Summary A total of twenty‐eight mycotoxins were surveyed in wine (red, white and rose), cider (white and rose) and their cork stoppers from eight countries. Toxins of different fungi genera were detected as follows: A lternaria ( AT s: alternariol – AOH ; alternariol methyl – AME ) and P enicillium/ A spergillus (ochratoxin A – OTA ; penicillic acid – PAC ). Toxins and levels varied with the sample types and country of origin. Wine presented contamination of OTA , AOH and AME . OTA was detected in forty‐one wine samples with levels ranging from 0.01 to 0.86 μg L −1 , below EU legislation. AOH and AME were detected in thirty‐three and eight of wines samples, respectively, at levels from 0.2 to 13.3 μg L −1 , while no contamination was detected in ciders up to the method LOQ s. Regarding the cork stoppers toxins detected, they were AOH , AME and PAC . Corks of red wine from different countries had levels of OAH and AME ranging from 5.0 to 101.0 and 2.5 to 5 μg g −1 , respectively. It is necessary to pay more attention on the corks processing and cork type used in the bottles as, different from the ordinary ones, the ground bark and compressed type did not have toxins detected.

The development of liquid chromatography-mass spectrometry (LC-MS) from a single mycotoxin confirmation technique into a multi-mycotoxin routine analytical method is described. LC-MS/MS not only has the advantage of measuring all regulated compounds in one single run, but also the possibility to discover unknown mycotoxins, or to find mycotoxins in matrices where they were never expected to be present or ever found before. After compilation of 45 methods published in the last eight years, the critical performance criteria in liquid chromatography and mass spectrometry are discussed. Typical problems like matrix effect, choice of internal standard and considerations concerning validation are worked out. Finally some future trends like Orbitrap, MALDI, DESI and the combination of multi-methods for different groups of chemical contaminants and residues are considered.

Mycotoxin analysis is usually carried out by high performance liquid chromatography after immunoaffinity column cleanup or in enzyme-linked immunosorbent assay tests. These methods normally involve determination of single compounds only. EU legislation already exists for the aflatoxins, ochratoxin A and patulin in food, and legislation will come into force for deoxynivalenol, zearalenone and the fumonisins in 2007. To enforce the various legal limits, it would be preferable to determine all mycotoxins by routine analysis in different types of matrices in one single extract. This would also be advantageous for HACCP control purposes. For this reason, a multi-method was developed with which 33 mycotoxins in various products could be analysed simultaneously. The mycotoxins were extracted with an acetonitrile/water mixture, diluted with water and then directly injected into a LC–MS/MS system. The mycotoxins were separated by reversed-phase HPLC and detected using an electrospray ionisation interface (ESI) and tandem MS, using MRM in the positive ion mode, to increase specificity for quality control. The following mycotoxins could be analysed in a single 30-min run: Aflatoxins B1, B2, G1 and G2, ochratoxin A, deoxynivalenol, zearalenone, T-2 toxin, HT-2 toxin, α-zearalenol, α-zearalanol, β-zearalanol, sterigmatocystin, cyclopiazonic acid, penicillic acid, fumonisins B1, B2 and B3, diacetoxyscirpenol, 3- and 15-acetyl-deoxynivalenol, zearalanone, ergotamin, ergocornin, ergocristin, α-ergocryptin, citrinin, roquefortin C, fusarenone X, nivalenol, mycophenolic acid, alternariol and alternariol monomethyl ether. The limit of quantification for the aflatoxins and ochratoxin A was 1.0 µg kg−1 and for deoxynivalenol 50 µg kg−1. The quantification limits for the other mycotoxins were in the range 10–200 µg kg−1. The matrix effect and validation data are presented for between 13 and 24 mycotoxins in peanuts, pistachios, wheat, maize, cornflakes, raisins and figs. The method has been compared with the official EU method for the determination of aflatoxins in food and relevant FAPAS rounds. The multi-mycotoxin method has been proven by the detection of more than one mycotoxin in maize, buckwheat, figs and nuts. The LC–MS/MS technique has also been applied to baby food, which is subject to lower limits for aflatoxin B1 and ochratoxin A, ergot alkaloids in naturally contaminated rye and freeze-dried silage samples.

A survey was conducted to determine the occurrence of mycotoxins in feedstuffs of dairy cows in the Netherlands and to estimate total dietary intakes of these compounds. Twenty-four dairy farms were visited twice and samples taken of all diet ingredients. Feed intake data were collected by means of questionnaires. A total of 169 feed samples were collected and analyzed for 20 mycotoxins using a liquid chromatography tandem mass spectrometry multimethod. Silage and compound feed were the main diet ingredients, representing on average 67 and 23% of dry matter intake, respectively. Deoxynivalenol (DON), zearalenone, roquefortine C, and mycophenolic acid were the mycotoxins with the highest incidence. The incidence of DON in silage, compound feed, and feed commodity samples was 38 to 54%. The incidence of zearalenone in silage, compound feed, and feed commodity samples was 17 to 38%. The DON and zearalenone had a low incidence in forage samples and were not detected in ensiled by-product samples. Roquefortine C and mycophenolic acid were only detected in silage and ensiled by-product samples (incidence 7 to 19%). Fumonisins B(1) and B(2) were detected in 2 compound feed samples and one feed commodity sample. Aflatoxins B(1), B(2), G(1), and G(2), ochratoxin A, T-2 and HT-2 toxin, 3-acetyl-DON, 15-acetyl-DON, diacetoxyscirpenol, sterigmatocystin, fusarenon-X, ergotamine, and penicillinic acid were not detected in any of the samples. Average concentrations of DON, zearalenone, roquefortine C, and mycophenolic acid in complete diets were 273, 28, 114, and 54 microg/kg, respectively. Maximum concentrations were 969, 203, 2,211, and 1,840 microg/kg, respectively. Calculated average daily intakes of these mycotoxins were 5.0, 0.5, 2.0, and 0.9 mg/animal, respectively, and maximum daily intakes 19.3, 3.5, 38.9, and 32.3 mg/animal, respectively. Corn silage was the major source of all 4 of these mycotoxins in the diet. Extremely high concentrations of roquefortine C and mycophenolic acid (up to 45 and 25 mg/kg, respectively) were detected in visibly molded areas in surface layers of corn silage. These areas appeared to be the main source of roquefortine C and mycophenolic acid in the diet. Because carry-over of DON, zearale-none, roquefortine C, and mycophenolic acid into milk is negligible, their occurrence in feedstuffs is not considered of significant concern with respect to the safety of dairy products for consumers. Potential implications for animal health are discussed.

The occurrence of mycotoxins in 140 maize silages, 120 grass silages and 30 wheat silages produced in the Netherlands between 2002 and 2004 was determined using a liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) multi-method. Deoxynivalenol (DON) was detected above the limit of quantification (LOQ) of 250 mu g kg(-1) in 72% of maize and 10% of wheat silages. Average DON concentrations were 854 and 621 mu g kg(-1), respectively, and maximum concentrations 3142 and 1165 mu g kg(-1), respectively. Zearalenone was detected above the LOQ of 25 mu g kg(-1) in 49% of maize and 6% of grass silages. Average zearalenone concentrations were 174 and 93 mu g kg(-1), respectively, and maximum concentrations 943 and 308 mu g kg(-1), respectively. The incidences and average concentrations of DON and zearalenone in maize silage were highest in 2004. The incidence of other mycotoxins was low: fumonisin B1 and 15-acetyl-DON were detected in 1.4 and 5% of maize silages, respectively, and roquefortin C in 0.8% of grass silages. None of the silages contained aflatoxins, ochratoxin A, T2-toxin, HT2-toxin, sterigmatocystin, diacetoxyscirpenol, fusarenon-X, ergotamine, penicillinic acid, or mycophenolic acid. This study demonstrates that maize silage is an important source of DON and zearalenone in the diet of dairy cattle. Since the carryover of these mycotoxins into milk is negligible, their occurrence in feed is not considered to be of significant concern with respect to the safety of dairy products for consumers. Potential implications for animal health are discussed.

A comparison was made between dry milling and slurry mixing as a comminuting step preceding mycotoxin analysis. Sample schemes of up to 30 kg are mandated by European Commission legislation. Cocoa, green coffee, almonds and pistachio samples of 10 kg were milled by a Romer analytical sampling mill and all three subsamples were analysed for aflatoxin B-1 or ochratoxin A content. The homogenization process was evaluated in terms of the analytical results, coefficients of variation for different mills and particle size distributions. Coefficients of variation for the comminuting step were higher for dry milling than for slurry mixing. This difference was explained based on measured particle size distributions for both milling types. Measurements also showed slight differences in mycotoxin content of samples based on milling procedures. This might lead to lots being wrongly accepted or rejected based on an erroneous subsample result. It was concluded that sample comminution was best performed by slurry mixing, which produced smaller particles and, consequently, homogeneous samples with lowest coefficients of variation. Additional data are given on analytical results in 10-kg subsamples that originate from the aggregate 30-kg sample as described in Commission Directive 98/53/EC.

Mycotoxin analysis is mainly carried out by high performance liquid chromatography after immunoassay clean-up or in enzyme-linked immunosorbent assay tests. These methods imply determination of single compounds. To maintain legal limits it would. be preferable to determine mycotoxins by routine analysis in different types of matrices in one single extract. This also holds for HACCP control purposes. Application of liquid chromatography-mass spectrometry made it possible to obtain such a method. Liquid chromatography separation of the Mycotoxins is done with a gradient of water and acetonitrile on a reversed-phase column within 25 minutes. The mass spectrometer is used with an electrospray ionisation interface in the positive mode. By multiple reaction monitoring it is possible to analyse fragment ions, to increase specificity for necessary quality control. Thus far, this multimycotoxin method has been developed to include aflatoxins B-1, B-2,G(1), and G(2) ochratoxin A, deoxynivalenol, nivalenol, fusarenon-X, 3 and 15-acetyl-deoxynivalenol, famonisins B-1, B-2 and B-3 diacetoxyscirpenol, zearalenone and derivatives, T-2 and HT-2 toxins, roquefortin, citrinin, cyclopiazonic acid, ergotamine, penicillic acid and sterigmatocystin. When the method was applied to food samples, i.e. peanut and cornflakes, a matrix effect showed up, which was expected due to the lack of clean-up. The matrix effect depends on the sample type. Validation data are presented for aflatoxins, ochratoxin A and deoxynivalenol in peanuts and cornflakes. The advantage of the multimycotoxin method has been proven by findings of more than one mycotoxin in maize, buckwheat, fig and pistachio samples. Recent developments are the application of the multimycotoxin method for baby food, which is subject to lower limits for aflatoxin B-1 and ochratoxin A, and for ergot alkaloids.

Sampling and sample preparation are issues that are generally underestimated in any analytical procedure. Tests can be as rapid as desired nowadays, whereas sampling and sample preparation are still time consuming, but nevertheless crucial steps, due to the fact that the judgement of a lot is based on the total procedure. In this contribution details of these aspects will be outlined in the field of primary agricultural and horticultural products, as to be precisely: mycotoxins, pesticides and genetically modified organisms (GMOs). From these diverse examples it will be clarified that these basic concepts can be applied to any type of other analytical problem.

At the end of April 2002, the Swedish Food Administration reported the presence of acrylamide in heat treated food products. Acrylamide has been shown to be toxic and carcinogenic in animals, and has been classified by the WHO/IARC among others as 'probably carcinogenic for humans'. The purposes of this study were firstly to analyse acrylamide contents of the most important foods contributing to such exposure, secondly, to estimate the acrylamide exposure in a representative sample of the Dutch population, and thirdly to estimate the public health risks of this consumption. We analysed the acrylamide content of foods with an LC-MS-MS method. The results were then used to estimate the acrylamide exposure of consumers who participated in the National Food Consumption Survey (NFCS) in 1998 (n=6250). The exposure was estimated using the probabilistic approach for the total Dutch population and several age groups. For 344 food products, acrylamide amounts ranged from <30 to 3100 microg/kg. Foods with the highest mean acrylamide amounts were potato crisps (1249 microg/kg), chips (deep-fried) (351 microg/kg), cocktail snacks (1060 microg/kg), and gingerbread (890 microg/kg). The mean acrylamide exposure of the NFCS participants was 0.48 microg/kg bw/day. Risk of neurotoxicity is negligible. From exposure estimations it appears that the additional cancer risk might not be negligible.

In April 1999 an amount of 2600 μg/kg DON was found in a sample breakfast cereals in the Netherlands. This event was the start of a lot of activities, which dealt with the prevention, control, health and consumer aspects of DON in food for human consumption. The Food Inspection Services started a monitoring program to measure DON in cereal products, flour and raw cereals. The National Institute of Public Health and the Environment, another part of the Ministry of Health in the Netherlands, was asked to carry out a risk analysis on DON. This was the basis for the Minister of Health to set an action limit for consumer products. She also informed Brussels and asked for a European limit. The Main Board on Agriculture set out to implement measures to be taken at harvesting, milling and bread baking industry. The Scientific Committee on Food of the EC expressed an opinion on DON in December 1999. Worldwide attention leads to discussion of a DON limit by JECFA in February 2001. In the period may 1999 until march 2002 a number of more than 1700 samples were analysed on DON. These originated from the cereal harvest of the years 1998 until 2001. The results showed a sharp decrease of DON content in samples of harvest 1999 when compared to 1998. This lower level was maintained in the 2000 and 2001 harvests. Apparently the measures taken to control the DON level succeeded to maintain values below the action limits. Despite these activities a smaller outbreak of DON appeared in 2001 in pasta products at a lower extent. This indicated that control should be done systematically, not sporadically, and at a European level, which is made possible since EC has set a limit in July 2000. Analytical results of the measurements are presented, together with the chronological order of the associated activities of national, EU and worldwide bodies on human health control. Special attention is paid to DON in bread, related to the level in flour.

A method was developed for accurate measurement of aflatoxin B1 in the edible portion of pistachio nuts. Twenty-nine samples of kernels with and without their shells were slurried with a Mega Ultra Turrax. A subsample of the homogenate was extracted with water-methanol, defatted with petroleum ether, purified with a silica solid-phase extraction column, and redissolved in methanol. After separation on an octadecyl column and postcolumn reaction with on-line electrochemically generated bromine, the aflatoxin B1 derivative was detected fluorometrically. The shells contained less than 1% of the aflatoxin B1 found in the edible kernel, and they accounted for 41.7-46.8% of the weight of the whole pistachio. These observations indicate it is possible to analyze an entire sample, up to 25 kg, as a whole and still be able to judge whether it meets the legal tolerance limit of 5 micrograms aflatoxin B1/kg edible part, as set by the Dutch Food Act.

Die von Hammett, Taft und Nieuwdorp et al. vorgeschlagenen Gleichungen werden durch Faktoranalyse unter Zugrundelegung der drei Variablen Lösungsmittel, Reaktionstyp und Substituent weiterentwickelt.

Retention volumes of monosubstituted benzenes, benzoic acids, phenols and anilines have been measured in reversed-phase liquid chromatography. Buffered acetonitrile-water and tetrahydrofuran-water eluents were used with an octadecylsilylsilica adsorbent. From the net retention volumes a substituent interaction effect was calculated and described with the linear free energy relationship developed by Taft. The positive sign of the values of the ρ-parameters, figuring in this relationship, was interpreted in terms of hydrogen bonding between the solutes and the eluent.

The equations, proposed by Hammett, Taft, and Nieuwdorp et al., respectively, for the simultaneous description of the influence of reaction type and substituent on equilibrium and reaction rate constants are discussed. The latter equation represents an example of factor analysis. This mathematical–statistical technique has also been applied to describe simultaneously the influence of solvent and substituent and the influence of solvent and reaction type. It is thus a logical step to classify equilibrium and reaction rate constants with respect to three modes, solvent, reaction type, and substituent, and to try to describe the influence of these three variables by three-mode factor analysis. Two examples of the application of this technique to literature data are given. The first concerns data on ionization constants for 15 series of substituted compounds in three solvents. The second example concerns data on phase equilibrium constants of six series of substituted compounds in nine two-phase systems. The two-phase systems comprise gas–liquid as well as liquid–liquid and solid–liquid systems. The precision of the fit of the observations and the precision of the prediction of the missing data are discussed. In the first example 237 data are missing. Among them are 90 data that cannot be predicted at all by the Hammett, Taft, or Nieuwdorp equations (viz., for reactions on which no measurements at all are available in a particular solvent). The standard deviation of the prediction of the latter data by three-mode factor analysis ranges from 0.1 to 0.2. In the second example nearly all missing data are for reactions on which no measurements at all are available in a particular solvent. They can be predicted by three-mode factor analysis with a standard deviation that ranges from 0.09 to 0.13. Further, it is shown that the number of parameters that is required to fit the observations by three-mode factor analysis is far less than the number of parameters in the corresponding regression analysis model, viz., the Taft model.

It is shown that the Snyder equation is not quite satisfactory for fitting retention data in normal-phase high-performance liquid chromatography (HPLC) on chemically bonded phases. This equation is a special case of the mathematical—statistical three-mode factor analysis model. This model, in its general form, has been used to fit two sets of literature data on the retention in normal-phase HPLC for 19 solutes on six adsorbents with two eluents, and for 39 solutes on three adsorbents with two eluents, respectively. This study represents the first application of three mode factor analysis with missing data, and also the first application of three-mode factor analysis in the field of the natural sciences. The accuracy of the fit of the observations and of the prediction of the missing data, for various numbers of extracted factors, is discussed.