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International audience; Premi®Test contains viable spores of a strain of Bacillus stearothermophilus which is sensitive to antimicrobial residues such as beta-lactams, tetracyclines, macrolides, sulphonamides, ... The growth of the strain is inhibited by the presence of antimicrobial residues in muscle samples. PremiÒTest was validated according to AFNOR rules (French Association for Normalisation). The AFNOR validation was based on the comparison of a reference method (French Official method (Four Plate Test) and the STAR protocol (5 plate test)) with the alternative method (PremiÒTest). A preliminary study was conducted in an expert laboratory (Community Reference Laboratory CRL) on both spiked and incurred samples (field samples). Several criteria (sensitivity, specificity, relative accuracy) have been estimated and discussed, in addition to detection capabilities. Adequate agreement was found between alternative and reference method. However, PremiÒTest was more sensitive to beta-lactams and sulphonamides than the FPT. Later on, a collaborative study was organised by the CRL with 11 laboratories. Blank and spiked juicemeat juice samples were sent to the participants. The expert laboratory (CRL) statistically analysed the results. In conclusion, Premiâ Test could be used for the routine determination of antimicrobial residues in muscles from different animal origins 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.

This study was performed in order to determine the stability of antibiotics belonging to eight families in solution or biological matrix. Knowledge of the stability of antibiotics has to be demonstrated during method development or validation. The stability of stock standard solutions of 53 antibiotics was assessed after determining the appropriate conditions of dissolution and storage. The stability of the same 53 antibiotics after addition to negative control cow milk or pork muscle tissue stored at –18 and –70°C was also assessed. Our concern was to obtain information concerning the stability of antibiotic residues in fortified biological matrixes in order to make easier the implementation of a routine screening method for antibiotic residues within the framework of the French monitoring program. Antibiotic solutions and fortified samples were analyzed using an LC/MS/MS method previously validated for screening purposes and for which it was checked that all pertinent criteria to obtain interpretable stability results were fulfilled. The design for testing the stability of antibiotics in solutions and matrix samples is described, as well as the rules of decision that were observed. Term periods for the stability study ranged from 1 month to 1 year, depending on the class of compounds. The results presented in this article will be useful and time-saving for many reference and field laboratories because LC/MS/MS methods are more and more commonly used for screening purposes.

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).

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.

International audience; The screening of antimicrobial residues in eggs is especially an important subject. Three different commercial kits for the screening of sulphonamides and other antimicrobials in eggs have been validated in accordance with the decision 2002/657/EC (EC 2002): one ELISA kit multi-sulphonamides (from RAISIO Diagnostics) and 2 microbiological tests (PremiÒTest from DSM and ExplorerÒ kit from Zeu-Inmunotec). The specificity was very satisfactory for all kits (false positive rates were lower than 2 % for all kits). The detection capabilities (CCb) have to be as low as possible for banned substances and lower than MRL when MRLs were set. The sensitivity of Premi®Test was better than sensitivity of Explorer® test, probably because of the eggs dilution before Explorer® test. The CCb values towards most of the tested sulphonamides were satisfactory with Premi®Test (£ 100 ng g-1µg kg-1). The participation to a proficiency test for the detection of sulphonamides in eggs with Premi®Test confirmed these results. The detection capability of tetracycline and doxycycline were at the level of MRL or 2 times the MRL maximum. The detection capabilities of chlortetracycline and oxytetracycline were higher (4 to 6 times the MRL). The detection capabilities for amoxicillin, neomycin, tylosin and erythromycin were lower than their respective MRLs. The participation to a proficiency test with Premi®Test gave very satisfactory results. Detection capabilities for sulphonamides were much lower for ELISA kit than for microbiological tests. The ELISA kit (from RAISIO Diagnostics) could be recommended for the targeted screening of sulphonamides in eggs. On the other hand, Explorer® test and Premi®Test could be used as wide screening tests which allowed to detect most of the antimicrobial families.

A proficiency test for the determination of nitrofuran metabolites in shrimp tissue was organized in the first half of 2003. This test was intended to allow the participants to use their routine method and to assess their competence on this specific analysis. The participation in this proficiency test was offered to all the National Reference Laboratories (NRLs) of the European Union (EU) in charge of the analysis of nitrofurans, to Official Laboratories of the then 10 Candidate Countries for entry in EU and to some countries exporting food to the EU. The participants (20) analysed nitrofuran metabolites in eight randomly coded frozen samples including three blank samples. All participants performed a confirmatory method using liquid chromatography/mass spectrometry to detect total nitrofuran metabolite residues. Both qualitative and quantitative analyses of the results were investigated. Qualitatively, 16 laboratories out of 20 gave the correct interpretation of the results in term of compliant/non-compliant sample. Quantitatively, laboratory performance was evaluated by calculating the z-scores.

International audience