Rapid antibiotic detection for illegal and unlicensed substances in animal feedingstuffs

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Development of a multi-antibiotic EIA for the detection of virginiamycin, spiramycin, tylosin, bacitracin, and olaquindox. After preparation of four separate prototype EIAs for the detection of virginiamycin, bacitracin, spiramycin/tylosin and olaquindox, a single multi-antibiotic prototype EIA was constructed. Multi residue kit for the detection of 5 antibiotics. Introduction: Strips of a microtiter plate are coated with 4 different antibodies directed against 5 different antibiotics (virginiamycin, spiramycin/tylosin, bacitracin and olaquindox. For each antibiotic to be tested a zero standard (B max) and a cut-off standard has to be used in duplicate. Sample, standard solution and conjugate are added to the wells of the microtiter plate and incubated overnight at 4C. The conjugate contains the immunogens (virginiamycin, spiramycin/tylosin, bacitracin and olaquindox) individually conjugated to HRP. After incubation, the plate is washed followed by a substrate incubation of 30 minutes. The reaction is stopped by the addition of sulphuric acid. The colour intensity is measured in a spectrophotometer at 450 nm. Interpretation of the results: Results are calculated using the following equation: Result = (Neg - Abs)/(Neg - Pos) x 100% (threshold) Neg = absorbance of standard 0 (negative control) Pos = absorbance of cut-off standard (positive control) Abs = absorbance of sample Sample is positive at a threshold > 80% Sample is negative at a threshold < 80% Ring test. The ring test performed by 15 laboratories as part of the RADIUS project has shown the developed prototype ELISA test to be a very reliable guide for detecting the presence of a range of antibiotics present in animal feeds.

TNO has tested and validated the chemical method developed by the Gent University by analysing different types of feeds (poultry, pig and cattle). During the validation study different types of LC-MS instruments were used. Finally the type of instrument used by Gent University proved to yield the best results. After testing the chemical method, the description of the method (SOP) was improved in a dedicated meeting by Gent University, Queens University and TNO in Zeist (NL). At the same time, details of the preparation of the ring test were discussed. As part of the preparation of the ring test, the feed samples specifically prepared were quantitatively analysed to obtain data on the incorporated levels and the homogeneity of the material. Not all, but sufficient medicated feeds were found to be suitable to be used a ring test material. During the ring test, TNO also participated as participant. Overall, the scores obtained in the ring test proved that the method developed is suitable as confirmatory method for the detection of banned antibiotics in different types of animal feeds.

The banned antibacterial growth promoters were extracted from 2.5 g of feed by shaking with 10 mL of 70:30 (v:v) methanol:water + 2 % formic acid after the addition of about 20 mg of sodium sulphide. The obtained extract was submitted to a clean-up procedure by diluting 3 mL of this extract with 27 mL of water and applying this diluted extract to an OASIS HLB column. Finally, the antibacterial compounds were eluted from the SPE cartridge with 2 mL of 50:50 (v:v) acetonitrile:water and this eluent is further diluted with water until an acetonitrile content of 30 % was reached. Of this extract 20 µL was injected onto the HPLC column. Separation of the different compounds was achieved on a fully end-capped high purity silica Kromasil C18 column. Because of better peak shapes, a gradient program with acetonitrile as organic phase was chosen. The mass spectrometer was operated in ESI+ mode. This method was in-house validated in accordance with Commission Decision 2002/657/EC for three different feed types (poultry, pig and cattle). For most compounds CCα and CCβ was not influenced by the type of feed. Moreover, the values obtained were more or less comparable for all compounds, with a detection capability of about 0.5 mg/kg, which is half of the in advanced determined MRPL. A complete discription of the developed method and the validation results is published by Van Poucke et al. (Analytica Chimica Acta, 483, 2003, 99-109) During a European collaborative trial (11 participants / 7 different European Countries) the method was further validated. Results indicated that the confirmatory LC-MS/MS method for the detection and confirmation of banned antibacterial agents is able to distinguish between truly contaminated and non-contaminated samples and is at least able to identify the origin of the present antibacterial growth promoter, i.e. if it was caused by cross-contamination of the compound in the feedmill or was deliberately added to the feed.

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