Chapter 10
Recent Developments in Trichothecene Analysis 1
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Michelangelo Pascale , Vincenzo Lippolis , Chris M. Maragos , and Angelo Visconti 1
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Institute of Sciences of Food Production, National Research Council (ISPA-CNR), Via G. Amendola 122/O, 70126 Bari, Italy Mycotoxin Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604 2
Trichothecenes are a group of toxic metabolites mainly produced by several Fusarium species occurring in cereals. Gas-chromatographic methods (GC-ECD or GC-MS) are widely used for quantitative determination of the more toxic type-A trichothecenes, i.e., T-2 and HT-2 toxins. Deoxynivalenol (DON), the main type-B trichothecene frequently occurring in wheat, is commonly detected by liquid chromatography (LC)-UV with good accuracy and precision. Recently, at ISPA-CNR a new method has been developed for the simultaneous determination of T-2 and HT-2 toxins in cereal grains at ppb levels using immunoaffinity column clean-up, labeling with 1-anthroylnitrile, and L C with fluorescence detection (FD). Moreover a fluorescence polarization (FP) immunoassay has been developed at USDAARS-NCAUR for rapid quantification of deoxynivalenol (DON) in wheat. Results of recent researches on the improvement of methods for the determination of T-2 and HT2 toxins in cereals by LC-FD and new labeling reagents (i.e., 1-naphthoyl chloride, 2-naphthoyl chloride, pyrene-1-carbonyl cyanide) and by capillary electrophoresis-laser induced fluorescence (CE-LIF) detection are presented, together with the optimization of the FP immunoassay for rapid screening of DON in common wheat, durum wheat, semolina, and pasta.
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© 2008 American Chemical Society
In Food Contaminants; Siantar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.
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Introduction Trichothecenes are a group of secondary metabolites produced by several fungal genera including Fusarium, Trichoderma, Cephalosporium, Myrothecium, Trichothecium and Stachybotrys (/). Trichothecenes produced by Fusarium species belong to two main categories: 'type-A' which are characterized by a functional group other than a carbonyl at C-8, and 'type-B' which have a carbonyl group at C-8. T-2 toxin and HT-2 toxin are type-A trichothecenes, while deoxynivalenol (DON), also known as vomitoxin, is a type-B trichothecene. These toxins are commonly found in cereals including wheat, barley, maize, oats, rye, and derived products, particularly in Europe and in cold and wet climatic regions. A recent survey on the occurrence of Fusarium toxins in food in the European Union (EU), in order to assess the dietary intake by the population of EU member states, showed an incidence of positive samples of 57% out of 11,022 samples analyzed for DON and 20% and 14% out of 3,490 and 3,032 samples analyzed for T-2 and HT-2 toxins, respectively. Wheat and maize were the cereals most frequently contaminated by DON; maize, wheat and oats by T-2 and HT-2 toxins (2). Similar results were reported on globally by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in the fifty-sixth report, which was on the safety evaluation of mycotoxins in food (3, 4). Toxicological studies have shown that T-2 toxin, HT-2 toxin and DON have strong inhibitory effects on protein, DNA, and RNA synthesis and cause toxic effects on cell membranes. In addition, T-2 toxin, which is considered the most toxic trichothecene, has immunosuppressive and cytotoxic effects, and has been shown to cause hémorragie diseases and necrosis and inflammation of the skin and mucosal surfaces. The little direct information on the toxicity of HT-2 toxin which is available indicates that it induces adverse effects similar to T-2 toxin. DON has also been shown to cause vomiting, haematic, and anorexic syndromes as well as neurotoxic and immunotoxic effects in mammals (5-5). Due to their toxic effects, the contamination of cereals and cereal-based products by trichothecene mycotoxins represents a real risk for human and animal health. In order to protect human health from exposure to these mycotoxins through the consumption of cereal-based food products, several countries have established regulatory or guideline limits in raw materials and foods intended for human consumption. In particular, a few dozen countries have set limits for DON, whereas Armenia, Belarus, Bulgaria, Estonia, Hungary, Latvia, Moldova, the Russian Federation, Slovakia, and Ukraine have fixed limits for T-2 toxin (6). The US Food and Drug Administration (FDA) has issued an advisory level of 1.0 ng/g for DON in milled wheat products (e.g., flour, bran, and germ) that may be consumed by humans (7). Harmonized regulations for DON in food, including official protocols for sampling and analysis, were implemented on July 1 2006 within the European Union, whereas permissible levels of T-2 and HT-2 toxins in cereal-based products are currently under discussion (8, 9). st
In Food Contaminants; Siantar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.
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194 Analytical methods for rapid, sensitive, and accurate determination of these mycotoxins in cereals and cereal-based products are required in order to protect consumers from the risk of exposure, to allow monitoring programs (rapid screening of materials in the food/feed production chain) and to ensure that regulatory levels fixed by the EU or other international organizations are met. Gas chromatographic (GC) methods based on electron-capture (ECD), flame ionization (FID), and mass spectrometric (MS) detection are the most widely used methods for quantitative determination of T-2 and HT-2 toxins. DON is commonly measured at ppb levels by high performance liquid chromatographic (LC) methods based on ultraviolet (UV) and MS detection. These methods require preliminary clean-up of extracts in order to obtain good sensitivity, are time-consuming, expensive, and unsuitable for screening purposes. Enzymelinked immunosorbent assay (ELISA) methods using polyclonal or monoclonal antibodies have been developed for rapid screening of trichothecenes in cereals. However, ELISA methods show strong cross-reactivity against mycotoxin analogues (i.e., acetylated-DON), involve multiple washing steps, and require long incubation times for complete antigen-antibody reaction (10, 11). Recently, a variety of emerging immunoassays have been proposed for the rapid analysis of DON or T-2 toxin in several matrices. They are based on novel technologies including lateral flow devices (LFD), membrane-based flow-through enzyme immunoassay,fluorescencepolarization (FP) immunoassay, and surface plasmon resonance (SPR) biosensors (12-14). This paper summarizes results of some of the recent research carried out at the Institute of Sciences of Food Production of the National Research Council (ISPA-CNR, Bari, Italy) and at the Mycotoxin Research Unit of the USDAARS-NCAUR (Peoria, IL, USA) aimed at improving determination of T-2 toxin, HT-2 toxin, and DON in cereals and cereal-based products.
LC with Fluorescence Detection for T-2 and HT-2 Toxins Intake estimates indicate clearly that the presence of mycotoxins in the diet at low levels can be of some concern for public health. Recently, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the Scientific Committee on Food (SCF) of the European Commision pointed out that data on the presence of T-2 and HT-2 toxins in food are limited and of low quality and they should not be used for intake estimations. In addition, the sensitivity of the analytical methods used was poor in many cases which can lead to an overestimation of the intake. Therefore the development of a sensitive method for T-2 and HT-2 toxins and collection of more data on their presence in cereal and cereal-based products, in particular in oats and oat products, are of high priority for reliable dietary exposure assessments (4, 8). Several chromatographic (TLC, GC-ECD, GC-FID, GC-MS and LC-MS) and enzyme-linked immunoassay (ELISA) methods have been developed for the determination of T-2 toxin, alone or in combination with other trichothecenes (10, 15). At present, GC with ECD or MS detection are the techniques most used for
In Food Contaminants; Siantar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.
Downloaded by PENNSYLVANIA STATE UNIV on July 1, 2012 | http://pubs.acs.org Publication Date: October 20, 2008 | doi: 10.1021/bk-2008-1001.ch010
195 quantification of type-A trichothecenes, although a pre-column derivatization step of the extracts is necessary to increase volatility of the toxins and provide higher sensitivity. Recently, a comparative collaborative study on method performance for analysis of trichothecenes (including T-2 and HT-2 toxins) by GC methods clearly showed that method improvements are needed with respect to recovery, accuracy, and precision of the measurements. The main problems are derived from matrix interferences that induced enhancement of the trichothecene response, with consequent overestimation of the contamination levels (16). LC with fluorescence detection (FD) gives high sensitivity, selectivity, and repeatability of measurements, but is not applicable to trichothecene detection due to the lack of appropriate chromophores in their structure. Although specific labeling reagents are commercially available for hydroxyl groups to form fluorescent esters, their use in trichothecene analysis showed low reaction yield or interfering peaks in the chromatogram. A simple procedure for the simultaneous determination of T-2 toxin, HT-2 toxin, T-2 triol, and T-2 tetraol by LC-FD has been developed (17). It involved the synthesis of the labeling reagent, coumarin-3-carbonyl chloride, an optimized derivatization reaction and a procedure for clean-up of the reaction mixture to remove excess reagents and by-products. Recently, the derivatizing reaction with coumarin-3-carbonyl chloride was used for developing a method for the determination of T-2, HT-2 toxin, neosolaniol, and diacetoxyscirpenol in cereal cultures of Fusarium sporotrichioides on maize, rice, and wheat by LC-FD after solid phase extraction (SPE) column clean-up. Although the method had good sensitivity, its applicability to cereal samples showed low toxin recoveries (18, 19). Two commercially availablefluorescentreagents for modifying alcohols, 9anthroylnitrile (9-AN) and 1-anthroylnitrile (1-AN) (Figure 1), were tested for labeling T-2 toxin in order to make it detectable by LC-FD. Due to its high efficiency for a variety of acylation reactions, the base 4-dimethylaminopyridine (DMAP) was used as catalyst for the reaction. The derivatization conditions were optimized by investigating different reaction solvents, reagent molar ratios, temperatures, and reaction times. Both 1-AN and 9-AN reagents reacted with the hydroxyl group of T-2 toxin under mild conditions to form the corresponding esters. The T-2-(l-AN) derivative gave up to 15 times higher fluorescence intensity than the T-2-(9-AN) derivative in all experiments performed. Experiments to test the stability of T-2 anthroyl ester showed no decrease of intensity offluorescenceof T-2 derivative up to 5 days for solutions stored in the dark and under light. The derivatizing reaction has been used to develop a sensitive, reproducible, and accurate method for the determination of T-2 toxin in wheat, corn, barley, oats, rice, and sorghum (20). The method uses immunoaffinity columns (IACs) specific for T-2 toxin for clean-up of cereal extracts, pre-column derivatization with 1-AN, and LC-FD for T-2 toxin determination. Recoveries from different cereals spiked with the mycotoxin at levels ranging from 50 ¿ig/kg to 1,500 ¿ig/kg were from 80% to 99%, with within-laboratory relative standard deviation (RSD ) lower than 6% for all spiking levels. The limit of detection was 5 ^g/kg, based on a signal-to-noiseratio of 3:1. The use of IACs allowed rapid clean-up and provided clean extracts r
In Food Contaminants; Siantar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.
Downloaded by PENNSYLVANIA STATE UNIV on July 1, 2012 | http://pubs.acs.org Publication Date: October 20, 2008 | doi: 10.1021/bk-2008-1001.ch010
196 to be analyzed by LC due to the specificity of the antibody. The method allowed the determination of T-2 toxin at ppb (ng/kg) levels in various cereals with good accuracy and precision, enabling measurement of the toxin at levels that can occur in naturally contaminated cereal samples (20). By applying the same derivatization procedure, a stable fluorescent derivative was obtained also with HT-2 toxin (75). In order to develop a method based on LC-FD and IAC clean-up for the simultaneous determination of T-2 and HT-2 toxins in cereal grains, the specificity of the anti-T-2 toxin antibody was evaluated against different structurally related trichothecene mycotoxins: deoxynivalenol (DON), nivalenol (NIV), T-2 toxin, T-2 triol, T-2 tetraol, HT-2 toxin, and acetyl T-2 toxin. The antibody cross-reacted 100% with T-2 and HT-2 toxins, and 90% with T-2 triol and acetyl T-2 toxin. The high cross-reactivity of the antibody with T-2 triol and acetyl T-2 should not be a problem for the LC determination of T-2 and HT-2 toxins after pre-column derivatization with 1AN. In particular, acetyl T-2, which lacks free hydroxyl groups, should not react with 1-AN, while T-2 triol, carrying three hydroxyl groups, should react with 1A N to form derivatives with polarity quite different from (and therefore not interfering with) the T-2 and HT-2 anthroylnitrile derivatives. Derivatization with 1-AN and IAC clean-up were successfully applied to the analysis of T-2 and HT-2 toxins in wheat, maize, and barley. Recoveries from spiked samples with toxin levels from 25 to 500 jig/kg ranged from 70 to 100%, with RSD lower than 8%. The limit of detection of the method was 5 ng/kg for T-2 toxin and 3 ¿ig/kg for HT-2 toxin, based on a signal-to-noise ratio of 3:1. The analytical method did not allow the determination of HT-2 toxin in oats because of interfering chromatographic peaks occurring at the retention time of HT-2-(lAN) derivative (15). The method was also applied to the determination of T-2 and HT-2 toxins in eggs (21). r
COCN
COCN
9-anthroylnltrlle (9-AN)
1-anthroylnltrile (1-AN)
O
II C—CN
1-naphthoyl chloride (1-NC)
2-naphthoyl chloride (2-NC)
pyrene 1-carbonyl cyanide (PCC)
Figure 1. Fluorescence labeling reagents for T-2 and HT-2 toxins (commercially available) In Food Contaminants; Siantar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.
197 In order to provide an improved LC-FD method with lower detection limits (LOD) for T-2 and HT-2 toxins in foodstuffs, including oats, three different commercially availablefluorescentreagents were tested (Figure 1). 1-naphthoyl chloride (1-NC), 2-naphthoyl chloride (2-NC) and pyrene-1-carbonyl cyanide (PCC) reacted with the hydroxyl groups of T-2 toxin and HT-2 toxin under mild conditions to form the corresponding esters (22). Excitation and emission spectra of the fluorescent derivatives were recorded and maximum excitation and emission wavelengths were selected. A wide linear range (10-1000 ng for either T-2 or HT-2 derivatized toxin), good repeatability (RSD
