During the past years the use of antibiotics as feed additives has been recognised as a major risk for human health by the European legislator mostly because the presence of antibiotic residues in foodstuffs could cause toxic effects, directly in sensitive individuals such as allergic reactions and also indirectly because their widespread usage could be responsible for the promotion of resistant strains of bacteria. Following a ban of a number of antibiotics as feed additives from 1 January 2006 , no antibiotics other than coccidiostats and histomonostats can be marketed and used as feed additives within the European Union. Coccidiostats constitute the main choice to fight against coccidiosis. Coccidiosis is a major disease in poultry as well as in many other hosts. Commission Regulations authorise the use and the conditions of use of specific coccidiostats as feed additives, which are listed in the Community Register of Feed Additives. The conditions of use specify individually for each additive important aspects such as the target animal, the inclusion level of the active substance in the feed and – in the case of cocciodiostats – the duration of the period (withdrawal period) before slaughter when the use of these substances is prohibited.

The development, validation and implementation of reliable analytical methods are therefore of key importance to enforce the provisions laid down in the authorisation regulations of coccidiostats. In particular, the analytical methods should allow for measuring these substances at inclusion but also at trace level in order to cope with the unavoidable cross-contamination from target feed to non-target feed, occurring on the production lines. These methods should preferably be designed as multi-analyte method, in order to facilitate their use in the frame of official control. One of the main challenges is to provide simple, reliable and sensitive methods including the sample preparation.

In this presentation an overview of recent developments of analytical methods for the simultaneous determination of authorised coccidiostats will be given. These developments of quantitative and confirmatory methods include chromatographic based methods as well as an flow cytometry based multiplex immunoassay developed in the frame of the large European Collaborative Project CONffIDENCE* within the 7th Framework Programme of the European Community coordinated by RIKILT – Institute of Food Safety, Wageningen, The Netherlands. For the chromatographic methods, a new and fit to purpose multi-analyte method for the determination of the eleven authorised coccidiostats (monensin A, salinomycin, narasin, lasalocid, semduramicin, maduramicin, decoquinate, robenidine, nicarbazin, diclazuril and halofuginone) at trace levels in poultry, cattle, pig and calf compound feed by liquid chromatography tandem mass spectrometry (LC-MS/MS) has been developed and single-laboratory validated. The concentration level of the target analytes at which the validation experiments have been carried out varied between 0.5% and 6% of the authorised dose of the coccidiostats depending on the tested matrix.

In the multiplex assay, the target analytes are lasalocid, monensin, narasin, nicarbazin, diclazuril and salinomycin in eggs and in non-targeted feeds in the frame of a potential cross-contamination from feed produced with the highest authorised dose of the coccidiostats (e.g. broiler feed) into the afterwards produced non-targeted feed (e.g laying hens feed), taking into account hypothetical carry over rates of 2%, 5% and 10 %.

Keywords: coccidiostats; feed; Liquid chromatography–tandem mass spectrometry; multiplex immunoassay, validation

* CONffIDENCE has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 211326.

Websites :

http://irmm.jrc.ec.europa.eu/