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International audience; Biocide disinfectants are used in agro-food industries in order to limit the development of pathogens present in environment or on surfaces in contact with food for human or animal consumption. Biocide residues remaining on food surfaces may constitute a toxicological risk for the consumer. Very little feedback from the field on the rates of biocide residues in the industry exist due to a lack of simple, fast and responsive self-checking methods. The development of biosensors for the detection of biocide disinfectants represents a promising way to explore this, but most of this research remains limited.

International audience; The administration of nitrofurans to livestock to treat or prevent animal diseases has been banned in the EU for the production of food of animal origin. The corresponding marker residues are tissue-related metabolites AMOZ, AHD, SEM, and AOZ. The MRPL (minimum required performance limit)/RPA (Reference point for action) was set at 1 µg kg(-1) in the EU. Thus, all the laboratories involved in the control of nitrofuran metabolites must detect at least at this analytical limit of performance. The objectives of the work reported here were to evaluate the performance of ELISA kits from two different manufacturers (R-Biopharm, Germany; Europroxima, the Netherlands) for the individual screening of the four nitrofuran metabolites (AOZ, AMOZ; AHD; and SEM) in aquaculture products (fish, shrimps), and then to validate the kits according to the European Decision EC/2002/657 and to the European guideline for the validation of screening methods. The false positive rates were below 9 % for the kits from both manufacturers. The detection capabilities CCβ determined were all below the current RPA (1 µg/kg). However, regarding the updated RPA at 0.5 µg/kg that shall apply in 2022, the AMOZ and SEM kits from R-Biopharm and the SEM kit from Europroxima will not be able to reach it.

International audience; Colistin is a polypeptide antibiotic mainly used in porcine and poultry to treat gastrointestinal infections. It has been included by the World Health Organisation (WHO) in the list of critically important human antibiotics of high priority for antimicrobial resistance since 2017. Therefore, it is necessary to develop specific and sensitive screening methods for this molecule. Screening for colistin with immunoassays is an interesting alternative to LC-MS/MS screening methods. The performance of three commercially available ELISA kits was evaluated in poultry and porcine muscles for the detection of colistin in regards to its European maximum residue limit (MRL) (150 µg/kg). The applicability of the three ELISA kits to the detection of colistin at or below the MRL in porcine and poultry muscles was demonstrated. The detection capabilities (CCβ) of two kits were or lower than or equal to the MRL (150 µg/kg). The lowest detection capability (30 µg/kg) was achieved with the third ELISA kit. The specificity of the three kits was very satisfactory (false positive rates 0%). The three kits are very specific for the detection of colistin (colistin A and B) and polymyxin B.

International audience; The Infiniplex for milk(R) (IPM) kit is a quick method for the simultaneous and qualitative detection of more than 100 molecules including antibiotic residues, mycotoxins, anti-inflammatories and antiparasitic drugs into a single test that does not require milk treatment. The IPM(R) kit was validated according to the European decision EC/2002/657 and according to the European guideline for the validation of screening methods (2010). Our validation was focused only on antibiotic residues. The washing step was identified as the most critical step of the assay. Insufficient washes could cause a significant background noise that prevents imaging. Positive controls have to be freshly prepared each day (insufficient stability). The method was specific with a low false-positive rate of 1.7% on 5 discrete test regions (DTR) ((beta-lactams, lincomycin, virginiamycin, quinolones and sulphonamides)) and a false-positive rate of 0% on the 26 other DTR. During our validation, the 42 determined detection capabilities CCbeta for 12 antibiotic families (aminoglycosides, cephalosporins, lincosamides, macrolides, miscellaneous antibiotics, penicillins, phenolated polymixins, polypeptide antibiotics, quinolones, sulphonamides, tetracyclines) were at between once and twice the decision levels stated by the manufacturer. Forty CCbeta determined were lower than the respective regulatory limits (i.e. MRL, RC, MRPL) in milk, except for tilmicosin (1.5 times the MRL) and neospiramycin (>1.25 times the MRL). The estimated CCbeta of thiamphenicol, cloxacillin, danofloxacin, sulphathiazol, ceftiofur and sulphamonomethoxine were lower than or at the MRL. However, it was difficult to approach an accurate CCbeta with only qualitative results. It is impossible to know whether or not we were close to the cut-off value. The software could be improved by differentiating between low-positive and high-positive results. The results of our participation in three qualitative proficiency tests in 2016 and 2017 for the detection of quinolones, tetracyclines and sulphonamides in cows' milk were very satisfactory.

International audience; Different types of contaminants (e.g., pesticides, veterinary drugs) can be found in food products intended for human consumption (e.g., water, food from animal or plant origin). The risks for the consumers' health are multiple (e.g., endocrine disruptor's effect, carcinogenic effect, allergic risks). The detection of contaminants in food products is the first critical step of the control. Conventional screening methods could be bioassays, immunological methods, or physicochemical methods. Biosensors are identified as innovative screening methods. Electrochemical biosensors make it possible to develop a promising and economically interesting approach. Technological advances (e.g., nanomaterials, microfluidics) improved the methods' sensitivity.

Introduction Chemical (eg pesticides, veterinary drugs, etc.) and bacteriological contaminants (eg. foodborne pathogens) could contaminate animal and plant derived food products for human consumption. Some antibiotic residues (eg. chloramphenicol, nitrofuran metabolites, dyes) are banned in foodstuffs of animal origin (eg. milk, meat, eggs, etc.) in European Union because of toxicological risks for the consumer. The European Regulation has set Minimum Required Performance Limits (MRPL) [1] or Reference Point for Action (RPA) for banned substances [2]. Food containing residues of substances at or above the MRPL or RPA are declared non-compliant and consignments are rejected from the consumer’s market. Screening methods are the first stage of food control and so are essential for food safety monitoring. Conventional screening methods are microbiological methods (eg. plate tests, tube tests), immunological methods (eg. ELISA, radioimmunoassays) or physico-chemical methods (Thin Layer Chromatography, High Performance Liquid Chromatography (HPLC), liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)). These methods sometimes lack of sensitivity or specificity; they also could be time and money consuming. There is thus a need to develop novel screening methods for antibiotic residues detection, preferably with the potential for the field-testing (eg. farm control, self-control). Electrochemical biosensors make it possible to develop a promising and economically interesting approach. Electrochemical immunosensor An innovative electrochemical method based on disposable Screen Printed Carbon Electrodes (SPCE), coupled to magnetic beads (MB), allowing the simultaneous detection of 3 families of antibiotics in milk, was published by a Spanish academic team [3]. This technique presents major advantages: low cost (eg. disposable electrodes, potentiostat), promising detection limits, portability, and possible automatisation. Our laboratory has evaluated the transferability of the method. An electrochemical immunosensor has been developed for the detection of chloramphenicol residues in milk as a proof of concept. The matrix effect (milk samples) was high and so sample preparation has to be improved to reduce matrix effects. The objective is to develop an amperometric bead-based immunosensor for the multiplex detection of banned antibiotics (eg. chloramphenicol, nitrofuran metabolites, dyes) in bovine milk. Method Antibodies (Abs) against antibiotics are grafted on the surface of magnetic beads (MBs). Milk samples and antibiotic conjugated with Horseradish peroxidase (HRP) are mixed with MBs-Abs. A competition occurs between the HRP conjugates and the antibiotic residues if present in the sample, for the binding to the antibody. The MBs are washed to remove free antibiotics and conjugates. Then, a Screen Printed Carbon Electrode (SPCE) with MBs on its surface (maintained by a magnet) is soaked into a buffer solution containing hydroquinone; when adding hydrogene peroxide (H2O2) to the solution, an amperometric signal is produced, due to the enzymatic activity of HRP and measured. The amperometric signal is inversely proportional to the antibiotic concentration in the sample. Results and Conclusions Screening methods for the detection of veterinary drugs in food products have to be validated according to the European regulation [1] and to the European guideline for the validation of screening methods [4]. After the development and the optimization of the analytical parameters (eg. sample preparation, HRP concentration, incubation time, applied potential, etc), the methods developed for single compounds will be evaluated and validated according to the European regulations. Then the single compound methods will be merged into one multiplex method if possible. The results will be presented to the conference, discussing the advantage sand drawbacks of amperometric biosensors for the screening of antibiotic residues in food products. References 1. Commission Decision (EC) N° 2002/657 of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and interpretation of results. 2002: Official Journal of European Communities. p. 8-36. 2. Commission Regulation (EC) N° 470/2009 laying down Community procedures for the establishment of residue limits of pharmacologically active substances in foodstuffs of animal origin 2009, The European parliement and the Council: Official Journal of the European Union p. 11-22. 3. Conzuelo F, Ruiz-Valdepeñas Montiel V, Campuzano S, Gamella M, Torrente-Rodríguez RM, Reviejo AJ, Pingarrón JM. 2014. Rapid screening of multiple antibiotic residues in milk using disposable amperometric magnetosensors. Anal. Chim. Acta. 820:32-38. 4. CRL, Guideline for the validation of screening methods for residues of veterinary medicines (initial validation and transfer). 2010: Available from:: Guideline_Validation_Screening_en.pdf. p. 1-18.

International audience; Screening methods are used as a first-line approach to detect the presence of antibiotic residues in food of animal origin. The validation process guarantees that the method is fit-for-purpose, suited to regulatory requirements, and provides evidence of its performance. This article is focused on intra-laboratory validation. The first step in validation is characterisation of performance, and the second step is the validation itself with regard to pre-established criteria. The validation approaches can be absolute (a single method) or relative (comparison of methods), overall (combination of several characteristics in one) or criterion-by-criterion. Various approaches to validation, in the form of regulations, guidelines or standards, are presented and discussed to draw conclusions on their potential application for different residue screening methods, and to determine whether or not they reach the same conclusions. The approach by comparison of methods is not suitable for screening methods for antibiotic residues. The overall approaches, such as probability of detection (POD) and accuracy profile, are increasingly used in other fields of application. They may be of interest for screening methods for antibiotic residues. Finally, the criterion-by-criterion approach (Decision 2002/657/EC and of European guideline for the validation of screening methods), usually applied to the screening methods for antibiotic residues, introduced a major characteristic and an improvement in the validation, i.e. the detection capability (CCbeta). In conclusion, screening methods are constantly evolving, thanks to the development of new biosensors or liquid chromatography coupled to tandem-mass spectrometry (LC-MS/MS) methods. There have been clear changes in validation approaches these last 20 years. Continued progress is required and perspectives for future development of guidelines, regulations and standards for validation are presented here.

International audience; Efficient screening methods are needed to control antibiotic residues in eggs. A microbiological kit (Explorer® 2.0 test (Zeu Inmunotech, Spain)) and an immunobiosensor kit (Microarray II (AM® II) on Evidence Investigator™ system (Randox, UK)) have been evaluated and validated for screening of antibiotic residues in eggs, according to the European decision EC/2002/657 and to the European guideline for the validation of screening methods. The e-reader™ system, a new automatic incubator/reading system, was coupled to the Explorer 2.0 test. The AM II kit can detect residues of six different families of antibiotics in different matrices including eggs. For both tests, a different liquid/liquid extraction of eggs had to be developed. Specificities of the Explorer 2.0 and AM II kit were equal to 8% and 0% respectively. The detection capabilities were determined for 19 antibiotics, with representatives from different families, for Explorer 2.0 and 12 antibiotics for the AM II kit. For the nine antibiotics having a maximum residue limit (MRL) in eggs, the detection capabilities CCβ of Explorer 2.0 were below the MRL for four antibiotics, equal to the MRL for two antibiotics and between 1 and 1.5 MRLs for the three remaining antibiotics (tetracyclines). For the antibiotics from other families, the detection capabilities were low for beta-lactams and sulfonamides and satisfactory for dihydrostreptomycin (DHS) and fluoroquinolones, which are usually difficult to detect with microbiological tests. The CCβ values of the AM II kit were much lower than the respective MRLs for three detected antibiotics (tetracycline, oxytetracycline, tylosin). Concerning the nine other antibiotics, the detection capabilities determined were low. The highest CCβ was obtained for streptomycin (100 µg kg–1).

International audience; Honey could be contaminated by antibiotic residues. There is still a great need for a cheap and single multi-residue method. The Evidence Investigator™ system is a biochip and semi-automated system. The microarray kit I (AM I) for the detection of sulphonamide and trimethoprim (TMP) residues in honey was evaluated, and then validated according to the European decision EC/2002/657 and to the European guideline for the validation of screening methods for veterinary medicines (2010). The method was found to be rapid and able to screen simultaneously 14 sulphonamides and TMP in honey with a very easy sample preparation procedure. The false–positive rate of 4% maximum is very satisfactory. All detection capabilities (CCβ) were well below the recommended concentration (RC) of 50 µg kg−1. The applicability of AM I kit with different types of honey (monofloral or multifloral, liquid or solid, different floral origins, etc.) has been proved.

International audience; The main chemicals used against varoa are acaricides, and the antibiotics used for the control of bee bacterial diseases are mainly tetracyclines, streptomycins, sulfonamides and chloramphenicol. No maximum residue limits (MRLs) have been set for any antibiotics in honey. Therefore, in the European Union, minimum recommended concentrations (RC) for the analytical performance of methods to control a certain set of these non-authorised chemicals in honey were published by the European Union Reference Laboratory (EU-RL) in 2007. Concerning the strategy for the control for antibiotic residues in honey, there is still a great need for a cheap and single multi-residue method. Biochip array technology is an innovative assay technology for the multi-analyte screening of biological samples in a rapid and easy-to-use format. A multi-array system, called Evidence Investigator™ (Randox, Crumlin, Co., Antrim, UK), was evaluated in our laboratory. It is a semi-automated biochip system designed for research, clinical applications and veterinary use. A competitive chemiluminescent immunoassay is employed for the detection of antimicrobials. The MicroArray II kit (AM II) dedicated to the screening of six different families of antibiotic residues was validated according to the European guideline for the validation of screening methods for residues of veterinary medicines. The specificity was proven to be very satisfactory, and applicability to different kinds of honey was demonstrated. The detection capabilities (CCβ) of six antibiotic residues were determined and were below the RCs when exist. The AM II kit could detect at least six quinolones, four tetracyclines and three epimers, three aminoglycosides, three macrolides, thiamphenicol, florfenicol and ceftiofur along with one of its stabilised metabolites, the desfuroylceftiofurcysteine disulfide (DCCD).

International audience; The Evidence Investigator™ system (Randox, UK) is a biochip and semi-automated system. The microarray kit II (AM II) is capable of detecting several compounds belonging to different families of antibiotics: quinolones, ceftiofur, thiamphenicol, streptomycin, tylosin and tetracyclines. The performance of this innovative system was evaluated for the detection of antibiotic residues in new matrices, in muscle of different animal species and in aquaculture products. The method was validated according to the European Decision No. EC/2002/657 and the European guideline for the validation of screening methods, which represents a complete initial validation. The false-positive rate was equal to 0% in muscle and in aquaculture products. The detection capabilities CCβ for 12 validated antibiotics (enrofloxacin, difloxacin, ceftiofur, desfuroyl ceftiofur cysteine disulfide, thiamphenicol, florfenicol, tylosin, tilmicosin, streptomycin, dihydrostreptomycin, tetracycline, doxycycline) were all lower than the respective maximum residue limits (MRLs) in muscle from different animal origins (bovine, ovine, porcine, poultry). No cross-reactions were observed with other antibiotics, neither with the six detected families nor with other families of antibiotics. The AM II kit could be applied to aquaculture products but with higher detection capabilities from those in muscle. The detection capabilities CCβ in aquaculture products were respectively at 0.25, 0.10 and 0.5 of the respective MRL in aquaculture products for enrofloxacin, tylosin and oxytetracycline. The performance of the AM II kit has been compared with other screening methods and with the performance characteristics previously determined in honey.

International audience; The screening of antimicrobial residues in eggs is an especially important subject. Three different commercial kits for the screening of sulphonamides and other antimicrobials in eggs were validated in accordance with Decision 2002/657/EC: one enzyme-linked immunoabsorbant assay (ELISA) kit multi-sulphonamides (from RAISIO Diagnostics) and two microbiological tests (a Premi® test from DSM and an Explorer® kit from Zeu-Inmunotec). The false-positive rates were lower than 2% for all kits. The detection capabilities (CCβ) have to be as low as possible for banned substances and lower than the maximum residue limit (MRL) when MRLs have been set. The sensitivity of the Premi® test was better than that of the Explorer® test, probably because of the dilution of the eggs before the Explorer® test was used. The CCβ values towards most of the tested sulphonamides were satisfactory with the Premi® test (≤100 μg kg-1). Performance in a proficiency test for the detection of sulphonamides in eggs with the Premi® test confirmed these results. The detection capabilities of tetracycline and doxycycline were at the level of the MRL or twice the MRL maximum. The detection capabilities for chlortetracycline and oxytetracycline were higher (four to six times the MRL). The detection capabilities for amoxicillin, neomycin, tylosin and erythromycin were lower than their respective MRLs. Detection capabilities for sulphonamides were much lower for the ELISA kit than for microbiological tests. The ELISA kit could be recommended for the targeted screening of sulphonamides in eggs. On the other hand, the Explorer® and Premi® tests could be used as wide screening tests allowing the detection of most of the antimicrobial families.

Two microbiological kits based on Bacillus stearothermophilus (Eclipse 50® and Premi®Test) have been evaluated and validated according to the European guideline for the validation of screening methods (January 2010) and in relation to the concentrations recommended by the EU-RL in 2007. Both tests are robust, a fast method and easy to implement. Both tests are applicable to a very large variety of honeys from different floral and geographical origins (rosemary, lavender, scrub, heath, alder, forest, lemon, acacia, chestnut, raspberry, mountain and flowers) as well as honey of different colours (from blank honey to brown honey, including yellow and orange honey). A satisfactory false-positive rate of 5% was obtained for the Eclipse 50® test. The observed detection capabilities CCβ of the Eclipse 50® kit were: chlortetracycline (>75 µg kg(-1)), oxytetracycline (≤200 µg kg(-1)), tetracycline (>100 µg kg(-1)), cloxacillin (≤40 µg kg(-1)), tylosin (≤200 µg kg(-1)), desmycosin (>400 µg kg(-1)), sulfadiazine (≤300 µg kg(-1)), sulfadimethoxine (≤250 µg kg(-1)), sulfamerazine (>300 µg kg(-1)), sulfamethazine (>1000 µg kg(-1)), sulfamethizole (>75 µg kg(-1)), sulfamethoxazole (≤25 µg kg(-1)), sulfanilamide (≫1000 µg kg(-1)), sulfaquinoxaline (>75 µg kg(-1)), sulfathiazole (≤250 µg kg(-1)) and lincomycin (>1500 µg kg(-1)). These levels were all higher than the recommended concentrations where they exist. Due to its lack of sensitivity, it cannot be recommended for reliable routine use. The observed CCβ of the Premi®Test kit were: chlortetracycline (10 µg kg(-1)), oxytetracycline (>10 µg kg(-1)), tetracycline (≤10 µg kg(-1)), cloxacillin (≤5 µg kg(-1)), tylosin (≤10 µg kg(-1)), desmycosin (≤15 µg kg(-1)), sulfadiazine (≤25 µg kg(-1)), sulfadimethoxine (≤25 µg kg(-1)), sulfamerazine (≤25 µg kg(-1)), sulfamethazine (≤25 µg kg(-1)), sulfamethizole (≤25 µg kg(-1)), sulfamethoxazole (≤10 µg kg(-1)), sulfanilamide (≤25 µg kg(-1)), sulfaquinoxaline (≤10 µg kg(-1)), sulfathiazole (25 µg kg(-1)) and lincomycin (≤25 µg kg(-1)). The Premi®Test kit could be recommended for reliable use in routine control due to its low detection capabilities (except for aminoglycosides), but the disadvantage is a high false-positive rate of 14%.

Antibiotics are mixed with the food of bees to fight against diseases. No maximum residue limits have been set for honey. Recommended concentrations (RCs) have been published by European Union Reference Laboratories for tylosin and streptomycin. The objective of this project was to select and validate enzyme-linked immunosorbent assay (ELISA) kits for the screening of tylosin and streptomycin/dihydrostreptomycin residues to be implemented in the French honey control plan. Four ELISA kits for tylosin and five ELISA kits for streptomycin were evaluated. At the end, one kit each was selected and validated for tylosin (TECNA AB620) and streptomycin (Europroxima). Both ELISA kits for tylosin and streptomycin are specific, robust, fast and easy-to-use tests. The detection capability CC β of tylosin A was less than or equal to 10 µg kg(-1) (half the RC). The CC β of desmycosin (the hydrolysed product of tylosin A in acidic conditions) is approximately 200 µg kg(-1), which is five times the RC for tylosin (20 µg kg(-1)). Thus, this kit is fit for the screening of tylosin A but is unsuitable to detect desmycosin. The detection capability CC β of streptomycin was less than or equal to 10 µg kg(-1) (one fourth the RC). The cross-reactivity with dihydrostreptomycin was equal to 136%. Both ELISA kits were applicable to a wide variety of honey (single flower and multiflower, different floral origins, different geographic origins, different consistencies [liquid or solid] and different colours).

International audience; The Sulfasensor® Honey kit is a receptor test dedicated to the screening of sulphonamide residues respectively in different matrices. The aim of this project was to evaluate and validate this kit according to the CRL guideline for the validation of screening methods European regulation to achieve the French control plan in honey. The test is robust, quick (90 minutes for 40 samples), easy to perform and easy to read. The false positive rate was estimated to 12.5 %. The detection capabilities CC of the Sulfasensor® Honey kit were lower than or equal to 25 µg kg-1 for sulfamethazineSMZ, sulfamerazine, sulfathiazole, sulfapyridine, and between 25 to 50 µg kg-1 for sulfadiazine and sulfadimethoxine, 150 µg kg-1 for sulfaquinoxaline and 1000 µg kg-1 for sulfamethoxazole and sulfamethizole. Sulfanilamide is not detected by the kit. The kit is applicable to a wide variety of honeys (different floral and geographical origins, liquid or solid). This kit was implementing for the French control plan for the detection of antibiotic residues in honey in 2010 in parallel with an HPLC method. However, in 2011, the Sulfasensor® Honey kit has been replaced by a LC-MS/MS method for the screening and the confirmation of sulfonamide residues in honey, which detect all the sulfonamides of interest.

A rapid receptor test dedicated to the screening of tetracycline (TTC) residues, the Tetrasensor? kit in different matrices, has been evaluated and validated according to the European regulation to achieve the French control plan in honey. The detection capabilities (CC?s) were included between 5 and 15 µg kg?1 for chlortetracycline (CTC), oxytetracycline (OTC), TTC and doxycycline (DOXY). The cross-reactions of both kits towards other families of antibiotics were negligible and there was no masking effect of these antibiotics on the detection of tetracyclines. The false positive rate was equal to 9.4%. This kit was applicable to a wide variety of honeys [monofloral and multifloral, different floral origins, different geographical origins and different consistencies (liquid or solid), different colours]. Finally, the Tetrasensor? test is a robust method, quick, easy to perform and easy to read which is now applied to implement the French control plan for the detection of tetracyclines in honey. A rapid receptor test dedicated to the screening of tetracycline (TTC) residues, the Tetrasensor? kit in different matrices, has been evaluated and validated according to the European regulation to achieve the French control plan in honey. The detection capabilities (CC?s) were included between 5 and 15 µg kg?1 for chlortetracycline (CTC), oxytetracycline (OTC), TTC and doxycycline (DOXY). The cross-reactions of both kits towards other families of antibiotics were negligible and there was no masking effect of these antibiotics on the detection of tetracyclines. The false positive rate was equal to 9.4%. This kit was applicable to a wide variety of honeys [monofloral and multifloral, different floral origins, different geographical origins and different consistencies (liquid or solid), different colours]. Finally, the Tetrasensor? test is a robust method, quick, easy to perform and easy to read which is now applied to implement the French control plan for the detection of tetracyclines in honey.

International audience; The STAR protocol is a Five Plate Test (FPT) developed several years ago at the Community Reference Laboratory (CRL) for the screening of antimicrobial residues in milk and muscle. This paper presents the validation of this method according to the European Decision 2002/657/EC and to an internal guideline for validation. A validation protocol based on "simulated tissues" and on a list of 16 representative antimicrobials to be validated has been implemented in our laboratory during several months for the STAR protocol. The performance characteristics of the method have been determined (specificity, detection capabilities CCβ, applicability, ruggedness). In conclusion, the STAR protocol is applicable to a broad spectrum detection of antibiotic residues in muscles of different animal species (pig, cattle, sheep, poultry). The method has good specificity (false positive rate 4%). The detection capabilities have been determined for 16 antibiotics from different families in relation to their respective Maximum Residue Limit (MRL): beta-lactams (penicillins and cephalosporins ≤ MRL), tetracyclines (≤MRL and ≤ 2.5 MRL), macrolides (2 MRL), quinolones (≤ 2 MRL), some sulphonamides (≤ 3 MRL), trimethoprim (2 MRL). However, the sensitivity of the STAR protocol towards aminoglycosides (> 8 MRL) and florfenicol (≤ 10 MRL) was unsatisfactory (>> MRL). The 2 objectives of this study have been met: first validate the STAR protocol according to the European decision 2002/657/EC, then demonstrate that the validation guideline (Anonymous 2010) developed to implement this decision is applicable to microbiological plate tests even for muscle. The use of "simulated tissue appeared as a good compromise between spiked discs with antibiotic solutions and incurred tissues. In addition, the choice of a list of representative antibiotics allowed to reduce the scope of the validation, which was already costly in time and effort.

International audience; A multiresidue method was developed for monitoring antibiotic residues in milk using liquid chromatography coupled to a tandem quadrupole mass spectrometer (LC/MS-MS). Two very short extractions followed by two LC/MS-MS acquisitions allow the screening of 58 antibiotics belonging to 8 different families (penicillins, cephalosporins, sulfonamides, macrolides, lincosamides, aminoglycosides, tetracyclines and quinolones). This method is currently implemented in the laboratory in a qualitative way: i.e. monitoring the presence or absence of residue in a sample and identification of the analyte before the step of confirmation. In order to assess the performance of this method, a validation strategy described in an internal guideline for the validation of screening methods was applied. The aim of the validation was to prove the sufficient sensitivity of the method to detect all the targeted antibiotics at the level of interest (MRL) at least. According to the EU commission decision 2002/657/EC, the suiTable sensitivity of a screening method can be demonstrated when the CCbeta is below or equal to the MRL level and so the false compliant rate below or equal to 5 % at the MRL level. The validation scheme was established in order to take into account various variability factors: the apparatus response, the interday repeatability, the matrix effect, etc ... The results of the validation clearly demonstrate the suitability of this method for the detection and identification of more than 50 antibiotics and are in agreement with the results obtained in routine analysis.

Explorer® is a simple and fast new kit for the detection of inhibitory substances in raw meat. The test, a 96-well microtitre plate, is based on the inhibition of microbial growth (Geobacillus stearothermophilus spores). It was validated in accordance with European Commission Decision 2002/657/EC (2002). The specificity and detection capabilities for five compounds from major antimicrobial families and robustness were studied. The specificity of the test was assessed with four different animal species and was found to be very satisfactory (false-positive rates lower than 10%). The detection capabilities for amoxicillin (10 μg kg-1) and tylosin (100 μg kg-1) were at the maximum residue limit (MRL) level (50 and 100 μg kg-1, respectively) for both, for doxycycline (200 μg kg-1) and sulfathiazole (200 μg kg-1) at twice the MRL (100 μg kg-1 for each) and for cefalexin (500 μg kg-1) at 2.5 times the MRL (200 μg kg-1). Twenty-one samples were analysed in parallel with the Four Plate Test, the STAR protocol and the Explorer® test. One false-positive result and two false-negative results (samples containing oxytetracycline) were reported with the Explorer® test. In conclusion, the Explorer® test was shown to be robust and easily automated. Photometric reading allows informatic data storage and objective readings between technicians and days. The test can be used as a wide screening test because it enables detection of most of the antimicrobial families (penicillins, cephalosporins, tetracyclines, sulphonamides, and macrolides) in muscles from different animal species (porcine, bovine, ovine, poultry). © 2009 Taylor & Francis.

Premi Test contains viable spores of a strain of Bacillus stearothermophilus which is sensitive to antimicrobial residues, such as beta-lactams, tetracyclines, macrolides and sulphonamides. The growth of the strain is inhibited by the presence of antimicrobial residues in muscle tissue samples. Premi Test was validated according to AFNOR rules (French Association for Normalisation). The AFNOR validation was based on the comparison of reference methods (French Official method, i.e. four plate test (FPT) and the STAR protocol (five plate test)) with the alternative method (Premi Test). A preliminary study was conducted in an expert laboratory (Community Reference Laboratory, CRL) on both spiked and incurred samples (field samples). Several method performance criteria (sensitivity, specificity, relative accuracy) were estimated and are discussed, in addition to detection capabilities. Adequate agreement was found between the alternative method and the reference methods. However, Premi Test was more sensitive to beta-lactams and sulphonamides than the FPT. Subsequently, a collaborative study with 11 laboratories was organised by the CRL. Blank and spiked meat juice samples were sent to participants. The expert laboratory (CRL) statistically analysed the results. It was concluded that Premi Test could be used for the routine determination of antimicrobial residues in muscle of different animal origin with acceptable analytical performance. The detection capabilities of Premi Test for beta-lactams (amoxicillin, ceftiofur), one macrolide (tylosin) and tetracycline were at the level of the respective maximum residue limits (MRL) in muscle samples or even lower.

A semi-quantitative immunological method (BIACORE method) for detecting sulfamethazine in bovine milk was developed and validated using the total error approach. The acceptance limits were set at ±40% and the risk of procedure of (1−β) proportion measurements falling outside the acceptance limits was chosen at 5%. Different response functions were tested on the basis of the accuracy index (IA). The best model was a weighted (1/X2) quadratic regression and the simplest one was an unweighted quadratic regression. This approach identified the weak point of the method, which was precision. Finally this BIACORE method was able to detect positive samples containing sulfamethazine in the dosing range between 50 and 150 ng/ml.

Nicarbazin, a coccidiostat, is used as a feed additive in poultry but not in laying hens. Feed contamination may however occur resulting in residues being present in eggs. As a Maximum Residue Limit (MRL) does not exist for nicarbazin residues in eggs a "Differential Action Level" (DAL) of 100 microg/kg has been established by the Veterinary Medicines Directorate (VMD). We have studied a commercial ELISA kit validated to detect and quantify nicarbazin in eggs with a sensitivity of 3 microg/kg. We used the total error approach to assess the performance of and validate the kit at the DAL level. The accuracy profile has been successfully obtained for the ELISA kit. The method cannot however be validated as a semi-quantitative method and we have consequently determined a cut-off based on 5% false negative rate according to European Decision 2002/657 on blank and spiked samples (70 microg/kg). The cut-off value established was 20 microg/kg using the 95th percentile.