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Chemical assay development

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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