrix solid phase dispersion extraction (MSPDE), ultrasonication and homogenization extraction, with different mixtures of solvents. The best compromise for simultaneous 108

115 Chapter 2 Aflatoxins in maize quantitative extraction in a short time was reached by homogenizing in acetonitrile/water (80:20, v/v see later) a previously ground and sieved maize sample. After preliminary experiments, the methanol/water system was discarded because too many substances causing clean-up cartridge saturation were coextracted. Four different extraction mixtures were tested: acetonitrile/water 90:10 (v/v); acetonitrile/water 80:20 (v/v); acetone/water 60:40 (v/v); and acetone/water 50:50 (v/v). In every case the procedure was the same reported in the Experimental section. The extraction efficiency was evaluated by spiking, at level of 2 µg/kg, analyte-free samples before and after the extraction step and following remainder of the procedure reported in the Experimental section. In this way, the effect of extraction on the total recovery can be isolated and evaluated by comparing the peak areas for the same compound in samples spiked ante and post extraction. The acetonitrile/water 80:20 (v/v) mixture gave the highest recovery for the analytes, ranging from 89% (AFB2) to 97% (AFG2), while acetonitrile/water 90:10 (v/v) and acetone/water 60:40 (v/v) recoveries were from 68% to 78%, and acetone/water 50:50 (v/v) solution extracted about 65% of AFs present (see Table 2.1). 109

116 Chapter 2 Aflatoxins in maize Table 2.1. Aflatoxin recoveries from maize meal by changing the extraction mixture. Recovery % Extraction mixtures AFB1 AFB2 AFG1 AFG2 CH3CN/H2O (90:10, v/v) CH3CN/H2O (80:20, v/v) CH3COCH3/H2O (60:40, v/v) CH3COCH3/H2O (50:50, v/v) In addition, the proposed procedure was simple and fast (it needs about 15 min) only when the acetonitrile/water mixture was used, while the acetone/water mixture had the tendency to cause maize particle dispersion and filtration cartridge clogging. A C18 cartridge was used to filter the extract and retained most of the phospholipids and triglycerides; while washing maize twice, increased target compounds recoveries. The amount of substances coextracted from corn-based matrices was very high, and an efficient clean-up procedure was therefore essential. For the extract cleanup, Carbograph-4 was tested and chosen (see section). AFs were desorbed from Carbograph-4 by a dichloromethane/methanol/acetic acid (88:10:2, v/v) mixture. Using 250 mg of GCB to clean-up maize extract, the procedure gave low recoveries, particularly for AFBs. Experiments with solutions of standards diluted in water showed that larger volumes of the desorbing mixture (25 ml for AFGs and 50 ml for AFBs) were required to achieve recoveries >95%. By using 100 mg of Carbograph-4 packed in a 3 110

117 Chapter 2 Aflatoxins in maize ml cartridge, good recoveries with 15 ml of the eluting mixture were achieved. To avoid loss of the AFGs, the least retained target compounds, only 10 ml of the entire extract was submitted to the clean-up and methanol washing was limited to 2 ml. Recoveries from clean-up, evaluated by comparing analyte-free sample extract aliquots spiked at level of 2 µg/kg before and after clean-up were >93% for all aflatoxins (see Table 2.2). Table 2.2. Elution curves of aflatoxins adsorbed on 3 ml cartridge filled with 100 mg of Carbograph-4. CH2Cl2:CH3OH:CH3COOH (88:10:2 v/v/v) Recovery % Fraction (ml) AFB1 AFB2 AFG1 AFG nr nr nr nr Liquid chromatography-mass spectrometry LC mobile phase optimization by ammonium formate addition to enhance sensitivity has been discussed in section. The chromatographic column was kept in an oven to increase the retention time reproducibility. In this way, it was possible to divide the massspectrometry acquisition in periods (see Table 2.3), increasing the signal to noise ratio. 111

118 Chapter 2 Aflatoxins in maize Table 2.3. LC-MS/MS and precursor ion/product ion pairs for aflatoxin analysis in maize samples. Analyte Retention time±σ (min) Acquisition window (min) DP a (V) EP b (V) RCE (%) c Precursor ion (m/z) Product ions (m/z) AFM ± ; 259 AFG ± ; 257 AFG ± ; 283 AFB ± ; 287 AFB ± ; 241 a Declastering potential. b Entrance potential. c Relative collision energy, expressed as % respect to the maximum voltage difference value between the high pressure entrance quadrupole (Q0) and collisional cell quadrupole (RO2) (+/- 130 V) permitted by the instrument. The oven temperature was also changed from 30 to 50 C, using 5 C steps, in order to improve the IS separation from unknown matrix compounds that suppressed about 30% of its response, whereas for the other AFs trifling matrix effect was observed. This purpose was reached by setting the oven temperature at 45 C; using the devised conditions, the matrix effect, on IS too, was negligible. Therefore, parameters affecting the chromatographic separation were carefully evaluated with the aim of separating the target analytes into retention time windows. The gradient was adjusted to improve the 112

119 Chapter 2 Aflatoxins in maize separation of the analytes eluted. The preceding conditions allowed the separation of all target compounds, with the exception of the AFG1 and AFB2. Figure 2.2 shows the mass-chromatogram relative to the sum of the two selected transitions for each aflatoxin for an analytes-free sample spiked at 1 µg/kg level. The improvement, in terms of S/N ratio, with respect to the contemporaneous acquisition of all the transitions for the four AFs in the same window, was by a factor 2-3, depending on the specific compound. Figure 2.2. LC/ESI-MS/MS chromatogram in MRM mode resulting from an aflatoxins-free sample spiked at 1 µg/kg with AFB1, AFB2, AFG1 and AFG2. AFM1 was used as internal standard (IS). 113

120 Chapter 2 Aflatoxins in maize Recovery and precision Accuracy and precision were evaluated by recovery experiments. Recovery was evaluated at four spike levels: 0.5, 1.0, 2.0, and 10 µg/kg; the spiked samples were worked up as described in Experimental section, and analysed by LC-MS/MS. Considering that, for cereals ready for retail sale, the EU has fixed MRLs to 2 µg/kg for AFB1 and 4 µg/kg for the total of four aflatoxins, and the US FDA has set a MRL for total aflatoxins to 20 µg/kg, these concentrations cover the range of interest set by both legislations. For each concentration, six measurements were performed and data are reported in Table 2.4. Although the variance analysis showed a statistically (p<0.05) significant difference only for AFB2, there is some trend for recoveries to decrease at the lowest concentration. The recovery of the method was always above 81% for all compounds and the relative standard deviations ranged from 2-12%, and were near the quantification limits, but still acceptable. Table 2.4. Recovery and precision of the LC/ESI-MS/MS method for determining AFB1, AFB2, AFG1 and AFG2 in maize spiked at four concentration levels. RECOVERY (%) ± RSD a (%) Analyte 0.5 μg/kg 1 μg/kg 2 μg/kg 10 μg/kg AFB1 89 ± 9 93 ± 7 97 ± 3 98 ± 2 AFB2 81 ± 5 90 ± 4 93 ± 2 94 ± 2 AFG1 87 ± ± ± 6 96 ± 4 AFG2 92 ± 7 96 ± ± 2 98 ± 2 a RSD was calculated on six samples. 114

121 Chapter 2 Aflatoxins in maize Method performances Matrix-matched calibration regression lines were constructed as reported in section, for a set of six different analyte-free samples spiked after clean-up, and compared with those obtained using standard solutions (Table 2.5). The ESI-MS/MS responses were linearly related to injected amounts up to 40 ng, and all calibration curves showed good linearity with coefficients of determination R 2 not lower than The ratios between slopes (amatrix/astandard) were 0.91, 0.97, 0.95, and 0.92 for AFB1, AFB2, AFG1, and AFG2, respectively. The relative standard deviations (RSDs) of the slopes calculated from y-residuals [Miller 1993] were in the range of 1.5 to 2.8% and the regression lines differed significantly (p<0.05) only for AFG2. In spite of the small differences between external and matrixmatched calibration, the latter was used, with the purpose of improving accuracy. Table 2.5. Calibration regression lines. Regression equation a (standard solution) R 2 b Regression equation (sample) R 2 AFG2 y = x y = x AFG1 y = x y = x AFB2 y = x y = x AFB1 y = x y = x a Unweighted regression lines; y = peak area/is area, x = analyte amount. b Coefficient of determination. 115

122 Chapter 2 Aflatoxins in maize Analyte instrumental limits of quantification (LOQs) were estimated by the MRM LC-MS/MS chromatogram resulting by analysing 4 pg injected of each aflatoxin in standard solution. Method quantification limits (MQLs) and method identification limits (MILs) were then calculated by analysing a sample extract fortified at a 0.5 µg/kg level. These data are listed in Table 2.6. Table 2.6. Method performances. Analyte LOQ a pg inj MIL b μg/kg MQL c μg/kg AFB AFB AFG AFG a Instrumental limit of quantification. b Method identification limit (S/N=3 for the second most intense transition in MRM). c Method quantification limit (S/N =10) Effect of subsampling To ensure sample representativity, ten or more grams per sample are usually extracted [Razzazi-Fazeli 2002, Dall Asta 2004], and part of the extract is submitted to the subsequent analytical steps. Such amount is hardly compatible with the extraction procedure proposed, which makes use of a high solvent/sample ratio and a statical-dynamical extraction mode. Our rationale was that if a sample is homogeneous enough, a sub- 116

123 Chapter 2 Aflatoxins in maize sample as small as one gram may be representative. Thus, six 1 g subsamples of a naturally contaminated corn meal were independently processed and analysed for AFs analysis. The between sub-sample precision resulted in the range 2-12% (see table 2.4), and was then not significantly different from the between-day precision (p=0.05) of the method for the same analytes Real sample analysis The developed method was applied to the analyses of 48 actual samples: eight marketed maize meal samples and forty maize samples collected from field trials. Results are reported in Table 2.7. Samples showing levels under MIL for all AFs were not reported. 117

124 Chapter 2 Aflatoxins in maize Table 2.7. AFB1-2, AFG1-2 contamination levels in actual corn meal samples analysed by using LC/ESI-MS/MS. Concentration (μg/kg) Samples a AFB1 AFB2 AFG1 AFG2 Marketed samples Organic agriculture Conventional agriculture b nq c nq nq nq - Field trial samples d Caleppio (Lodi) Matea (2) Matea (1) nq Tevere (2) Tevere (1) nq Cotos (2) Cotos (1) nq Vigone (Turin) Eleonora (2) Rottaia (Pisa) Matea (2) a Only aflatoxin positive samples are reported. Total sample numbers: 8 for marketed samples and 40 for field trial samples. b Values under method identification limit (S/N=3 for the second most intense transition in MRM). c Not quantified, under method quantification limit (S/N =10). d Analysis of maize samples coming from 2005 harvest located in North of Italy field trials. Selected field locations: Caleppio (Lodi), Luignano (Cremona), Vigone (Turin), Rottaia (Pisa), and Palazzolo (Udine). Selected hybrids: Matea, Tevere, Cotos, and Eleonora, widely used for cultivation in Northern Italy and representing different maturity groups (from short-season to full season). Harvest time: (1) samples were harvested about 28 days after black layer maturity stage ; (2) samples were harvested in a second step, 43 days after black layer maturity stage.

125 Chapter 2 Aflatoxins in maize None of the samples gave evidence of mass spectral interference and the IS signal intensities were consistent with those obtained in matrixmatched calibration. As far as contamination level is concerned, only 1/8 marketed samples (coming from organic agriculture), and 4/40 samples coming from field trials, exceeded the MRL fixed by EU for AFB1. In field trials experiments, different AF levels were observed for hybrids farmed in the same manner in the five fields. Delayed harvesting seems to increase the risk of AF contamination; in fact all non-compliant samples come from deferred harvesting. In addition, samples showing the highest contamination came from a locality (Caleppio) where the temperature was over the seasonal mean during the kernel development. Although the samples that resulted most contaminated belonged to the 600 hybrid class (Cotos) and came from Caleppio, there was random contamination in the field samples collected. However, the few samples did not allow us to find significant correlations between hybrid type and aflatoxin content. Figure 2.3 shows the mass chromatogram of the most contaminated sample. 119

126 Chapter 2 Aflatoxins in maize Figure 2.3. LC/ESI-MS/MS chromatogram in MRM mode, obtained by analysing a field trial sample coming from 2005 harvest, Caleppio (Lodi) location, Cotos hybrid, harvested 43 days after the black layer maturity stage. The analyte concentration was: 15.6 µg/kg for AFB1 and 1.8 µg/kg for AFB2. AFM1 was used as internal standard (IS) Conclusions This LC/ESI-MS/MS multiresidue confirmatory method has been developed to identify and quantify simultaneously the major aflatoxins that can arise from fungal species A. flavus and A. parasiticus potentially colonizing Zea mays. The method has several advantages over the literature reported methods; it is sensitive, robust and selective. Moreover, it offers a valid clean-up alternative to immunoaffinity columns, which are expensive and unable to perform a multianalyte extraction. 120

127 Chapter 2 Aflatoxins in maize The results achieved and showed in this chapter suggest that it is important to monitor maize for the presence of AFs, especially during hot, dry conditions that encourage mould growth and aflatoxin production, and also that it may be possible to control toxin contamination with cultural practices that reduce plant stress. In view of this, the development and selection of cultivars of cereal and forage plants that are resistant to infection by toxigenic fungi should be the long-term objective of any effort to prevent contamination in future. It is an important aim because in the case of contemporaneous exposure to more than one toxin, even if at levels generally considered ineffective, there could be unexpected adverse effects, caused by an additive or synergic effect. 121

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129 Chapter 3 AFLATOXINS IN MILK 123

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131 Chapter 3 Aflatoxins in milk Chapter 3 AFLATOXINS IN MILK 3.1. Background Aflatoxin M1 (AFM1, milk toxin ) is the main monohydroxylated derivative of AFB1 (4-hydroxyderivative) forming in liver by means of cytochrome P450-associated enzymes [Sweeney 1998, Hsieh 1991] (see Figure 1.1). In ruminants, a considerable part of the ingested aflatoxin B1 is degraded in the rumen and does not reach systemic circulation. The absorbed fraction of aflatoxin B1 is extensively metabolised in the liver, resulting predominantly in aflatoxin M1, which enters the systemic circulation or is conjugated to glucuronic acid, and subsequently excreted via bile [EFSA 2004]. Circulating aflatoxin M1 can be excreted via the kidneys or secreted into milk in the mammary glands of dairy cows [Cathey 1994] that have consumed feeds containing AFB1 [Van Egmond 1989a,b, Galvano 1996, Veldman 1992]. It should be noted that aflatoxin M1 is not only found in dairy milk, but also in breast milk of nursing women who eat foods containing the toxin [JECFA 2001]. AFM1 is produced via hydroxylation of the fourth carbon in the AFB1 molecular. In addition to aflatoxin M1 other aflatoxin metabolites are excreted with milk, including aflatoxin M2 (the analogous metabolite of aflatoxin B2 resulting from hydroxylation of the fourth carbon in the AFB2 molecule) and aflatoxin M4, another hydroxy-metabolite of aflatoxin B1 [EFSA 2004]. Other aflatoxins of the M series found in milk include GM1, GM2, M2a and GM2a. They are hydroxylated derivatives of aflatoxins G1, G2, 125

132 Chapter 3 Aflatoxins in milk B2a and G2a, respectively [Schabort 1969]. All these metabolites occur in milk at much lower concentrations compared to aflatoxin M1 and thus are considered of less public health significance. Studies have clearly demonstrated AFM1 causes toxic and carcinogenic effects, similar or slightly less than that of aflatoxin B1 [Galvano 1996, Wood 1991], therefore this toxin, initially classified by IARC as a Group 2B human carcinogen [IARC 1993a], has now moved to Group 1 [IARC 2002]. Aflatoxin contaminated corn and cottonseed meals in dairy rations have resulted in AFM1 contaminated milk and milk products, including dry milk, cheese and yogurt [CAST 1989]. No evidence of AFM1 excretion in hen's eggs has been reported (the aflatoxin residues in eggs has been B1 rather than any of its known metabolites). The concentration of AFM1 in milk increases proportionally with the amount of AFB1 in the diet of the lactating cow. When ingestion is continuous, milk concentrations will increase until an equilibrium with intake is established. High producing cows converted AFB1 to AFM1 more efficiently than did low producing cows. However, the final concentration of AFM1 in milk was similar in both groups due to dilution by the greatest milk production in high-producing cows [Agag 2004]. Model calculations on the carry-over of aflatoxins present in feedstuff into milk revealed that under circumstantial maximum exposure from feed materials (albeit in compliance with the levels set for feed materials), milk obtained from high-yielding dairy cows and other milk producing animals, including small ruminants, buffalo and camels, might contain aflatoxin M1 levels exceeding the present statutory limits [EFSA 2004]. About % of aflatoxin B1 initially present in animal feed is transformed to aflatoxin M1 in milk (carry-over). A linear relationship has 126

133 Chapter 3 Aflatoxins in milk been found between intake of aflatoxin B1 in contaminated feed and the aflatoxin M1 content of milk, as follows: Concentration of aflatoxin M1 = [1.19 x aflatoxin B1 intake] (ng/kg of milk ) (µg/cow per day) Thus, production of milk containing aflatoxin M1 at 0.05 µg/kg (the EU limit) would require that the average intake of aflatoxin B1 by dairy cows be limited to approximately 40 µg per day. On the basis of a daily feed consumption of 12 kg of compound feeds per cow, application of a limit of 40 µg of aflatoxin B1 would mean that the content of aflatoxin B1 in the feed would have to be no more than 3.4 µg/kg in order to meet the limit of 0.05 µg/kg for aflatoxin M1 [WHO 2002]. On the other hand, it is noticeable that a limit of 5 µg/kg feed for dairy cattle is applied in the EU countries (see Table 1.1). Foreseeing carry-over is not so easy, in fact the transmission rate was shown to vary from animal to animal, from day to day, and from one milking to the next [Pittet 1998, Van Egmond 1986] as it is influenced by various (patho-)physiological factors, including the feeding regime, health status and individual biotransformation capacity, and finally by the actual milk production. AFM1 may or may not be present in dairy products in a particular year depending on the weather conditions for that period [Galvano 1996]. Numerous studies have been conducted on the effects of processing on the concentration of aflatoxin M1 in milk, the results of which are variable. AFM1 is relatively stable during pasteurization, sterilization and storage of milk and milk-based products [Galvano 1996, Rustom 1997]. Most studies 127

134 Chapter 3 Aflatoxins in milk show that the concentration is not appreciably reduced by heat or frozen treatment, nor by processing yoghurt, cheese, cream, milk powder or butter (although aflatoxin M1 is redistributed differentially in these products), then AFM1 intake, even at low concentrations, poses a significant threat to human health, especially to children who are the major consumers of milk. Cream separation can effect AFM1 distribution, since 80% is partitioned in the skim milk portion [Grant 1971] because of AFM1 binding to casein [Brackett 1982 b,d]. The behaviour of AFM1 in processes which involve fat separation may be explained by its semipolar character: it is a watersoluble compound binding with hydrophobic sides of casein, thus leading to a predominance in the non-fat fraction [Van Egmond 1986]. Aflatoxin M1 can be partially eliminated from milk by physical or chemical procedures, which include use of adsorbents (i.e. bentonite or hydrated sodium calcium aluminosilicates), ultraviolet radiation, bisulphites and hydrogen peroxide [Applebaum 1982b,c, Yousef 1985]. These treatments are not readily applicable by the dairy industry, however, and their safety has not been tested; moreover, the costs may be prohibitive for large-scale application. The most effective means for controlling aflatoxin M1 in the food supply is to reduce the amount of aflatoxin B1 in the feed of dairy cows. Recent studies have indicated that the prevalence of aflatoxin M1 in milk samples is often increased as a result of incorporating home-grown maize into the animal feed (RASFF 2003). Due to the present agricultural practice and the possibility of aflatoxin B1 to be present also in staple feeds grown in Europe, monitoring activity of aflatoxin M1 contamination of milk should be intensified and expanded to consumable milk from animal species other than dairy cows [EFSA 2004]. 128

135 Chapter 3 Aflatoxins in milk Specific regulations exist in many countries to control aflatoxin B1 in the animal feed supply, but it might be difficult to design an effective control programme in countries where cottonseed and maize are incorporated into animal feed, because of the heterogeneous distribution of aflatoxin in these commodities, which results in a high degree of sampling variability. The concentration of aflatoxin B1 in feed can be reduced by good manufacturing practice and good storage practices. If preventive measures fail, however, the aflatoxin B1 concentration in feed can be reduced by blending or by physical or chemical treatment. The physical treatments include application of heat, irradiation with microwaves, gamma-rays, X- rays or ultraviolet light, and adsorption on to hydrated sodium calcium aluminosilicate and other inert materials. The most successful chemical procedure for degrading aflatoxins in animal feed is treatment with ammonia. This procedure is used with agricultural commodities in various countries and leads to decomposition of 95 98% of the aflatoxin B1 present [WHO 2002] (for major details see Appendix 3). Because of potential health hazards for humans, worldwide monitoring of AFM1 in milk has been indicated and regulatory levels have been established. According to the U.S. Food and Drug Administration, AFM1 in milk should not exceed 0.5 µg/kg [FDA 2004b]. AFM1 level was set more restrictively to 0.05 µg/kg by the European Union (EU) in milk for adult consume [2003/2174/EC], while in baby-food products this level cannot be greater than µg/kg [2004/683/EC]. The 0.5 µg/kg limit for aflatoxin M1 has also been adopted by the Codex Alimentarius [Codex Alimentarius 2001]. Thus, the maximum permitted level of aflatoxin M1 in milk in the EU [2003/2174/EC] is among the lowest 129

136 Chapter 3 Aflatoxins in milk in the world, and is based on the ALARA (As Low As Reasonably Achievable) principle [EFSA 2004]. Accurate determination of AFM1 is also required to avoid human disease from AF exposure and to advance world-wide surveillance of food. For these reasons, it is important to devise accurate, specific and sensitive methods for determining AFM1 in milk. Immunological methods such as ELISA based assays are commonly used for screening purposes [López 2003, Magliulo 2005, Thirumala-Devi 2002] and besides them a new method based on a flow injection immunoassay system has been recently proposed [Badea 2004]. The most widespread analytical methods for quantitative purpose involve liquid chromatography, using either normalor reversed-phase separation, followed mainly by fluorescence detection [Dragacci 2001, Garrido 2003, Lin 2004, Manetta 2005, Simon 1998, Stubblefield 1986, Tuinstra 1993]. For clean-up, the use of immunoaffinity columns (IAC) is the most reported [Dragacci 2001, Garrido 2003, Lin 2004, Tuinstra 1993]. A European project has tackled the need to validate official methods at the low European regulatory limits, and an IAC-LC-FD method for determination of AFM1 in liquid milk has recently undergone collaborative study [Dragacci 2001]. In the last years, confirmation methods based on LC-MS(/MS) in compliance with the EU 2002 Decision [2002/657/EC] have been reported mainly for determining AFB1, AFB2, AFG1 and AFG2 in some foodstuffs [Abbas 2002, Blesa 2003,Takino 2004, Vahl 1998] but, LC-MS has been applied for AFM1 only in urine [Egner 2003, Walton 2001]. In this chapter the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection of AFM1 in milk is describe. Particular attention has been paid to finding a valid alternative to 130

137 Chapter 3 Aflatoxins in milk the expensive immunoaffinity columns for sample preparation. Besides, the present work focuses on the optimization and comparison of performances among LC-MS/MS methods using different ionization sources (ESI, APCI and APPI). The additional analytical capabilities offered by APPI-MS, with respect to ESI and APCI-MS, have been optimized to improve the detection limits of AFM1. At now, only one report on the coupling of LC with APPI-MS is present in literature for determining aflatoxins B1, B2, G1 and G2 in corn, peanuts and spices [Takino 2004] Experimental Samples Bottled and boxed milk samples were purchased from local retail markets. Different typologies of milk (pasteurized, ultra high temperature (UHT) treated, pasteurized high quality, skimmed) from different brands (both large and small manufacturers) were selected Sample preparation Five milliliters of a milk sample were poured into a screw cap 115 x 30 mm polycarbonate centrifuge tube. A 20 ml volume of acetone was added slowly (5-7 min) to the sample, the tube was tightly capped and vigorously shaken. The sample was centrifuged for 3 min at 5000 rpm to pellet insoluble matter. The whole supernatant was removed, diluted to 500 ml with water and passed through a Carbograph-4 cartridge at a flow 131

138 Chapter 3 Aflatoxins in milk rate of ml/min for clean-up step. Carbograph-4 cartridges were prepared by filling 6 ml polypropylene tubes with 250 mg of the adsorbent placed between two polyethylene frits. Before processing samples, these cartridges were attached to a vacuum manifold apparatus and washed sequentially with 10 ml of dichloromethane/methanol/acetic acid (88:10:2, v/v/v), 5 ml of methanol and 10 ml of Milli-Q water. After the sample was passed through, the cartridge was washed with 100 ml of water followed by 5 ml of methanol, the vacuum was adjusted to provide a flow rate of about 5 ml/min and AFM1 was then eluted from the cartridge with 10 ml of dichloromethane/methanol/acetic acid (88:10:2, v/v/v). The eluate was collected into a 1.4 cm i.d. round-bottomed glass vial, spiked with IS1 or IS2 solutions and evaporated at 40 C under a gentle flow of nitrogen. The residue was reconstituted with a suitable solvent (see later) and the obtained solution forced through a PTFE syringe filter (0.20 µm; 13 mm diameter) Recovery experiments For recovery studies, a milk sample containing 18 ng/kg of AFM1 was fortified with an appropriate volume of the standard solution; the spiked sample was left to equilibrate for two hours at room temperature in the dark and then extracted as described above and analysed LC-MS/MS analysis The purified samples were chromatographed on an Alltima C18 column (150 x 1 mm i.d. or 250 x 4.6 mm i.d., 5 µm particle size) from Alltech (Deerfield, IL, USA) with a Securityguard ODS, 4 x 2.1 mm i.d. precolumn 132

139 Chapter 3 Aflatoxins in milk (Phenomenex, Torrance, CA, USA) in reversed phase (RP) separation mode, and on a Supelcosil diol column (250 x 4.6 mm i.d., 5 µm particle size) supplied by Supelco in normal phase (NP) separation mode. A Q-TRAP linear ion trap mass spectrometer, coupled with a TurboIonSpray (TISP), a heated nebulizer (HN)-APCI or a PhotoSpray sources, was used. Q-TRAP linear ion trap mass spectrometer is based on a triple-quadrupole path (QqQ) in which the third quadrupole (Q3) can also be operated as a linear ion trap (QqLIT) with improved performance. In the QqLIT configuration the Q-TRAP can also operate in enhanced product ion scan (EPI) modes. Operating in EPI mode, LIT fill time was optimized at 50 ms and Q3 entry barrier at 8 V, while the scan rate was set at 4000 Th/s. An Analyst software version was employed for data acquisition and processing. Mass calibrations and resolution adjustments on the resolving quadrupoles were automatically performed as described in where also the tuning parameters independent of the ion source and chromatography selected for detection of target compound were reported in Table 1.2. RP-LC/ESI-MS/MS Column: C x 1 mm i.d.. Mobile phase A: acetonitrile/water (95:5, v/v), mobile phase B: water, both containing 2 mmol/l of ammonium formate. After an isocratic step at 20% A for 2 min, A was linearly increased to 85% in 7 min, then brought to 95% and held constant for 4 min. The flow rate was 50 µl/min. The sample was reconstituted in 400 µl of the starting mobile phase, filtered and 15 µl were injected. The ESI needle voltage was 133

140 Chapter 3 Aflatoxins in milk set at 5500 V; the curtain gas was set to 35 (arbitrary units), GS1 and GS2 were set, respectively, to 30 and 45 and probe temperature was set at 400 C. Nitrogen served both as turbo and collision gases. RP-LC/APPI-MS/MS Column: C x 4.6 mm i.d.. Mobile phase A: methanol/acetone (87:13, v/v), mobile phase B: water/acetone (87:13, v/v). After an isocratic step at 15% A for 1 min, A was linearly increased to 85% in 10 min, then brought to 95% and held constant for 2 min. The flow rate was 1.0 ml/min. The sample was reconstituted in 500 µl of the starting mobile phase, filtered and 100 µl were injected. The APPI source was equipped with a krypton discharge lamp, having a magnesium fluoride window that enabled transmission of the 10.0 and 10.6 ev photons emitted. The optimal source block voltage was 1400 V; the curtain gas was set to 30 (arbitrary units), GS1 and GS2 were set, respectively, to 70 and 30 and probe temperature was set at 375 C. High purity nitrogen was used as nebulizer, curtain, auxiliary and lamp gases. NP-LC/APPI-MS/MS Column: diol 250 x 4.6 mm i.d.. Mobile phase A: toluene/isopropanol (67:33, v/v), mobile phase B: toluene. After an isocratic step at 0% A for 4 min, A was linearly increased to 30% in 10 min, thereafter to 50% in 3 min and held constant at 50% for 7 min. The sample was reconstituted in 500 µl of toluene, filtered and 100 µl were injected. The column was thermostated at 45 C by a Timberland column thermostat (Alltech). Source parameters were the same as for RP-LC/APPI-MS/MS. 134

141 Chapter 3 Aflatoxins in milk 3.3. Results and Discussion Extraction and Clean-up The extraction procedure, as described in 3.2.2, was optimized after evaluating the performance of different SPE adsorbents. Low recovery due to the well-known interaction between AFM1 and milk proteins [Galvano 1996] and/or strong matrix effect due to coextracted and coeluted compounds [Bogialli 2003b] were probably responsible for initially failure. The hindrance was resolved to use a two-step sample preparation procedure: in the first step the protein fraction was precipitated and in the second step the supernatant was cleaned-up. Numerous procedures for protein precipitation were tested in terms of analyte recovery, reproducibility, simplicity and speed. Good results were obtained adding drop-by-drop 20 ml of acetone (in about 5-7 min) to 5 ml of milk sample at room temperature. Under these conditions the precipitate was constituted of small, easy-settling particles. Recovery, calculated by comparing samples spiked before and after precipitation at 50 ng/kg level, was 97±5%. When acetone was added all at once, the precipitate was formed of large, likely high-surface area particles, and recovery decreased to 86±10%. Moreover, in the case of relatively slow precipitation, centrifugation step could be very fast and the total drawing up of supernatant very easy. The choice of a GCB material for cleaning-up the extracts was explained in Preliminary experiments showed that AFM1 was totally retained from 2 L of water containing 20 ml of acetone by a cartridge filled with 250 mg of Carbograph-4. Moreover, 5 ml of methanol could be passed through the cartridge without any trace of the compound being eluted. 135

142 Chapter 3 Aflatoxins in milk AFM1 was desorbed from Carbograph-4 by a dichloromethane/methanol/acetic acid (88:10:2, v/v) mixture. In Table 3.1 the elution curves for AFM1 adsorbed on 250 mg of Carbograph-4 from 500 ml of water, by using different dichloromethane/methanol/acetic acid mixtures compared with the 90:10 (v/v) dichloromethane/methanol mixture, are reported. As can be seen, and as previously reported, although the compound is non-acidic by nature, only the addition of acetic acid to a mixture rich in dichloromethane allowed the total recovery of AFM1. This behaviour might be explained considering that acetic acid acts as competitor for dipolar interaction and hydrogen bonding between the OH group of AFM1 and the chemical heterogeneities on the Carbograph-4 surface. Table 3.1. The elution curves of aflatoxin M1 adsorbed on 6 ml cartridge filled with 250 mg of Carbograph-4 by using different dichloromethane/methanol/acetic acid mixtures. Fraction CH2Cl2:CH3OH:CH3COOH, v/v/v (ml) 68:30:2 78:20:2 88:10:2 93:5:2 90:10:0 Recovery (%) nd nd nd nd 3 nd = not detected The dilution of the acetonic extracts to 500 ml with water before the cleanup and washing the Carbograph-4 cartridge with 5 ml of methanol 136

143 Chapter 3 Aflatoxins in milk reduced matrix effect [Cavaliere 2005]. Recoveries from SPE step were obtained spiking milk samples before and after the Carbograph-4 extraction and following the remaining procedure. In this way, the effect of SPE on total recovery can be isolated and evaluated by comparing the absolute peak areas for the same compound in samples spiked ante and post extraction. Recoveries for AFM1 were 92±6% and 96±6% (n=5) at spiking level of 50 and 100 ng/kg, respectively Liquid chromatography-mass spectrometry The HN-APCI source was disregarded after preliminary experiments for lack of sensitivity. LC/ESI-MS/MS LC mobile phase optimization by ammonium formate addition to enhance sensitivity has been discussed in section. The fraction of extracted sample submitted to analysis is an important parameter in determining the method quantification limit (MQL). The injection of 15/400 of the extract reconstituted in the starting mobile phase for LC onto a 1 mm i.d. column was found as optimum. Under these conditions, the analyte and IS were well soluble, their peaks were not broadened and, comparing the slopes of calibration graphs constructed in standard solution and milk extract, matrix effect on signal intensity was negligible for both analyte and IS. Reducing the volume of solvent for sample reconstitution, the MQL did not improve significantly since chemical noise increased and matrix effect was not longer negligible. 137

144 Chapter 3 Aflatoxins in milk LC/APPI-MS/MS In this work the behaviour of AFM1 in the APPI source (both in RP- and NP-chromatography) was investigated deeply considering in particular the dopant effect to help ionization of the analyte. It is important to underline that in APPI, solvents must be carefully selected because they can heavily affect the response of analytes [Raffaelli 2003, Kauppila 2002]. RP-LC The water/methanol mixture offered much better ionization conditions than the water/acetonitrile one. This is probably due to the proton affinity of some of the isomeric forms of acetonitrile generated by photoionization as reported by Marotta et al. [Marotta 2003]. Three previously used dopants, such as acetone, toluene and anisole, added to the mobile phase at different percentages by means of a Harvard syringe pump connected to the auxiliary gas line with a capillary, were tested. Results are shown in Figure 3.1. As can be seen, the most intense signal was achieved by adding 20% toluene as dopant, but a good result was also obtained using acetone. Although rarely employed, acetone is amenable as organic modifier in RP- LC-MS [Liu 1995]; thus, to use as mobile phases water and methanol, both modified with 13% acetone (corresponding to 15% as external addition - that was the percentage giving the most intense signal with acetone) was tried. The rationale of this experiment was that a very homogeneous distribution of the dopant might improve the yield of proton transfer reaction to the analyte and reduce the noise. The experiment met with success, since an increment by a factor 2.5 in the signal-to-noise ratio (S/N) was obtained. 138

145 Chapter 3 Aflatoxins in milk Intensity (cps) toluene acetone anisolo 0 10% 15% 20% 25% 30% Dopant Figure 3.1. Effect of dopant agent addition on atmospheric pressure photoionization efficiency of aflatoxin M1 in a 6:4 (v/v) water/methanol mixture. APPI, as well as APCI, is a mass-flow dependent ionization technique. However, optimal flow rates of 200 µl/min [Yang 2002] or 500 µl/min [Takino 2004] have been reported. In Table 3.2 both the absolute intensity and signal-to-noise ratio obtained for AFM1 in the selected LC-MS/MS conditions at three different flow rates, such as 1.0, 0.5 and 0.2 ml/min, are shown. The last two flow rates were obtained by using a splitter. In this way, the amount of AFM1 that reaches the APPI source decreases as the splitted flow rate decreases. The aim of the experiment was to device the best column diameter (i.d.) to be employed considering that the maximum volume of sample that can be injected, as well as the optimum flow-rate, is depending on (i.d.) 2. Direct comparison can be easily done using the appropriate multiplicative factor. Although the highest signal was reached at a flow rate of 0.5 ml/min, better detection limit was achieved at

146 Chapter 3 Aflatoxins in milk ml/min due to very low background noise. This result does not mean that 1.0 ml/min is the optimal flow rate (and columns with i.d. = 4.6 mm the best choice) for LC/APPI-MS/MS in every case, because sometimes, as a consequence of injecting real samples, chemical noise, which is independent of the flow rate, may be predominant. Finally, 1/5 of the extract reconstituted in 500 µl of the starting mobile phase for LC could be injected without appreciable peak broadening and matrix effect on signal for both the analyte and IS. Table 3.2. The effect of flow rate on signal intensity and signal-to-noise ratio (S/N) of aflatoxin M1 analysed by RP-LC/APPI-MS/MS (see the text for chromatographic conditions). Flow rate (ml/min) Intensity (cps) S/N NP-LC Solvents like ethyl acetate, tetrahydrofurane and dichloromethane strongly suppressed the photoionization process. Thus, only the mixtures isoctane/isopropanol (with toluene as external dopant), isoctane/toluene/isopropanol and toluene/isopropanol were considered. In any case [M+H] + ion largely predominates over [M] + ; this behaviour can be considered in agreement with the results reported by Kauppila et al. [Kauppila 2002] for a molecule containing a carbonylic group (2-140

147 Chapter 3 Aflatoxins in milk acetonaphthone). The results reported in Figure 4.2 show that the highest S/N value was achieved for the mixture with lower isopropanol content. This finding clearly suggests using the least retentive among polar columns amenable for NP chromatography, such as the diol one. In addition, to improve efficiency, the column was thermostatted at 45 C and formononetin, a flavone, was preferred as IS because AFG1 signal in milk extract was irreproducible probably due to matrix effect. Also in this condition, 1/5 of the sample extract, reconstituted in 500 µl toluene, could be injected without noticeable matrix effect if the IS AFG1 was replaced with formononetin. S/N A = toluene:isopropanol:90:10 B = isoctane:isopropanol:toluene:33:33:33 C = isoctane:isopropanol:50:50 A B C Mobile phase Figure 3.2. Effect of different solvent mixtures suitable for normal-phase chromatography on signal-to-noise ratio (S/N) of aflatoxin M Recovery and precision Accuracy and precision were evaluated by spiking samples at three concentration levels, such as 0.05, 0.1 and 0.5 µg/kg. Precision was also measured for a milk sample found naturally contaminated at 18 ng/kg. 141

148 Chapter 3 Aflatoxins in milk Four samples per day were extracted and analysed randomly during a working week by LC/ESI-MS/MS. Results are reported in Table 3.3. Analyte recoveries were slightly dependent on spike level but in any case better than 90% and precision, expressed as RSD%, ranged 3-8%. Precision was also evaluated for LC/APPI-MS/MS with a sample found naturally contaminated at 14 ng/kg. RSD was 10% (n=5). Table 3.3. Recovery and precision of the LC/ESI-MS/MS method for determining aflatoxin M1 in milk spiked at three concentration levels. Concentration (µg/kg) Recovery (%) RSD a (%) b a n=5 b concentration of aflatoxin M1 in the milk sample used for recovery experiments Method performance comparison A comparison of performances among the three optimized LC-MS/MS methods for determining AFM1 in milk samples is summarized in Table 3.4. Slopes of regression lines measured in extracted samples did not differ significantly (p=0.05) from those obtained using standards in pure solvents; as an example, the regression lines for AFM1, and ISs behave in 142

149 Chapter 3 Aflatoxins in milk the same way are reported. Therefore, the methods did not appear to suffer from suppression phenomena due to coextracted and coeluted matrix compounds by virtue of the sample clean-up efficiency. Coefficients of determination (R 2 ) were also comparable. Noticeably, when acetone was added externally as dopant, the linear range for RP-LC/APPI- MS/MS was only The very low LOD (instrumental limit of detection, estimated as the amount of the analyte giving a S/N = 3) reached in LC/ESI-MS/MS was due to the sum of two factors: the effect of the ammonium formate and the choice of a microbore column. This LOD could not be totally exploited in terms of MQL (method quantification limit, estimated as the concentration of the analyte in the sample giving a S/N = 10) which was 12 ng/kg, since only a small fraction of the extract could be advantageously injected. RP- LC/APPI-MS/MS, taking advantage of an internal addition of the dopant, allowed obtaining a LOD three times higher than that for LC/ESI- MS/MS. However, since using LC/APPI a larger fraction of the extract that was suitable for LC/ESI could be injected, LC/APPI-MS/MS enabled a MQL of only 6 ng/kg. This result confirms the previous findings about the lower susceptibility to matrix effect of APPI respect to ESI ionization [Takino 2004]. 143

150 Table 3.4. Comparison among method performances. Regression equation a (standard solution) R 2 b Range (ng) Regression equation (sample) R 2 Intercept c (ng/µl) LOD d (ng) MIL e (ng/l) tr g MQL f (min) (ng/l) AFM1 IS RP-LC/ESI- MS/MS h y = x y = x NP-LC/APPI-MS/MS h y = x y = x RP-LC/APPI-MS/MS h y = x y = x a Weighted regression lines; y = peak area, x = analyte amount. b Coefficient of determination. c The intercept is due to the concentration originally present in the pooled extracts (n=5) used for the regression line. d Instrumental limit of detection. e Method identification limit (S/N= 3 for the second most intense transition in MRM). f Method quantification limit (S/N= 10). g Retention time. h For mobile phase compositions and gradients see the text.

151 Chapter 3 Aflatoxins in milk The only problem we experienced with the APPI source was sodium adducts instead of proton adducts formation, just for one working day, without any apparent reason. In Figure 3.3A and 3.3B are reported the chromatograms of milk extracts containing levels of AFM1 very close to the MQL for, respectively, LC/ESI-MS/MS and LC/APPI-MS/MS. As far as NP-LC/APPI-MS/MS is concerned, although the MQL was about half of EU legal limit, it did not offer any advantage. Furthermore, it made use of a relatively high volume of a toxic and dangerous solvent such as toluene. This result underlines that NP-LC/APPI-MS(/MS) can be favourably exploited only for those compounds which, like polycyclic aromatic hydrocarbons and di- and trichloroanilines, have very low affinity for the proton. Figure 3.3. Chromatograms showing method quantification limits for aflatoxin M1 in milk samples: (A) sample analysed by LC/ESI-MS/MS, calculated concentration 12.2 ng/kg; (B) sample analysed by LC/APPI-MS/MS, calculated concentration 6.3 ng/kg. 145

152 Chapter 3 Aflatoxins in milk Real sample analysis The applicability of the developed method was investigated by analysing ten milk samples of different typologies and brands commonly found in stores of Rome area. The samples were analysed in duplicate by RP-LC coupled to both ESI- and APPI- MS/MS; the concentrations obtained by the two methods correlated well and results are reported in Table 3.5. The levels of AFM1 found in all the analysed commercial samples had contamination levels under the maximum limit fixed by EU for adult human consume, but it is noticeable that in some samples the level of AFM1 found exceeds the value established for baby food products (25 ng/kg). This is really important because common commercial milk is often drunk by young children and sometimes by infants also. Table 3.5. Levels of aflatoxin M1 in ten samples of milk analysed by using LC/ESI-MS/MS and LC/APPI-MS/MS. Brand Milk typology aflatoxin M1 (ng/kg) ESI-MS/MS APPI-MS/MS 1 Pasteurized HQ a Pasteurized b Skimmed c Pasteurized HQ Pasteurized Skimmed UHT d Pasteurized Pasteurized Pasteurized <MQL 6.3 a High quality: fats >3.5%, proteins >3.2%. b Fats >3.5%, proteins >2.8%. c Fats %. d Ultra high temperature treated. 146

153 Chapter 3 Aflatoxins in milk Comparison between aflatoxins M1 and B1 Since determination of AFM1 in milk as well as AFB1 in feed operating in the same instrumental parameters may be of interest, the AFB1 behaviour under the best conditions selected for AFM1 was tested. Although structurally very similar, the two compounds behaved rather differently with both ESI and APPI sources. ESI In Figure 3.4A and 3.4B are reported, respectively, the Collisionally Induced Dissociation (CID) spectra of protonated AFB1 and AFM1 molecules, respectively, obtained at the same collision energy using the Q- TRAP mass spectrometer in the QqQ configuration. Under these conditions the fragmentation pattern seems indicate AFM1 predominant losses from difuranic hydroxylated ring (-18, -28, -56, -70, -100 Da), whereas fragmentation for AFB1 involves the whole structure (e.g. also -44 Da from lactonic ring) with successive losses, till the tropilium ion (91 m/z) stable structure is reached. Positive ion fragmentation processes upon low energy CID are charge-driven and it might be supposed that the hydroxyl group in the difuranic structure of AFM1 hinders, by internal hydrogen bond formation, the protonation of the basic oxygen in the lactonic structure. Whatever reason it may be, as a result the total ionic signal for AFM1 was less intense but also less widespread than that for AFB1. Under the most favourable conditions, such as 2 mmol/l ammonium formate in the mobile phase and low collision energy in Q2, instrumental LOD for AFM1 is only two times higher than that for AFB1. 147

154 Chapter 3 Aflatoxins in milk Figure 3.4. CID MS/MS spectra of aflatoxin B1 (A) and M1 (B). Q2 entrance potential 30 V. APPI The AFB1 response was also tested in the optimum RP-LC condition found for AFM1, and selecting the transition pairs and m/z. The LOD for the former compound was about 5 times lower than that for the latter one. 148

155 Chapter 3 Aflatoxins in milk Enhanced Product Ion (EPI) scan acquisition Triple-quadrupole instruments are generally used in MRM mode for accurate, precise and sensitive quantitation and confirmation of mycotoxins at trace levels in food matrices. Low detection limits can be achieved, but fragmentation pattern information is partially lost. It has been reported that, using the QqLIT mass spectrometer in EPI scan mode, full-scan spectra can be obtained for an extracted ion chromatogram (XIC) peak with a S/N about half that for the corresponding transition in MRM [Kim 2002, Xia 2003]. On the other hand, full-scan product ion spectra (EPI data) contain more structural information about the target compound. However, MRM acquisition mode is preferred for routine analyses. Unlike in a quadrupole, the LIT is able to trap and then accumulate ions by appropriate setting of the LIT fill time parameter, resulting in a requirement for relatively frequent cleaning of the LIT. A very high specificity and confidence for analyte identification at concentration level close to the MQL in MRM mode could be achieved operating the Q-TRAP mass spectrometer in the QqLIT configuration. In Figure 3.5 are reported the background-subtracted (ESI)EPI spectra of AFM1 obtained by injecting 0.15 ng of AFM1 standard solution (A) and a milk extract containing 14 ng/kg (B). As can be seen by comparing the spectra, the characteristic fragment ions (namely 301, 283, 273, 259, 229 and 203 m/z) are all present in the sample spectrum at a relative intensity in compliance with the Commission Decision 2002/657CE [Table 4 of 2002/657/EC]. This demonstrates that with the new hybrid quadrupole-linear ion trap instrument, more stringent confirmatory data can be achieved without narrowing linearity range in instrumental response. 149

156 Chapter 3 Aflatoxins in milk Figure 3.5. Background-subtracted Enhanced Product Ion (EPI) spectra of aflatoxin M1 obtained in the quadrupole linear ion trap configuration by injecting 0.15 ng of AFM1 standard solution (A) and a milk sample extract containing 14 ng/kg (B) Conclusions A two step isolation procedure (proteins precipitation and SPE by a Carbograph-4 cartridge) produced milk sample extracts amenable for accurate and precise determination by LC-MS/MS with external calibration of AFM1 levels in compliance with current EU regulations. Both positive ESI and APPI sources could be used. Although the APPI gave the best result in term of MQL, ESI may be preferred for three reasons: 1) MQL is still four times lower than the maximum level allowable and the other performances are similar; 2) the APPI source until now is not yet widespread; 3) the mechanism of the APPI process is still not completely understood and the response of a compound may differ remarkably for apparently very little or no change in the source conditions (we 150

157 Chapter 3 Aflatoxins in milk experienced for one working day sodium adducts instead of proton adducts formation without any apparent reason). An additional reason for preferring the ESI interface is that it was found to be much more rugged, maintaining sensitivity after many injections of dirty samples and requiring significantly less maintenance than the APPI interface. In spite of these faults, the APPI source shows very promising performances in the field of aflatoxin LC-MS/MS analysis. 151

158

159 Chapter 4 AFLATOXINS IN CHEESE 153

160

161 Chapter 4 Aflatoxins in cheese Chapter 4 AFLATOXINS IN CHEESE 4.1. Background As already said, AFM1 content in milk was shown to vary from animal to animal, from day to day, and from one milking to the next [Pittet 1998, Van Egmond 1986]. AFM1 is relatively stable in raw and processed milk products, and is slightly affected by pasteurization or processing into cheese [Sarımehmetoglu 2004, Yaroglu 2005]. In addition, the protein fraction of milk, in particular casein, binds AFM1 [Brackett 1982c], thus, if raw milk contains AFM1, cheese made from such milk will also contain AFM1 [Barbieri 1994, Blanco 1988, Yaroglu 2005]. Cream and butter contain lower concentrations of M1 than the milk from which these products are made, while, cheese contains higher concentrations of AFM1. Even if the presence of AFM1 in cheese seems to be very variable [López 2001], it has been observed that AFM1 concentrations was times higher in soft cheeses and times higher in hard cheeses than in the milk from which the cheeses were made [Yousef 1989]. These values can be ascribed to the condensation of the raw material occurring during cheese manufacture [Kaniou-Grigoriadou 2005]. In relation to cheese, the presence of aflatoxins may be fundamentally due to three possible causes: (i) presence of AFM1 in milk with which cheese are elaborated, as a consequence of carry-over of AFB1 from contaminated cow feed to milk, (ii) synthesis of AFs (B1, B2, G1 and G2) by fungi which grow on cheese such as Aspergillus flavus, A. parasiticus and A. nomius, though the scarce presence of carbohydrates does not make it a very suitable substrate 155

162 Chapter 4 Aflatoxins in cheese [Zerfiridis 1985], and (iii) the use of powdered milk with AFM1, to enrich milk employed in cheese elaboration [Blanco 1998]. Another aspect to be taken into account is the use of different processing methods for cheese manufacture. Several investigations on the partitioning of AFM1 during cheese manufacture reported a wide range of distribution of AFM1 between whey and curd, also because of starting with different milk contamination levels. Some authors observed that half or more of the AFM1 was in the whey [Blanco 1988, Grant 1971, Purchase 1972, Stoloff 1975, Stubblefield 1974, Wiseman 1983] In contrast, others reported that most of AFM1 was with the curd [El Deeb 1992], someone else that the amount in whey and curd is approximately the same as in the original milk [Yousef 1989]. These contrasting results can be ascribed to different factors such as different type and degree of milk contamination, differences in milk quality, type of cheese, presence of a small portion of curd in whey which could influence AFM1 concentration, cheese manufacturing procedures and the analytical methods employed [Blanco 1988, Sarımehmetoglu 2004]. Besides aflatoxin M1 seemed to occur predominantly with casein, it is highly probable that cheese curd contained a higher concentration than whey [Yousef 1989]. Contradictory data have also been published about AFM1 recovery after cheese preparation. Thus, the first studies showed variable losses of AFM1, during cheese manufacture [Grant 1971, López 2001, Purchase 1972, Stubblefield 1974]. On the other hand, subsequent research [Applebaum 1982d, Brackett 1982b,c,d, Van Egmond 1986] showed an increase in the AFM1 concentration after the process, so that cheese or curd made from 156

163 Chapter 4 Aflatoxins in cheese naturally contaminated milk contained a greater concentration of the toxin than was in the original milk [Brackett 1982a,b]. These differences may be due, not only to the use of different analytical methods for AFM1 determination but also to different recoveries of AFM1 related to the chemical composition of the cheeses [López 2001], cheese type, and amount of water eliminated during processing [Galvano 1996]. During the second phase of cheese manufacture, ripening, some discrepancies were found in the stability of aflatoxin M1, but, in general, it did not appear to be degraded during ripening. Examination of different types of cheese showed high stability of AFM1 during maturation and storage [Brackett 1982b, Applebaum 1982a]. The population can therefore be indirectly exposed to aflatoxins not only by the consumption of milk but also of milk products such as cheese [Dragacci 1996, Pietri 1997]. Hence, the detection and determination of AFM1 in dairy products, particularly in cheese (where there is an AFM1 enrichment factor), is of increasing interest [Kaan Tekinşen 2005]. As to cheese, Switzerland and Turkish have introduced a legal limit for AFM1 to 0.25 µg/kg [FAO 1997, Van Egmond 1989a, Turkish Food Codex Regulation 1997]. At present, European Union has not imposed a maximum tolerance level for aflatoxin in cheese, even if Italian Ministry of Health has posed a provisional limit to 0.45 µg/kg for AFM1 in hard cheeses [Italian Health Department 2004] (eg. Grana Padano and Parmigiano Reggiano type). This limit is higher than the Swiss one. However, it is provisional and it is only for matured hard cheese that, eventhough starts from the same initial milk contamination, reaches higher toxin concentration at final process because of loss of water. 157

164 Chapter 4 Aflatoxins in cheese Immunological methods such as enzyme-linked immunosorbent assay (ELISA) are commonly used for screening purpose [Aycicek 2005, Blanco 1988, Kaniou-Grigoriadou 2005, López 2001, Sarımehmetoglu 2004]. ELISA is not fully reliable due to cross-reaction interferences, especially at concentrations just lower than 0.05 µg/kg [Biancardi 1997]. To allow an effective control of the contamination of cow milk and dairy products by AFM1, very sensitive and reliable analytical methods have been developed. Many of them are based on solvent extraction followed by partition with hexane or heptane to eliminate fat share [Aycicek 2005, Blanco 1988, Dragacci 1995, Kaniou-Grigoriadou 2005, López 2001, Manetta 2005, Sarımehmetoglu 2004]. Clean-up is then obtained by solidphase extraction (SPE) or immunoaffinity chromatography in combination with reversed-phase HPLC and fluorescence detection with or without derivatization [Abdulkadar 2000, Barbieri 1994, Kaan Tekinşen 2005, Manetta 2005, Pietri 1997, Piva 1987, Prado 2000]. There are still very few published methods for AFM1 analysis in complex food matrices like cheese and only one utilized LC-MS as identification and quantification technique [Kokkonen 2005]; however, the limit of quantification (LOQ) achieved for AFM1 is higher (0.6 µg/kg) than the proposed limit. The matrix complexity can give rise to an unpredictable matrix effect so, although very selective, also LC-MS/MS may require time and labour intensive sample preparation steps. Matrix solid-phase dispersion (MSPD) is an extraction technique suitable for extracting selected analytes from liquid or soft solid samples [Barker 2000, Tolls 2003]. The use of hot water as an effective extractant for solid samples was proposed by Hawthorn et al. [Hawthorne 1994]. The polarity of water 158

165 Chapter 4 Aflatoxins in cheese decreases as the temperature is increased. This means that selective extraction of polar and medium polar compounds can be performed by suitably adjusting the water temperature. Recently, hot water extraction (HWE) has been proposed as an alternative extraction procedure for residue determination in food, in which sand as dispersing solid was employed [Bogialli 2003a]. The aim of work described in this chapter was to develop and evaluate a LC-MS/MS method for determining AFM1 in sample of Grana or Parmigiano cheeses (typical hard, long maturing cheeses) and mozzarella (a typical fresh cheese) at concentration below the provisional adopted limits. Two sample pre-treatment procedures that don t make use of immunosorbent cartridge were compared Experimental Samples Two different kinds of cheese were chosen: one was hard, cooked and long matured cheese, with water content of about 30-32%, protein 34% and fats 29%; the other was a fresh cheese whose medium composition is water 59%, protein 19% and lipidic content 20%. Several P.O.D. (Protected Designation of Origin) Italian hard cheeses (Parmigiano Reggiano and Grana Padano) [1996/1107/EC] and different brands of mozzarella were randomly purchased from local retail markets and drug stores in Rome. Sampling for hard cheeses was done by the aid of the retailer. A whole cheese (about 40 Kg) was split lengthwise and about 500 g piece was cut through the shape diameter, whereas for mozzarella a whole cheese ( g) was taken. The samples were purred in plastic bags (for food use) 159

166 Chapter 4 Aflatoxins in cheese and transferred to the laboratory in an ice box. In the laboratory, cheeses were stored in a refrigerator (4 ± 2 C). All samples were analysed within a week. From these global samples were obtained laboratory samples of about 200 and 50 g for hard cheese and mozzarella, respectively. Hard cheese sample was finely grated, whereas mozzarella was cut into small pieces and homogenized. Thereafter, sub-samples of about 1 g were taken for analysis Sample preparation Procedure I: Solvent Extraction/MSPD One gram of hard cheese was placed into a 50 ml polycarbonate tube followed by adding 10 ml of dichloromethane, homogenized twice for 10 s using a Polytron homogenizer. The homogenize was then placed on the top of a sand cartridge, prepared by filling 6 ml tubes with about 3 g of the solid placed between two frits, positioned in the vacuum manifold and filtered. The vacuum was adjusted to the maximum and the extract was collected into a 3 cm i.d. round-bottomed glass tube. The extraction vessel was washed with 10 ml of the extracting solvent, and this washing was also passed through the sand cartridge and collected in the tube. The same procedure was applied to mozzarella cheese with the exception of solvent used for extraction, consisting in acetone. The filtrate was evaporated near to dryness in a bath set at 40 C under a gentle stream of nitrogen. One gram of C18 was added to the oily residue and mixed with a small spatula until a homogeneous dry powder was obtained. Finally, the matrix dispersed sample was packed into a 6 ml 160

167 Chapter 4 Aflatoxins in cheese polypropylene cartridge pre-filled with 250 mg of the same C18 material, and a frit was placed above the packing material. C18 was washed immediately prior to use with 2 ml of methanol. The MSPD packed column was placed onto a vacuum manifold and the analyte was recovered by passing through the cartridge, at a flow rate of about 1-2 ml/min, a methanol/water mixture (80:20, v/v). Ten ml of eluate were collected in a graduate tube. Procedure II: HWE One gram of cheese was taken. Cheese was placed in a porcelain mortar with 4 g of sand and blended with the pestle, until homogeneous and dry material was obtained. This material was packed into the extraction cell, taking care to tap the tube to avoid loose packing of the particles. Any void space remaining after packing the solid material was filled with sand. Stainless steel frits were located above and below the packing. The tube was put into the oven and filled with a mixture of water/methanol (90:10, v/v) at room temperature till the first drop go out. At this point, the output needle valve was closed and the oven was heated at 150 C for 5 min. The valve was opened, keeping a 10 atm pressure, and the analyte was eluted from the cell by the water/methanol solution at 1 ml/min flow rate, collecting 20 ml in a calibrate glass tube. Clean-up The extracts coming from procedure I or procedure II were cleaned-up by a Carbograph-4 SPE cartridge prepared by filling 6 ml polypropylene tubes with 250 mg of the adsorbent placed between two polyethylene frits. Before processing samples, the Carbograph-4 cartridges were attached to a 161

168 Chapter 4 Aflatoxins in cheese vacuum manifold apparatus and washed sequentially with 10 ml of dichloromethane/methanol/acetic acid (88:10:2, v/v/v), 5 ml of methanol and 10 ml of Milli-Q water. The extracts, diluted to 500 ml with water, were passed through the cartridge at a flow rate of about ml/min; the cartridge was than washed with 100 ml of water followed by 5 ml of methanol for decreasing the residual water content. The vacuum was adjusted to provide a flow rate of about 5 ml/min, and AFM1 was eluted from Carbograph-4 with 10 ml of dichloromethane/methanol/acetic acid (88:10:2, v/v/v), the eluate was then spiked with 25 µl of a I.S. solution 0.04 µg/µl. After evaporation, the residue was reconstituted with 500 µl of starting mobile phase for LC and the obtained solution was forced through a PTFE syringe filter (0.2 µm; 13 mm diameter). A 20 µl aliquot of the final solution was analysed by LC/ESI-MS/MS Recovery experiments Before analysis, samples were checked to evaluate their contamination level applying procedure I. Only aflatoxin-free or low contamination level samples were selected. Recovery studies were therefore carried out on three samples of mozzarella cheese (two analyte-free and one at µg/kg), two Parmigiano Reggiano samples (one analyte-free and one contaminated at µg/kg) and one Grana Padano sample (analytefree). One gram of sample was placed in a flat amber glass vessel and artificially fortified with variable volumes of AFM1 working standard solution diluted in 1 ml of acetone, taking care to uniformly spread it on the sample. An intimate contact between the analytes and the sample was obtained by mixing with a spatula for some minutes. The samples were 162

169 Chapter 4 Aflatoxins in cheese allowed to air drying at 25 C in a ventilate oven, to eliminate the organic solvent. The spiked samples were then treated following both extraction procedure described above, and analysed. Analysis of spiked and unspiked samples was conducted in duplicate LC-MS/MS analysis The purified samples were chromatographed on an Alltima C18 column (150 x 1 mm i.d., 5 µm particle size) from Alltech (Deerfield, IL, USA) with a Securityguard ODS, 4 x 2.1 mm i.d. precolumn (Phenomenex, Torrance, CA, USA). A gradient elution with acetonitrile/water (95:5, v/v) as mobile phase A, and water as mobile phase B, both containing 2 mmol/l ammonium formate was used. After an isocratic step at 20% A for 2 min, A was linearly increased to 85% in 7 min, then brought to 95% and held constant for 4 min. The flow rate was 50 µl/min. A Q-TRAP linear ion trap mass spectrometer, coupled with a turboionspray (TISP) was used and an Analyst software version was employed for data acquisition and processing. Mass calibrations and resolution adjustments on the resolving quadrupoles were automatically performed as described in where also the tuning parameters selected for detection of target compounds were reported. The [M+H] + ions of the aflatoxins (m/z 329 for both compounds) were used as parent ions. The daughter ions detected were: m/z 273 and 259 for AFM1 and m/z 311 and 243 for AFG1. 163

170 Chapter 4 Aflatoxins in cheese Quantitation and statistical evaluation Linearity was evaluated as reported in and a linear range between quantification limit and 70 ng (R 2 = ) was found (see also Table 3.4). AFM1 was quantified using external calibration, matrix matched, or standard addition procedures (see 4.3 section). For the external calibration, standard solutions were made at five concentration levels ( pg/µl). Matrix matched calibration was also made at the same five concentration levels ( pg/µl), by fortifying extracts obtained from an analyte-free sample. Standard additions procedure was done at three levels of spiking after evaluating the original contamination level Results and Discussion Extraction and Clean-up As reported in the introduction, one of the goals of this study was to obtain, from cheese samples, extracts amenable for LC-MS/MS analysis, with a simple, time effective procedure, without using the expensive immunosorbent cartridges. In chapter 3 a sample preparation method for determining AFM1 in milk consisting in a protein precipitation step with acetone, dilution of the acetonic extract with water and clean-up by GCB was described. Using the same scheme for cheese was an useless attempt. Dilution of acetonic extracts with water resulted in a foggy aqueous samples that plugged the SPE cartridges. This effect was likely due to the coextracted lipidic fraction more abundant in cheese, especially hard, aged cheese, than in milk. A defatting step was necessary between extraction and Carbograph clean-up. The most widely used defatting method is 164

171 Chapter 4 Aflatoxins in cheese partition of fats by liquid-liquid extraction (LLE) [Aycicek 2005, Dragacci 1995, Kaniou-Grigoriadou 2005, Kokkonen 2005, López 2001, Manetta 2005, Sarımehmetoglu 2004] but several were shortcomings of this method. The extensive use of glassware may result in cumulative loss by adsorption on glass of hydrophobic analytes. LLE requires the use of relatively large amounts of highly purified solvents that are expensive as well as flammable and toxic. Vigorous shaking of solvent and liquid matrix may create serious emulsion problems, owing to the presence in the sample of natural surfactants. Emulsions can be eliminated only by additional time-consuming operations. MSPD can overcome these drawbacks because the analytical protocol is drastically simplified and shortened, the possibility of emulsion formation is eliminated and solvent consumption is substantially reduced. As an alternative, a more selective extraction step, based on the versatility of water as extracting solvent when the temperature is properly adjusted, was tested. Therefore, in this chapter, the development and evaluation of two different sample preparation procedures are described. In the first one, the analyte extraction was performed by homogenization with an organic solvent (dichloromethane or acetone); this extraction is very effective but not selective, therefore needs extensive clean-up. The second developed protocol was based on a more selective extraction, carried out with a hot water/methanol solution (90:10, v/v), followed by a simpler purification step. 165

172 Chapter 4 Aflatoxins in cheese Procedure I: Solvent Extraction/MSPD/Carbograph-4 clean-up Initial extraction with acetone gave the best recovery for a watery fresh cheese such as mozzarella whereas dichloromethane was a better extracting solvent for the hard cheese type tested. This fact can be easily explained considering the relative content of water and fats. One gram of C18 material was a sufficient quantity to obtain a dry matrix dispersion from the oily residue obtained after solvent removing, amenable for packing an extraction cartridge. The preloaded packing was necessary for optimal defatting, that was reached also by selective elution using an appropriate volume of methanol/water mixture (80:20, v/v). This eluate can be diluted with water and passed through the Carbograph-4 clean-up cartridge. Procedure II: HWE/Carbograph-4 clean-up The influence of extraction parameters versus the analyte recovery was studied. All parameters were evaluated with triplicate experiments by comparing a hard cheese sample aflatoxin-free spiked before and after extraction with AFM1 at 0.45 µg/kg. Briefly, initial extraction experiments were performed by using pure water heated at 120 C as extractant and the recovery of AFM1 was 52±19%. Increasing the extraction temperature to 150 C improved recovery to 68±16% and using an extraction temperature of 180 C did not improved significantly AFM1 recovery, this was likely due to some thermal decomposition [Laganà 2000]. Therefore, to enhance the extraction efficiency without thermal degradation, 10% of methanol was added to water operating at 150 C: this modification was enough to increase recovery to 85±12%. Doubling methanol percentage, AFM1 amounts removed from cheese sample did not significantly 166

173 Chapter 4 Aflatoxins in cheese increase, but the extract appeared to contain a greater amount of matrix components compared with one obtained with 10% of methanol. Extraction with hot liquids can be performed in static mode, dynamic mode, or a combination of them [Bruno 2002]. Using 20 ml of a water/methanol solution (90:10, v/v) at 150 C, the effect of the static extraction period duration on analyte extracted amounts was investigated, varying the time from 0 to 10 min. When the static extraction step was not performed, lower recoveries were obtained and more failed extraction happened, due to clogging of the extraction cell. Static extraction time exceeding 5 min did not yield higher efficiency. Thus, the extraction was performed, as reported in the experimental section, using 20 ml of a water/methanol solution (90:10, v/v) at 150 C and with a static extraction time of 5 min. This extract was amenable for Carbograph-4 clean-up after dilution with water Method comparison Matrix effect Matrix effect, for both procedures, was evaluated by comparing analytefree samples spiked with a solution containing AFG1 and AFM1, after clean-up, with a reference solution. Practically, two Mozzarella cheese samples and two hard cheese samples (one Parmigiano Reggiano and one Grana Padano) were extracted as reported under the two Experimental Procedures. Before evaporation, the SPE-eluates were spiked with AFM1 and AFG1 at 0.45 µg/kg and 1 µg/kg level, respectively. Quantification of two compounds was performed by comparing their absolute peak areas to those obtained injecting a standard solution. The results are reported in 167

174 Chapter 4 Aflatoxins in cheese Table 4.1. As can be seen the procedure II exhibits a more prominent matrix effect, especially analysing hard cheese, this demonstrate the effectiveness of MSPD step included in procedure I. Table 4.1. Matrix effect obtained using the two sample preparation procedures (n=4). Procedure I: Solvent Extraction/MSPD Procedure II: HWE Mozzarella Cheese Hard Cheese Mozzarella Cheese Hard Cheese Relative peak area a (RSD) AFM (5%) 0.84 (9%) 0.81 (7%) 0.52 (11%) AFG (6%) 0.89 (7%) 0.80 (8%) 0.61 (9%) a Peak area of the analyte injected from a cheese extract relative to that of the analyte injected from a standard solution. Recovery and precision Recovery experiments were performed on artificially fortified cheese samples, at two contamination level: 0.25 µg/kg and 0.45 µg/kg which were, respectively, AFM1 Switzerland and Turkish legal limit, and provisional limit in hard cheeses of Italian Minister of Health. Recoveries were calculated by comparison with samples fortified after clean-up, excluding in this way matrix effect. Results of these experiments are summarized in Table 4.2. Recoveries for method I ranged between 81 and 92% with relative standard deviation not larger than 7%, while method II 168

175 Chapter 4 Aflatoxins in cheese seems to provide a little lower performance, although none of data were significantly different at p=0.05. Table 4.2. Recoveries and precision (n=6). Procedure I: Solvent Extraction/MSPD Procedure II: HWE 0.25 μg/kg 0.45 μg/kg 0.25 μg/kg 0.45 μg/kg R% (RSD) Mozzarella Cheese Hard Cheese 92 (5%) 90 (3%) 79 (7%) 82 (8%) 83 (6%) 81 (7%) 84 (11%) 80 (15%) Quantification limit Instrumental LOQ was estimated from the LC-MS MRM chromatogram resulting from analysis of AFM1 standard solution at 0.1 pg/µl concentration (2 pg injected) and its value was about 0.6 pg. Method Quantification Limits (MQLs) for both procedures were calculated analysing real contaminated samples, at a concentration close to 10 times signal to noise, and correcting the obtained values for calculated procedure recovery. Data are listed in Table 4.3. In the same table, in order to well compare the increase of MQLs as regards LOQ, due to recovery and matrix effect, LOQ values are reported as concentration, rather than as injected amount. 169

176 Chapter 4 Aflatoxins in cheese Table 4.3. Comparison among method quantification limit using the two sample preparation procedures. Procedure I: Solvent Extraction/MSPD Procedure II: HWE LOQ a (ng/kg) MIL b (ng/kg) MQL c (ng/kg) MIL (ng/kg) MQL (ng/kg) Mozzarell a Cheese Hard Cheese a Instrumental limit of quantification (S/N =10). b Method identification limit (S/N =3 for the second most intense transition in MRM). c Method quantification limit (S/N =10). As can be seen, although both procedures have MQLs below the lower current legal limits, the first procedure gave better MQLs than the second one. This is due to an enhanced purification that minimizes matrix effect and chemical noise. This could be important when, fixed a legal limit, there is the need of a confirmatory method having well defined performance criteria. For example, a recent decision of European Union [2002/657/EC] establishes that for undoubted compound identification, an S/N>3 should be achieved, at a maximum tolerable level, for the worst transition. In Figure 4.1 are reported the extracted ion current profiles for the second most intense transition of AFM1 relative to a Parmigiano Reggiano sample analysed after procedure I (A) and II (B). The contaminant level found was 0.34 µg/kg and S/N ratios were140 and 17, respectively. 170

177 Chapter 4 Aflatoxins in cheese A B Figure 4.1. Chromatograms showing the extracted ion current profiles for the second most intense transition of AFM1 (0.34 µg/kg level) relative to a Parmigiano Reggiano sample analysed after sample preparation I (A) and II (B) Real sample analysis Some cheeses purchased from Italian retail markets were analysed. For mozzarella extracts prepared with the procedure I, for which no matrix effect was evidenced, the external calibration was applied. In a first attempt, for all other samples, matrix-matched calibration was used. Analysing some hard cheeses aged more than 20 months, very different values with the two procedures were found. This was due to the quantification method adopted: for hard cheeses matrix effect can vary with aging, and this outcome is more evident for the procedure II. In this case only the standard addition calibration gives right values and, as reported in Table 4.4, a good accordance with the two extraction procedures was obtained. 171

178 Chapter 4 Aflatoxins in cheese Moreover, very low contamination was found for mozzarella cheese, while for hard, aged cheeses almost all samples contained AFM1. This finding demonstrates that a careful control of AFM1 contamination in milk used for making cheese is necessary, particularly for cheese type intended for aging. 172

179 Chapter 4 Aflatoxins in cheese Table 4.4. Aflatoxin M1 levels in real sample. AFM1 (μg/kg) Sample Procedure I: Solvent Extraction/MSPD Procedure II: HWE Mozzarella nq a nq b Mozzarella a nq b Mozzarella nq a nq b Mozzarella a b Grana Padano (16 months ageing) Parmigiano Reggiano (18 months ageing) nq b nq b nq c nq c Grana Padano (20 months ageing) Grana Padano (20 months ageing) Parmigiano Reggiano (24 months ageing) Parmigiano Reggiano (24 months ageing) Grana Padano (24 months ageing) Parmigiano Reggiano (36 months ageing) Parmigiano Reggiano (36 months ageing) b c b c b c b nq c b c b c b nq c nq = not quantified a Quantified using external calibration. b Quantified using matrix-matched calibration. c Quantified using standard addition procedures. 173

180 Chapter 4 Aflatoxins in cheese 4.4. Conclusions Extraction procedures adopted in many analytical methodologies for determining contaminants in solid matrices are both time and solvent consuming. The LC/ESI-MS/MS confirmatory method described in this chapter has been developed to identify and quantify AFM1 in complex matrices such as cheese offering a valid clean-up alternative to expensive immunoaffinity columns and to liquid-liquid extraction for defatting method. Two different sample preparation procedure have been described and compared, both having MQL lower than the current legal limits. 174

181 Chapter 5 APPENDICES 175

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183 Chapter 5 Appendix 1 APPENDIX 1 Bioterrorism Mycotoxins can be used as chemical warfare agents. As a number of mycotoxins, which may be lethal in relatively low doses, may be cultured and grown on a wide variety of grains, the possibility of deliberate mycotoxin contamination of commodities and/or foods should be recognized by the food industry when developing defense plans. The impact of an intentional act of mycotoxin contamination could be severe, with potential public health outcomes involving high mortality and devastating economic consequences stemming from the corresponding impact on the healthcare system, public fear, and avoidance of affected products [Murphy 2006]. There is considerable evidence that Iraqi scientists developed aflatoxins as part of their bioweapons program during the 1980s. Toxigenic strains of Aspergillus flavus and A. parasiticus were cultured, and aflatoxins were extracted to produce over 2,300 litres of concentrated toxin. The majority of this aflatoxin was used to fill warheads; the remainder was stockpiled [Stone 2001, Zilinskas 1997]. Unlike the aflatoxins, trichothecenes can act immediately upon contact, and exposure of living beings to a few milligrams of T-2 is potentially lethal. In 1981, the Secretary of State Alexander Haig of the United States accused the Soviet Union of attacking Hmong tribesman in Laos and Kampuchea with a mysterious new chemical warfare agent (now known as nivalenon, deoxynivalenon, and T-2), thereby violating the 1972 Biological Weapons Convention. The symptoms exhibited by purported 177

184 Chapter 5 Appendix 1 victims included internal hemorrhaging, blistering of the skin, and other clinical responses that are caused by exposure to trichothecenes. The purported chemical warfare agent came to be known as yellow rain [Bennett 2003]. Prior to September 11, 2001, there was little concern pertaining to defence against intentional contamination. Because of this, grain storage and delivery systems, as well as food manufacturing plant security systems, deserve attention and crisis plans should be in place to deal with possible biological and chemical terrorism incidents. Where appropriate, these efforts should include mycotoxins. 178

185 Chapter 5 Appendix 2 APPENDIX 2 Environmental factors affecting mycotoxin production Mould growth and mycotoxin production are related to weather extremes (causing plant stress or excess hydration of stored feedstuffs), to inadequate storage practices, to low feedstuff quality and to faulty feeding conditions. In general, environmental conditions heat, water and insect damage cause plant stress and predispose plants in the field to mycotoxin contamination. Moulds grow over a temperature range of C, a ph range of 4-8 and above 0.7 aw (water activity - equilibrium relative humidity expressed as a decimal instead of a percentage). Moulds can grow on a dry surface, but can also grow on feeds containing more than 12-13% moisture [Whitlow 2002]. Mycotoxins can occur both in tropical areas and in temperate regions of the world, depending on the species of fungi. Both fungal growth and mycotoxin production are dependent on environmental factors, with the limits for mycotoxin production usually being narrower than those for growth only [Frisvad 1991]. The factors influence the fungus interacting with each other, either increasing or decreasing growth and mycotoxin production. The relevant factors for mycotoxin production are: Temperature. Mycotoxin production is greatly influenced by temperature and water activity. Usually mycotoxin production occurs at the same temperatures as the optimal growth. Penicillium grows well and produces mycotoxins at lower temperatures than Aspergillus. Aflatoxin synthesis can happen from C and is optimal from C. Both, A. flavus and A. 179

186 Chapter 5 Appendix 2 niger are able to grow between 8 and 45 C. Aflatoxin B1 production is stimulated by higher temperatures relative to aflatoxins G1. Optimal AFB1 production occurred between C whereas 23 C is optimal for AFG1 formation. Low temperatures (8-10 C) induce production of approximately equal amounts of aflatoxins B and G, however, total production is lowered and more time required. At 5 C Aspergillus cannot produce aflatoxins and ochratoxin anymore, whereas Penicillium and Fusarium are able to produce mycotoxins [Weidenbörner 2001a]. Water content. The water content of a substrate is given as water activity (aw) or as water content in percent (%). But using the second is problematic, as it includes also the bound water, which is unavailable for fungi. For this reason aw is the most commonly used value. Water activity is a measure of the availability of the water in the sample and not the water content [WHO 2002]. The aw is defined as the ratio of the vapor pressure of water in a material (p) to the vapor pressure of pure water (p0) at the same temperature: aw = p/p0. There are several factors, which control aw in a system. These factors are osmotic and matrix effects, that reduce the relative humidity as compared to pure water. aw is also temperature dependent. Most of food borne fungi grow at a minimal aw of 0.8, which is lower than the aw needed for bacterial growth (0.9). Xerophilic moulds grow at minimal aw from and can spoil low water activity products, for example grain, nuts, herbs, jam, dried fish and fruits. Foodstuffs with aw 0.6 are protected from microbial spoilage. The optimal aw for moulds are usually close to 1, for xerophilic fungi the values range from [Weidenbörner 2001a]. Mycotoxin production occurs at higher water contents than needed for growth. 180

187 Chapter 5 Appendix 2 Aspergillus species normally grow at lower water activities (aw of 0.78) and at higher temperatures than the Fusarium species. Therefore, Aspergillus flavus and aflatoxin are favoured by the heat and drought stress associated with warmer climates [Whitlow 2002]. Penicillium species grow at relatively low water activities and low temperatures and are widespread in occurrence. Because both Aspergillus and Penicillium can grow at low water activities, they are considered storage fungi [Christensen 1977], also if climate tropicalization makes these species to be field fungi too, so they can proliferate before harvest. ph. Most food borne fungi develop from ph 2.5 to ph 9.5 with an optimal ph from 4.5 to 6.5. Mycotoxin production usually takes place at a different ph optimum than fungal growth [Weidenbörner 2001a]. Atmosphere. According to Frisvad & Samson [Frisvad 1991] the concentrations of oxygen and carbon dioxide in the atmosphere and especially of dissolved oxygen in the substrate strongly influence growth and mycotoxin production by various moulds. The required amounts differ from species to species. Generally a combination of low oxygen content and high carbon dioxide inhibits growth and mycotoxin production of Aspergillus and Penicillium species. Taniwaki et al. [Taniwaki 2001] showed that in 40% CO2 and 1% O2 the growth of A. flavus in cheese is reduced by 65%, and the level of aflatoxin B1 production is insignificant. Also in the most favourable atmosphere studied (20% CO2 and 5% O2) the aflatoxin B1 production is reduced by a factor of 1000 compared to production in air. The level of cyclopiazonic acid production by Penicillium commune in 20% CO2 and 5% O2 decreased to 8% of that in air. 181

188 Chapter 5 Appendix 2 Substrate composition. Mould fungi are heterotroph organisms and therefore need organic compounds as glucose, maltose, saccharose and other water-soluble carbohydrates. Moulds cause mainly spoilage of carbohydrate-rich substrates, sometimes very specific to a certain composition. For example Penicillium crustosum, P. commune and P. echinulatum are common only on nuts and other lipid- and protein-rich substrates like meat and cheese [Frisvad 1991]. Microbial Competition. The presence of competing microorganisms can restrict fungal growth and mycotoxin production. For example Aspergillus niger, Rhizopus stolonifer or lactic bacteria decrease or inhibit aflatoxin production [Weidenbörner 2001a]. 182

189 Chapter 5 Appendix 3 APPENDIX 3 Using aflatoxin-contaminated corn [Vincelli 1995] Cleaning/screening Aflatoxins are often present in highest concentration in broken and cracked kernels. Cleaning grain with a gravity table or rotary screen can reduce the aflatoxin concentration of a corn lot. This is a practical option that is sometimes successful in reducing aflatoxin contamination. For some producers, cleaning it offers a first line of defence for dealing with contamination. However, research shows that aflatoxin levels are not always reduced following cleaning, especially in highly contaminated lots. This is because aflatoxins can be present at high levels in kernels that appear sound and undamaged. Cleaning has the best chance of significantly reducing aflatoxin content for lots with high levels of broken corn-foreign matter content and moderate levels of aflatoxins (below 100 ppb). Cleaning does offer the advantage of preventing the accumulation of fines and trash in the centre of the bin, which can improve air movement through the grain in storage. The method is legal and can be very effective, but it is important to remember that the screenings will then contain a potentially high level of aflatoxin contaminated material and should be used with care and should not be fed to any livestock. Feeding Feeding to appropriate livestock is probably the best use of most aflatoxincontaminated corn. On-farm feeding, or sale to a livestock operation instate or out-of-state, are all acceptable uses of the corn. Before feeding 183

190 Chapter 5 Appendix 3 contaminated corn to livestock, it is important to obtain one or more accurate estimates of the level of aflatoxins in the lot. There are no clear-cut safe levels for different animal species regarding resistance or tolerance to aflatoxins. However, aflatoxin-contaminated feed can be tolerated by some animals, particularly mature ones. Obviously, the higher the level of contamination, the greater the risk in feeding to animals. Furthermore, continued proper storage is essential so that aflatoxin levels do not continue to increase in the grain or prepared feed. Recognize that there may be other unidentified mycotoxins in corn invaded by Aspergillus or other fungi. Predicting the precise effects of utilizing feeds of known analytical composition is still difficult. Ethanol Production Aflatoxins do not appear in distilled alcohol, even when the corn has relatively high levels of toxin. The toxins are not degraded during fermentation and distillation but simply are concentrated in the spent grain. Thus, ethanol plants can utilize aflatoxin-contaminated corn, although they may prefer not to, because of a desire to use the spent grain as livestock feed. Blending One method of reducing moderate levels of aflatoxin contamination is to blend contaminated grain with clean grain. For reasons explained below, blending is intended only for on-farm use. If not done properly, blending poses the risk of contaminating clean corn with unacceptable levels of aflatoxins. Accurate sampling is essential if blending is to be successful. If contamination levels in one lot are much higher than measured, the entire 184

191 Chapter 5 Appendix 3 blended lot may become unacceptable. To ensure uniform mixing, lots to be blended should be fed into a common auger at rates needed to obtain the desired blend. Blending is not an approved for interstate commerce and blended lots are considered to be adulterated. Blending is a practice intended to reduce aflatoxin to acceptable levels in small lots only for on-farm use. In many cases, the best option for using aflatoxin-contaminated corn may be to segregate contaminated corn and feed it appropriately as described above, rather than blending. Detoxifying Binding agents Recent research has shown that the toxicity of aflatoxins may be influenced by dietary supplements. The addition of non-nutritive binding agents, such as the zeolite clays (sodium bentonite), and aluminosilicates have been shown to be effective protectants against aflatoxin toxicity. The basic mechanism for their action appears to involve aflatoxin chemisorption in the gastrointestinal tract of animals, resulting in a major reduction in aflatoxin bioavailability (see enterosorption at section). Furthermore, these chemical compounds decreased the level of aflatoxin M1 in the milk of lactating dairy cows (see also 3.1 section). However, these compounds have not been approved on aflatoxin binding claims. These compounds will not stop mould growth in the feed. Because there are no practical methods of economically decontaminating large volumes of aflatoxin-contaminated grain, the use of the chemical feed additives which bind the mycotoxin provides an option for using contaminated corn. 185

192 Chapter 5 Appendix 3 Ammoniation Detoxifying the grain with anhydrous ammonia is an alternative where the corn is to be used on the farm. Ammonia, applied as either a gas (anhydrous ammonia) or liquid (aqua-ammonia), reacts with the aflatoxin molecule to destroy its toxicity. Proper treatment can reduce aflatoxin concentrations by 95% or more. Swine and poultry may be reluctant to eat the treated grain if an ammonia smell is present, but otherwise no problems have been reported from feeding treated corn to livestock. While potentially valuable in some instances, ammoniation poses several problems. Ammoniation is not an FDA approved practice for corn in interstate commerce. Thus, treated corn must be used on-farm. In Europe this practice can t be use for corn. Concentrated anhydrous ammonia is hazardous to humans and livestock, explosive, and corrosive to equipment and storage bins. Ammoniation also discolors the grain, turning it a light caramel color which may be objectionable to buyers. Others Roasting corn at F (about C) can reduce in aflatoxin content ranging by 40% to 80%, with higher temperatures resulting in greater reductions. However, the temperatures required to reduce aflatoxin content are higher than those used in a normal corn roasting process. Some loss in feed value can be expected when using these temperatures for roasting. Ensiling of contaminated high-moisture corn does not adequately degrade aflatoxins. 186

193 Chapter 5 Appendix 4 APPENDIX 4 Liquid chromatography-mass spectrometry The development of liquid chromatography-mass spectrometric (LC-MS) techniques in the last few decades has made possible the analysis of trace amounts of analytes from complicated matrices. With LC, the analytes of interest can be separated from each other as well as from the interfering matrix, after which they can be reliably identified thanks to the sensitivity and specificity of MS. Mass spectrometers work by ionizing molecules and then sorting and identifying the ions according to their mass-to-charge (m/z) ratios. Two key components in this process are the ion source, which generates the ions, and the mass analyser, which sorts the ions. Several different types of ion sources are commonly used for LC-MS. Each is suitable for different classes of compounds. Several different types of mass analysers are also used. Each has advantages and disadvantages depending on the type of information needed. The most ideal way to analyse a specific compound depends on its mass, its polarity and the degree to which the molecule will break down during ionization. It is possible to construct an idealized diagram showing molecular weight on one axis and polarity on the other (Figure A.1). In this diagram there are three separate regions indicated: APPI, APCI and ESI (the most modern LC-MS sources). The three methods work by doing the analyte molecules ionization and separation from the mobile phase at atmospheric pressure. Hence they are known as Atmospheric Pressure 187

194 Chapter 5 Appendix 4 Ionization, or API techniques. The analyte ions are then mechanically and electrostatically separated from neutral molecules. Figure A.1. Idealized diagram showing the modern methods used for MS detection of organic molecules based on molecular weight and relative polarity. Electrospray Electrospray ionization (ESI) is the most commonly used of the API techniques and can work with an extremely large range of compound polarities. Electrospray relies in part on chemistry to generate analyte ions in solution before the analyte reaches the mass spectrometer. In ESI, gasphase ions of the analytes are formed by using a high electric field. Best ionization is achieved when the analytes are already charged in solution, and therefore ESI is best suited for the analysis of polar and ionic compounds. 188