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In the combat against bacterial resistance, there is a clear need to check the use of antibiotics in animal husbandry, including poultry breeding. The use of chicken feathers as a tool for the detection of use of antibiotics was investigated. An extraction method for the analysis of oxytetracycline (OTC) from feathers was developed and was tested by using incurred feathers obtained from a controlled animal treatment study. The use of McIlvain-ethylenediaminetetraacetic acid buffer only in combination with acetone gave the highest extraction yield, indicating the need of an organic solvent for feather extraction. By using the developed method, it was found that after a withdrawal time, the OTC concentration in feathers is in the mg kg(-1) range, far higher than that in muscle and liver tissue. Based on the analysis of individual segments of feathers from OTC-treated chicken, evidence was found supporting the hypothesis of secretion of antibiotics through the uropygial gland and external spread over feathers by grooming behaviour. It was also found that part of the administered OTC is built into the feather rachis. Finally, we provide the first evidence that the analysis of individual segments of the rachis can be used as a tool to discriminate among different treatment strategies, for example, therapeutic versus subtherapeutic. As a result, we concluded that the analysis of feathers is an extremely valuable tool in residue analysis of antibiotics.

Recombinant bovine somatotropin (rbST) can be used to enhance milk production in dairy cattle. This is permitted in several countries but unauthorized in the European Union. Antibodies, which are produced endogenously in response to rbST administration, can be detected as a biomarker for indicating rbST (ab)use. For the first time, a fast and easy-to-perform screening assay for anti-rbST antibodies has been developed and applied to raw milk samples. This flow cytometric immunoassay (FCIA) is capable of discriminating between milk from rbST-treated animals and untreated animals. In accordance with literature, 67% of the rbST-treated animals responded positively with antibody production, whereas 94% of the untreated animals did not. The analysis of simulated tank milk samples showed more than 95% of the milk mixtures as truly positive for rbST treatment, indicating that the 33% physiologically non-responding cows will not be a problem when pooled tank milk samples are considered. FCIA biomarker responses in raw milk were specific for rbST and also obtained in pasteurized milk of rbST-treated animals. Using milk as a sample matrix for detection has the advantages of non-invasive sampling, and for tank milk analysis at the farm only one milk sample is needed to screen the whole farm for rbST (ab)use.

A liquid chromatography–tandem mass spectrometry method for the analysis of seven antiviral drugs, zanamivir, ribavirin, oseltamivir, oseltamivir carboxylate, amantadine, rimantadine and arbidol, in poultry muscle is reported. The antiviral drugs were extracted from the homogenized poultry muscle sample using methanol. The extract was purified using tandem solid-phase extraction combining a cation exchange cartridge and a phenylboronic acid cartridge. To prevent excessive matrix effects, the analytes were separated from the matrix constituents using a column-switch liquid chromatography system combining a reversed-phase and a Hypercarb analytical column. Detection was carried out using tandem mass spectrometry. The method was fully validated according to 2002/657/EC [1] and proved to be adequate for quantification and confirmation of zanamivir and ribavirin at 10 μg kg−1, oseltamivir, oseltamivir carboxylate, amantadine and rimantadine at levels below 1.0 μg kg−1 and for qualitative confirmatory analysis of arbidol at levels below 1 μg kg−1. Electronic supplementary material The online version of this article (doi:10.1007/s00216-011-5581-3) contains supplementary material, which is available to authorized users.

Administration of recombinant bovine somatotropin (rbST) to enhance milk production in dairy cows is banned within the European Union. Therefore, methods for pinpointing rbST abuse are required. Due to the problematic detection of rbST itself in serum, methods are also focused on detecting changes in rbST-related biomarkers. In this study, a fast and easy-to-perform microsphere-based flow cytometric immunoassay (FCIA) for detection of rbST-induced antibodies in serum was developed. Until now, detection of rbST-induced antibodies was also problematic due to non-specific binding of serum proteins resulting in a high rate of false positive results. Therefore, five different sample preparation methods, i.e. dilution, octanoic acid precipitation, filtration, protein G purification, and a previously described generic FCIA sample preparation were critically compared to overcome non-specific binding to the microspheres. Only the generic FCIA sample pretreatment was effective in reducing non-specific binding. As a result, an absolute decision level for detecting rbST antibodies in serum of dairy cows was determined and its applicability was demonstrated. In accordance with biological expectations from literature, rbST antibodies were induced in three out of four rbST-treated dairy cows. These rbST-induced antibodies were successfully detected for up to 4¿weeks after the last rbST treatment, whereas no false positive results were obtained for 27 untreated dairy cows. This is the first method, able to overcome the interference of serum proteins and therefore, can be applied with high confidence for screening unknown herds of cattle for rbST antibodies, an important biomarker for pinpointing at rbST abuse in cattle

Persistent organic pollutants (POPs) are environmental and food-related contaminants of global public health concern and known to be carcinogenic and endocrine disruptors. Their monitoring is essential, and an easy-to-use, rapid, and affordable multianalyte screening method with simplified sample preparation can be a valuable tool prior to instrumental analysis. For this purpose, a flow cytometric immunoassay (FCIA), based on a spectrally encoded microbeads technology, was developed for the multiplex detection of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (BDEs) in buffer and fish extracts. The sensitivities of the assays in the three-plex FCIA format were similar to the individual FCIAs for the marker compounds benzo[a]pyrene (BaP), 3,3',4,4'-tetrachlorobiphenyl (PCB77), and 2,2',4,4'-tetrabromodiphenyl ether (BDE47) in buffer with IC(50) values of 0.4, 20, and 2 μg L(-1), respectively. Apart from the three markers, we could detect at least 14 other POPs. Extracts of fish with different fat content, prepared with a simplified extraction and cleanup procedure, had an insignificant influence on the overall three-plex FCIA performance, with the exception of some impact on the PAHs detection. The performance of the three-plex FCIA, in combination with the simple extraction procedure, is adequate for regulatory control in accordance with the required limits.

The influence of natural organic matter on the screening of pharmaceuticals in water was determined by using high resolution liquid chromatography (HRLC) combined with full scan mass spectrometry (MS) techniques like time of flight (ToF) or Orbitrap MS. Water samples containing different amount of natural organic matter (NOM) and residues of a set of 11 pharmaceuticals were analyzed by using Exactive Orbitrap (TM) LC-MS. The samples were screened for residues of pharmaceuticals belonging to different classes like benzimidazoles, macrolides, penicillins, quinolones, sulfonamides, tetracyclines, tranquillizers, non-steroidal anti-inflammatory drugs (NSAIDs), anti-epileptics and lipid regulators. The method characteristics were established over a concentration range of 0.1-500 mu g L(-1). The 11 pharmaceuticals were added to two effluent and two influent water samples. The NOM concentration within the samples ranged from 2 to 8 mg L(-1) of dissolved organic carbon. The HRLC-Exactive Orbitrap (TM) LC-MS system was set at a resolution of 50,000 (FWHM) and this selection was found sufficient for the detection of the list of pharmaceuticals. With this resolution setting, accurate mass measurements with errors below 2 ppm were found, despite of the NOM concentration of the different types of water samples. The linearities were acceptable with correlation coefficients greater than 0.95 for 30 of the 51 measured linearities. The limit of detection varies between 0.1 mu g L(-1) and 100 mu g L(-1). It was demonstrated that sensitivity could be affected by matrix constituents in both directions of signal reduction or enhancement. Finally it was concluded that with direct shoot method used (no sample pretreatment) all compounds, were detected but LODs depend on matrix-analyte-concentration combination. No direct relation was observed between NOM concentration and method characteristics. For accurate quantification the use of internal standards and/or sample clean-up is necessary. The direct shoot method is only applicable for qualitative screening purposes. The use of full scan MS makes it possible to search for unknown contaminants. With the use of adequate software and a database containing more than 50,000 entries a tool is available to search for unknowns.

This paper describes the discrimination of eight different isomers of chloramphenicol (CAP), an antibiotic banned for use in food producing animals, by reversed phase and chiral liquid chromatography in combination with tandem mass spectrometric detection. Previously, by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) the presence of CAP was confirmed in some grass and herb samples collected on Mongolian pastures up to concentrations of 450 μg kg −1 . It was not possible to establish the cause of CAP residues which has initiated research on the natural occurrence of this drug. CAP occurs in the para -configuration and in the meta -configuration and contains two chiral centers thus eight different isomeric configurations exist, namely four (RR, SS, RS, SR) meta -stereoisomers and four para -stereoisomers. It is known that only RR- p -CAP has antimicrobial properties. To find out if the CAP detected in the plant material samples is the active configuration, a high resolution reversed phase LC-MS/MS system was tested for its ability to separate the different isomers. This system proved to be able to discriminate between some isomers, but not between RR- p -CAP and SS- p -CAP, also called dextramycin. Despite a detailed elucidation of the product ions and the fragmentation patterns of all isomers, MS/MS did not add sufficient specificity for full discrimination of the isomers. Therefore a chiral liquid chromatographic separation with MS/MS detection that is able to distinguish all isomers was developed and finally the isomeric ratio of non-compliant plant material samples and some CAP formulations was determined using this system. This showed that Mongolian grass and herb samples only contain the biological active isomer of CAP as do the obtained formulations. Therefore the CAP present in the plant material might origin from the production by soil organisms or from a manufactured source.

Pollen of the European and Asian white birch (Betula pendula and B. platyphylla) causes hay fever in humans. The allergenic potency of other birch species is largely unknown. To identify birch trees with a reduced allergenicity, we assessed the immunochemical characteristics of 15 species and two hybrids, representing four subgenera within the genus Betula, while focusing on the major pollen allergen Bet v 1. Antigenic and allergenic profiles of pollen extracts from these species were evaluated by SDS-PAGE and Western blot using pooled sera of birch-allergic individuals. Tryptic digests of the Bet v 1 bands were analyzed by LC-MS(E) to determine the abundance of various Bet v 1 isoforms. Bet v 1 was the most abundant pollen protein across all birch species. LC-MS(E) confirmed that pollen of all species contained a mixture of multiple Bet v 1 isoforms. Considerable differences in Bet v 1 isoform composition exist between birch species. However, isoforms that are predicted to have a high IgE-reactivity prevailed in pollen of all species. Immunoblotting confirmed that all pollen extracts were similar in immune-reactivity, implying that pollen of all birch species is likely to evoke strong allergic reactions.

Interest in nanoparticles (NPs) has increased explosively over the past two decades. Using NPs, high loadings of vitamins and health-benefit actives can be achieved in food, and stable flavors as well as natural food-coloring dispersions can be developed. Detection and characterization of NPs are essential in understanding the benefits as well as the potential risks of the application of such materials in food. While many such applications are described in the literature, methods for detection and characterization of such particles are lacking. Organic NPs suitable for application in food are lipid-, protein- or polysaccharide-based particles, and this review describes current analytical techniques that are used, or could be used, for identification and characterization of such particles in food products. We divide the analytical approaches into four sections: sample preparation; separation; imaging; and, characterization. We discuss techniques and reported applications for NPs or otherwise related particle compounds. The results of this investigation show that, for a successful characterization of NPs in food, at least some kind of sample preparation will be required. While a simple sample preparation may be satisfactory for imaging techniques for known analytes, for other techniques, a further separation using chromatography, field-flow fractionation or ion-mobility separation is necessary. Subsequently, photon-correlation spectroscopy and especially mass spectrometry techniques as matrix-assisted laser desorption/ionization combined with time-of-flight mass spectrometry, seem suitable techniques for characterizing a wide variety of organic NPs.

The official control of animal proteins in feed is focused on the prevention of Bovine Spongiform Encephalopathy (mad cow disease). The current legislation of the European Union is planned to avoid the feeding of animal by-products to the same species as its origin (ban of cannibalism, or species-to-species ban). With respect to the official control, the circumscription of the term species in legislation should be defined, and species-specific markers should be available. Markers will include primer sets, antibodies, near-infrared profiles or visual characteristics. The method of classical light microscopy is currently the only accepted method in the framework of the official detection of animal proteins. Besides the necessary development of complementary methods, either as stand alone methods or in combination, the visual characteristics used for a microscopic examination of meat and bone meal particles should be fully explored. Multivariate analysis of a range of characteristics of lacunae in bone fragments revealed that discrimination is possible between mammalian and avian bone fragments. Translation to features for every day practical use should be carried out very carefully, and only comprehensively collected information on a range of features will give a first indication of the source. Characteristics of hairs and feather filaments can be used to identify the origin of animal particles. An in situ identification method has been developed for antibody conjugation with troponin I in muscle fibers on a microscopic slide. A proof of principle is presented. Interlaboratory transferability and validation have still to be achieved. The development and testing of light microscopy markers in the framework of the SAFEED-PAP project revealed that a fine tuning of existing microscopic characteristics appears to be possible. Keywords. Microscopic method, feed, marker, hair, feather, bone, meat and bone meal.

A multi-mycotoxin immunoassay-using the MultiAnalyte Profiling (xMAP) technology-is developed and evaluated. This technology combines a unique color-coded microsphere suspension array, with a dedicated flow cytometer. We aimed for the combined detection of aflatoxins, ochratoxin A, deoxynivalenol, fumonisins, zearalenone and T-2-toxin in an inhibition immunoassay format. Sets of six mycotoxin-protein conjugates and six specific monoclonal antibodies were selected, and we observed good sensitivities and no cross-interactions between the assays in buffer. However, detrimental effects of the feed extract on the sensitivities and in some cases on the slopes of the curves were observed and different sample materials showed different effects. Therefore, for quantitative analysis, this assay depends on calibration curves in blank matrix extracts or on the use of a suitable multi-mycotoxin cleanup. To test if the method was suitable for the qualitative detection at EU guidance levels, we fortified rapeseed meal, a feed ingredient, with the six mycotoxins, and all extracts showed inhibited responses in comparison with the non-fortified sample extract. Contaminated FAPAS reference feed samples assigned for a single mycotoxin showed strong inhibitions in the corresponding assays but also often in other assays of the multiplex. In most cases, the presence of these other mycotoxins was confirmed by instrumental analysis. The multiplex immunoassay can be easily extended with other mycotoxins of interest, but finding a suitable multi-mycotoxin cleanup will improve its applicability.

Tetracyclines are extensively used in veterinary medicine. For the detection of tetracycline residues in animal products, a broad array of methods is available. Luminescent bacterial biosensors represent an attractive inexpensive, simple and fast method for screening large numbers of samples. A previously developed cell-biosensor method was subjected to an evaluation study using over 300 routine poultry samples and the results were compared with a microbial inhibition test. The cell-biosensor assay yielded many more suspect samples, 10.2% versus 2% with the inhibition test, which all could be confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Only one sample contained a concentration above the maximum residue limit (MRL) of 100 microg kg(-1), while residue levels in most of the suspect samples were very low (

In this manuscript we provide evidence for the seed transmission of Pepino mosaic virus (PepMV) in tomato. Fruit was harvested from a tomato crop artificially infected with both European and CH2 genotypes of PepMV and more than 100,000 seeds were extracted and cleaned using an enzymatic treatment without disinfection. Infection assays using indicator plants confirmed the presence of viable virus on the seeds. Seeds were distributed to ten different laboratories in three separate batches, where they were germinated and the young plants tested by ELISA. In total over 87,000 plants were tested and 23 positives detected, indicating an overall transmission rate of 0.026%. However, the observed seed transmission rates varied from 0.005% to 0.057%, depending on the seed batch used. Results clearly showed that PepMV can be transmitted from seeds contaminated with virus to seedlings, highlighting the risk of using seeds from PepMV-infected plants and the potential for seed transmission to contribute to the further spread of PepMV.

This report of the Dutch National Reference Laboratory (NRL) for feed additives (RIKILT Wageningen UR) descreibes the activities employed in 2012. The main tasks of the NRL are: providing assistance to the European Union Reference Laboratory (EURL) on their request, as well as providing advice and support to the competent authority, which is the Dutch Ministry of Economic Affairs - Department 'Plantaardige Agroketens en Voedselkwaliteit'.

This report of the Dutch National Reference Laboratory (NRL) for feed additives (RIKILT Wageningen UR) descreibes the activities employed in 2012. The main tasks of the NRL are: providing assistance to the European Union Reference Laboratory (EURL) on their request, as well as providing advice and support to the competent authority, which is the Dutch Ministry of Economic Affairs - Department 'Plantaardige Agroketens en Voedselkwaliteit'.

This report describes the activities employed by RIKILT regarding the functions as: - the National Reference Laboratory (NRL) for feed additives; - advice regarding temporary use exemptions, other advice and support of EL&I. This report also presents the activities by the NRL to keep up expertise on the analysis of feed additives like participation in proficiency tests and presenting (posters, abstracts, publications)analytical research.

Administration of recombinant bovine somatotropin (rbST) to enhance milk production in dairy cows is banned within the European Union. Therefore, methods for pinpointing rbST abuse are required. Due to the problematic detection of rbST itself in serum, methods are also focused on detecting changes in rbST-related biomarkers. In this study, a fast and easy-to-perform microsphere-based flow cytometric immunoassay (FCIA) for detection of rbST-induced antibodies in serum was developed. Until now, detection of rbST-induced antibodies was also problematic due to non-specific binding of serum proteins resulting in a high rate of false positive results. Therefore, five different sample preparation methods, i.e. dilution, octanoic acid precipitation, filtration, protein G purification, and a previously described generic FCIA sample preparation were critically compared to overcome non-specific binding to the microspheres. Only the generic FCIA sample pretreatment was effective in reducing non-specific binding. As a result, an absolute decision level for detecting rbST antibodies in serum of dairy cows was determined and its applicability was demonstrated. In accordance with biological expectations from literature, rbST antibodies were induced in three out of four rbST-treated dairy cows. These rbST-induced antibodies were successfully detected for up to 4¿weeks after the last rbST treatment, whereas no false positive results were obtained for 27 untreated dairy cows. This is the first method, able to overcome the interference of serum proteins and therefore, can be applied with high confidence for screening unknown herds of cattle for rbST antibodies, an important biomarker for pinpointing at rbST abuse in cattle

Feed materials were analysed for the mycotoxins deoxynivalenol, ochratoxin A, zearalenone, aflatoxin B1, fumonisin B1 and B2, and HT-2- and T-2-toxins. In this report trends in the average content during the period 2001-2009 are reported for these mycotoxins. Monitoring data from the National Feed monitoring program and from the Commodity Board Animal Feed are used for this study.

This report of the National Reference Laboratory (NRL) for residues of veterinary drugs and growth promoting agents in products of animal origin according to Council Directive 96/23/EC describes the activities employed in 2011. The main tasks of the NRL are the communication with Routine Field Laboratories (RFL), the preparation of quality control samples and the advisory function for the Competent Authority and RFL. Furthermore the NRL organizes comparative tests, ensure that the RFL observes the residue limits, disseminates information obtaines from the European Union Reference Laboratories (EURLs) and participate in training organized by the Commission or by the EURLs.

Feed materials were analysed for the mycotoxins deoxynivalenol, ochratoxin A, zearalenone, aflatoxin B1, fumonisin B1 and B2, and HT-2- and T-2-toxins. In this report trends in the average content during the period 2001-2009 are reported for these mycotoxins. Monitoring data from the National Feed monitoring program and from the Commodity Board Animal Feed are used for this study.

This report describes the activities employed by RIKILT regarding the functions as: - the National Reference Laboratory (NRL) for feed additives; - advice regarding temporary use exemptions, other advice and support of EL&I. This report also presents the activities by the NRL to keep up expertise on the analysis of feed additives like participation in proficiency tests and presenting (posters, abstracts, publications)analytical research.

Dit rapport beschrijft de resultaten van het onderzoek naar antibiotica in consumptievlees. Het onderzoek is door RIKILT in opdracht van Stichting Natuur en Milieu uitgevoerd. In totaal zijn 41 monsters varkensvlees (hamlappen) en 40 monsters kippenvlees (filets) onderzocht op de aanwezigheid van tetracycline, sulfonamide en beta-lactam antibiotica. In drie monsters vlees zijn antibiotica aangetroffen. Het betreft hier één monster varkensvlees met doxycycline, één monster varkensvlees met sulfadiazine en één monster kippenvlees met sufadimidine.

Different veterinary pharmaceuticals are used in agricultural livestock becoming a source of environment contamination. Furthermore, no regulation exists for the concentration limits of pharmaceuticals in soil or water. Monitoring programs for environment contamination with pharmaceuticals are needed, requiring new sensitive and selective screening methods. The present study focuses on developing a method for the simultaneous scanning of forty-two compounds (pharmaceuticals, azole biocides and fungicides) in soil and plant material samples. For extraction purposes the use of ultrasonic assisted and accelerated solvent extraction (ASE) were compared. The extract was purified and concentrated by applying a solid phase extraction step followed by ultra-high-performance-chromatographic separation and accurate-mass spectrometric detection using Exacte Orbitrap technology (FWHM 50,000). The effects of the different extraction solvents and conditions on the extraction efficiency were tested. Although both extraction approaches are applicable the optimal extraction efficiency was obtained by applying accelerated solvent extraction using solvent mixtures containing acetone for soil and methanol for plant samples. An ASE process has been validated for the determination of selected pharmaceuticals and fungicides in soil and in plant material. The recoveries from soil samples were >70% for more than 68% of the compounds. The levels of detection were =10 µg kg-1 for 93% of the compounds tested. The recoveries from plant material were >70% for 64% of the compounds tested. The levels of detection were =10 µg kg-1 for 66% of the compounds. The developed method was used to screen soil and plant material collected throughout the Netherlands and oxytetracycline residues were detected.

Dit rapport beschrijft de resultaten van het onderzoek naar antibiotica in consumptievlees. Het onderzoek is door RIKILT in opdracht van Stichting Natuur en Milieu uitgevoerd. In totaal zijn 41 monsters varkensvlees (hamlappen) en 40 monsters kippenvlees (filets) onderzocht op de aanwezigheid van tetracycline, sulfonamide en beta-lactam antibiotica. In drie monsters vlees zijn antibiotica aangetroffen. Het betreft hier één monster varkensvlees met doxycycline, één monster varkensvlees met sulfadiazine en één monster kippenvlees met sufadimidine.

This report of the National Reference Laboratory (NRL) for residues of veterinary drugs and growth promoting agents in products of animal origin according to Council Directive 96/23/EC describes the activities employed in 2011. The main tasks of the NRL are the communication with Routine Field Laboratories (RFL), the preparation of quality control samples and the advisory function for the Competent Authority and RFL. Furthermore the NRL organizes comparative tests, ensure that the RFL observes the residue limits, disseminates information obtaines from the European Union Reference Laboratories (EURLs) and participate in training organized by the Commission or by the EURLs.

The use of cephalosporin antibiotics in veterinary practice is likely to play an important role in the development of ß-lactam-resistant bacteria. To detect off-label cephalosporin antibiotic usage, an analytical method is needed that, besides the native compound, also detects their active metabolites. In this paper, the applicability of three approaches for the quantitative analysis of ceftiofur using LC–MS/MS is assessed, viz. (A) analysis of ceftiofur, desfuroylceftiofur and/or desfuroylceftiofur cystein disulfide, (B) derivatisation of ceftiofur metabolites to desfuroylceftiofur acetamide and (C) chemical hydrolysis using ammonia, to produce a marker compound for ceftiofur. We found that approach A was not suited for quantitative analysis of total ceftiofur concentration or for effectively detecting off-label use of ceftiofur. Approach B resulted in adequate quantitative results, but was considered a single compound method because it depends on cleavage of a thioester group, which is present in only a limited number of cephalosporin antibiotics. Approach C showed adequate quantitative results but, in contrast to approach B, it is applicable to a range of cephalosporin antibiotics. Therefore, it is applicable as a broad quantitative screening of cephalosporin compounds in poultry tissue samples to indicate off-label use of cephalosporins in poultry breeding. Based on this study, it was concluded that approach C is the most suitable to detect off-label use of a range of cephalosporin antibiotics.

Shellfish products may be contaminated with marine biotoxins which, after consumption, may lead to human illness. The Netherlands has a regular monitoring programme for marine biotoxins and the possible toxic phytoplankton in shellfish production waters. The aim of the current study was to evaluate the presence of potential toxic phytoplankton species and marine biotoxins in Dutch production waters over the last decade, and to analyse the relationship between toxin levels and abundance of possible causative phytoplankton species. The results of the monitoring programme of the period 1999-2009 were used. The presence of Alexandrium spp. were negligible, but Pseudo-nitzschia spp. and phytoplankton causing diarrhetic shellfish poisoning (DSP toxin-producing phytoplankton) were present in nearly all three main production areas and years. The main DSP toxin-producing species was Dinophysis acuminata followed by D. rotundata and Prorocentrum lima. Toxins causing paralytic shellfish poisoning (PSP) and amnesic shellfish poisoning (ASP) were present in only a few individual shellfish samples, all at low levels. At the end of 2002, an episode of DSP toxicity was recorded, based on the rat bioassay results. Of the samples that were chemically analysed for DSP toxins in 2007 and 2008, about half of the samples in 2007 contained these toxins, although levels were low and no positive results were obtained using the rat bioassay. There was a slight positive correlation between concentrations of DSP toxin-producing phytoplankton and levels of DSP toxins in 2007. Increased DSP toxin levels were found up to 5 weeks after the peak in DSP toxin-producing phytoplankton. This positive, but weak, relationship needs to be confirmed in future research using more samples and chemical methods to quantify the presence of DSP toxins. If this relationship is further substantiated and quantified, it could be used within the current monitoring programme in the Netherlands to predict the risk areas regarding DSP toxicity in shellfish.

Chloramphenicol is a broad-spectrum antibiotic with, apart from its human medicinal use, veterinary abuse in all major food-producing animals. Chloramphenicol occurs in four stereoisomers (all para-nitro substituted) and furthermore four meta-nitro analogs of chloramphenicol exist. In this paper these are referred to as eight chloramphenicol isomers. According to EU regulations an analytical method should be able to discriminate the analyte from interfering substances that might be present in the sample, including isomers. For the first time a quantitative method for the analysis of trace levels of eight chloramphenicol isomers in urine by chiral liquid chromatography in combination with tandem mass spectrometric detection is reported. The separation of the isomers on the analytical column, the clean-up of urine and the selectivity of the monitored product ions turned out to be critical parameters. To obtain reproducible retention isocratic elution on a chiral AGP column was applied. For urine samples matrix compounds present in the final extract caused decreased retention of the isomers on the chiral stationary phase and a lack of chromatographic resolution. Therefore an extended clean-up procedure that combines solid phase extraction and liquid–liquid extraction had to be developed. The final method was fully validated and showed satisfactory performance for all isomers with decision limits (CCa) ranging from 0.005 to 0.03 µg L-1 and within-laboratory reproducibility of all isomers below 20% at the minimum required performance limit level of 0.3 µg L-1.

A prototype imaging surface plasmon resonance-based multiplex microimmunoassay for mycotoxins is described. A microarray of mycotoxin-protein conjugates was fabricated using a continuous flow microspotter device. A competitive inhibition immunoassay format was developed for the simultaneous detection of deoxynivalenol (DON) and zearalenone (ZEN), using a single sensor chip. Initial in-house validation showed limits of detection of 21 and 17 ng/mL for DON and 16 and 10 ng/mL for ZEN in extracts, which corresponds to 84 and 68 μg/kg for DON and 64 and 40 μg/kg for ZEN in maize and wheat samples, respectively. Finally, the results were critically compared with data obtained from liquid chromatography-mass spectrometry confirmatory analysis method and found to be in good agreement. The described multiplex immunoassay for the rapid screening of several mycotoxins meets European Union regulatory limits and represents a robust platform for mycotoxin analysis in food and feed samples.

The gut contents and fatty acid composition of 49 fish belonging to five Antarctic demersal families (Nototheniidae, Macrouridae, Channichtyidae, Bathydraconidae and Artedidraconidae) sampled at two stations at the Southern Ocean shelf and deep sea (600 and 2150 m) were analysed in order to identify their main food resource by linking trophic biomarkers with the dietary items found in the fish guts. Main food items of most fish analysed were amphipod crustaceans (e.g. in 63% of Trematomus bernachii guts) and polychaetes (e.g. in 80% of Bathydraco sp. guts), but other food items including fish, other crustaceans and gastropods were also ingested. The most prominent fatty acids found were 20:5(n−3), 16:0, 22:6(n−3) and 18:1(n−9). The results of gut content and fatty acid analyses indicate that all fish except the Channichthyidae share similar food resources irrespective of their depth distribution, i.e. benthic amphipods and polychaetes. A difference of the dietary spectrum can be observed with ontogenetic phases rather than between species, as high values of typical calanoid copepod marker fatty acids as 22:1(n−11) indicate that younger (smaller) specimens include more zooplankton in their diet.

This report of the National Reference Laboratory (NRL) for feed additives describes the activties employed in 2010. The main tasks of the NRL are: giving assistance to the European Union Reference Laboratort (EU-RL) on their request and advice and support the competent authority, the Dutch Ministry of Economic Affairs, Agriculture and Innovation - Department of Food, Animal and Consumer (EL&I-VDC)

The official control of animal proteins in feed is focused on the prevention of Bovine Spongiform Encephalopathy (mad cow disease). The current legislation of the European Union is planned to avoid the feeding of animal by-products to the same species as its origin (ban of cannibalism, or species-to-species ban). With respect to the official control, the circumscription of the term species in legislation should be defined, and species-specific markers should be available. Markers will include primer sets, antibodies, near-infrared profiles or visual characteristics. The method of classical light microscopy is currently the only accepted method in the framework of the official detection of animal proteins. Besides the necessary development of complementary methods, either as stand alone methods or in combination, the visual characteristics used for a microscopic examination of meat and bone meal particles should be fully explored. Multivariate analysis of a range of characteristics of lacunae in bone fragments revealed that discrimination is possible between mammalian and avian bone fragments. Translation to features for every day practical use should be carried out very carefully, and only comprehensively collected information on a range of features will give a first indication of the source. Characteristics of hairs and feather filaments can be used to identify the origin of animal particles. An in situ identification method has been developed for antibody conjugation with troponin I in muscle fibers on a microscopic slide. A proof of principle is presented. Interlaboratory transferability and validation have still to be achieved. The development and testing of light microscopy markers in the framework of the SAFEED-PAP project revealed that a fine tuning of existing microscopic characteristics appears to be possible. Keywords. Microscopic method, feed, marker, hair, feather, bone, meat and bone meal.

In July 2010 a rapid alert was issued by Spain concerning the presence of blood plasm in milk formula ("KUOVO") for calves. The blood plasma was detected by a microscopic staining method using tetramethylbenzidine (TMB). RIKILT investigated the suspect research sample by applying the standard research method for animal proteins and several additional staining methods, including TMB (macroscopic and microscopic).

In July 2010 a rapid alert was issued by Spain concerning the presence of blood plasm in milk formula ("KUOVO") for calves. The blood plasma was detected by a microscopic staining method using tetramethylbenzidine (TMB). RIKILT investigated the suspect research sample by applying the standard research method for animal proteins and several additional staining methods, including TMB (macroscopic and microscopic).

The EC has introduced maximum inclusion levels of copper and zinc salts in animal diets from 1970 onwards and reduced these levels in recent years. In this report historical values are used to give insight into trends in levels of copper and zinc in compound feeds for animals in the Netherlands. The results of these analyses will enable the nVWA (Dutch Food and Consumer Product Safety Authority) to develop a more risk-directed sampling strategy in the National Feed Monitoring program. Over 2000 feed samples are analysed for this report. The data for this analysis are from the period between 2001 and 2009. The copper and zinc data-set contains mostly feeds for piglets, (older) pigs, sheep and to a lesser extent bovine and other species.

Anabolic steroids are banned in food producing livestock in Europe. Efficient methods based on mass spectrometry detection have been developed to ensure the control of such veterinary drug residues. Nevertheless, the use of “cocktails” composed of mixtures of low amounts of several substances as well as the synthesis of new compounds of unknown structure prevent efficient prevention. New analytical tools able to detect such abuse are today mandatory. In this context, metabolomics may represent new emerging strategies for investigating the global physiological effects associated to a family of substances and therefore, to suspect the administration of steroids. The purpose of the present study was to set up, assess and compare two complementary mass spectrometry-based metabolomic strategies as new tools to screen for steroid abuse in cattle and demonstrate the feasibility of such approaches. The protocols were developed in two European laboratories in charge of residues analysis in the field of food safety. Apart from sample preparation, the global process was different in both laboratories from LC-HRMS fingerprinting to multivariate data analysis through data processing and involved both LC-Orbitrap-XCMS and UPLC-ToF-MS-MetAlign strategies. The reproducibility of both sample preparation and MS measurements were assessed in order to guarantee that any differences in the acquired fingerprints were not caused by analytical variability but reflect metabolome modifications upon steroids administration. The protocols were then applied to urine samples collected on a large group of animals consisting of 12 control calves and 12 calves administrated with a mixture of 17β-estradiol 3-benzoate and 17β-nandrolone laureate esters according to a protocol reflecting likely illegal practices. The modifications in urine profiles as indicators of steroid administration have been evaluated in this context and proved the suitability of the approach for discriminating anabolic treated animals from control ones. Such an approach may therefore open a new way for the screening of anabolic steroid administration through targeted monitoring of relevant biomarkers highlighted as a result of the metabolomics study.

The EC has introduced maximum inclusion levels of copper and zinc salts in animal diets from 1970 onwards and reduced these levels in recent years. In this report historical values are used to give insight into trends in levels of copper and zinc in compound feeds for animals in the Netherlands. The results of these analyses will enable the nVWA (Dutch Food and Consumer Product Safety Authority) to develop a more risk-directed sampling strategy in the National Feed Monitoring program. Over 2000 feed samples are analysed for this report. The data for this analysis are from the period between 2001 and 2009. The copper and zinc data-set contains mostly feeds for piglets, (older) pigs, sheep and to a lesser extent bovine and other species.

The stability of an antibiotic is a very important characteristic, especially in the field of antibiotic residue analysis. During method development or validation, the stability of the antibiotic has to be demonstrated no matter if the method is used for screening, confirmation, qualitative or quantitative analysis. A procedure for testing the stability of antibiotics in solutions and food samples using LC-MS/MS is described. The procedure is based on the assumption that the antibiotics are stable when stored at -70 °C. Representative solutions or spiked samples containing the antibiotic were stored at the temperature to be tested (-18 or 4 °C) and at -70 °C. After a selected storing time samples were moved from the chosen storage temperature to -70 °C. At the end of the study, all samples--per class of antibiotic--were analysed in one batch. By applying statistical models, it was finally concluded in which circumstances the antibiotic is stable. The stability of 60 antibiotics belonging to the classes of tetracyclines, sulphonamides, quinolones, penicillins, macrolides and aminoglycosides were tested. The stability of solutions containing tetracyclines and penicillins is only guaranteed for 3 months while stored at -18 °C. Solutions of all other antibiotics tested are stable for at least 6 or 12 months when stored at 4 °C. In muscle tissue stored at -18 °C no severe degradation of the tested antibiotics was observed, with the exception of the penicillins. The stability data reported here are useful as a reference for laboratories carrying out validation studies of analytical methods for antibiotic (residue) detection. The data should save the time needed for long-term stability testing of solutions and samples.

This report of the National Reference Laboratory (NRL) for feed additives describes the activties employed in 2010. The main tasks of the NRL are: giving assistance to the European Union Reference Laboratort (EU-RL) on their request and advice and support the competent authority, the Dutch Ministry of Economic Affairs, Agriculture and Innovation - Department of Food, Animal and Consumer (EL&I-VDC)

Pyrrolizidine alkaloids are toxins present in many plants belonging to the families of Asteraceae, Boraginaceae and Fabaceae. Particularly notorious are pyrrolizidine alkaloids present in ragwort species (Senecio), which are held responsible for hepatic disease in horses and cows and may lead to the death of the affected animals. In addition, these compounds may be transferred to edible products of animal origin and as such be a threat for the health of consumers. To investigate the possible transfer of pyrrolizidine alkaloids from contaminated feed to milk, cows were put on a ration for 3 weeks with increasing amounts (50-200 g day(-1)) of dried ragwort. Milk was collected and sampled twice a day; faeces and urine twice a week. For milk, a dose-related appearance of pyrrolizidine alkaloids was found. Jacoline was the major component in milk despite being a minor component in the ragwort material. Practically no N-oxides were observed in milk, notwithstanding the fact that they constituted over 80% of the pyrrolizidine alkaloids in ragwort. The overall carry-over of the pyrrolizidine alkaloids was estimated to be only around 0.1%, but for jacoline 4%. Notwithstanding the low overall carry-over, this may be relevant for consumer health considering the genotoxic and carcinogenic properties demonstrated for some of these compounds. Analysis of the faeces and urine samples indicated that substantial metabolism of pyrrolizidine alkaloids is taking place. The toxicity and potential transfer of metabolites to milk is unknown and remains to be investigated.

Dioxins and dioxin-like polychlorinated biphenyls (DL-PCBs) are hazardous toxic, ubiquitous and persistent chemical compounds, which can enter the food chain and accumulate up to higher trophic levels. Their determination requires sophisticated methods, expensive facilities and instruments, well-trained personnel and expensive chemical reagents. Ideally, real-time monitoring using rapid detection methods should be applied to detect possible contamination along the food chain in order to prevent human exposure. Sensor technology may be promising in this respect. This review gives the state of the art for detecting possible contamination with dioxins and DL-PCBs along the food chain of animal-source foods. The main detection methods applied (i.e., high resolution gas-chromatography combined with high resolution mass-spectrometry (HRGC/HRMS) and the chemical activated luciferase gene expression method (CALUX bioassay)), each have their limitations. Biosensors for detecting dioxins and related compounds, although still under development, show potential to overcome these limitations. Immunosensors and biomimetic-based biosensors potentially offer increased selectivity and sensitivity for dioxin and DL-PCB detection, while whole cell-based biosensors present interpretable biological results. The main shortcoming of current biosensors, however, is their detection level: this may be insufficient as limits for dioxins and DL-PCBs for food and feedstuffs are in pg per gram level. In addition, these contaminants are normally present in fat, a difficult matrix for biosensor detection. Therefore, simple and efficient extraction and clean-up procedures are required which may enable biosensors to detect dioxins and DL-PCBs contamination along the food chain.