Re-validation of a method for the determination of diclazuril in poultry feed by collaborative study

The European Union Reference Laboratory for Feed Additives (EURL-FA), hosted by the Institute for Reference Materials and Measurements (IRMM) of the Joint Research Centre (JRC), a Directorate General of the European Commission, is, among other tasks, mandated to provide scientific support to National Reference Laboratories (NRLs) in the field of the control of the authorised feed additives [1]. In this frame the EURL-FA has been contacted in June 2013 by an official control laboratory and its related National Reference Laboratory regarding severe technical problems when implementing an official method established by Commission Regulation (EC) No 152/2009 [2] for the determination of a specific coccidiostat, diclazuril, authorised as feed additive by the EU legislation [3]. The EURL-FA first conducted a survey among all NRLs for feed additives control & authorisation to gather their experiences with the Community method for the determination of diclazuril at authorised additive level in feed. The information provided by the NRL's hinted at a major flaw in a specific part of the method description of the Commission Regulation. Following the survey and reporting the issue to the Directorate General for Health and Food Safety (DG SANTE), it was decided to implement the method described in the Commission Regulation (EC) No 152/2009 [2] at the premises of the EURL-FA Control in order to identify the technical reason for this problem. The study performed at the EURL-FA Control confirmed the lack of fitness for purpose reported by the laboratories and due to a significant error in the method description as given in the European Commission Regulation [2]. The method was therefore modified at the EURL-FA Control and the results from the single-laboratory validation of this method showed satisfactory performance characteristics. Moreover, a modification of Commission Regulation (EC) No 152/2009 was considered necessary, to remove the error in the corresponding method description. In the 2014 annual workshops (EURL-FA Control and EURL-FA Authorisation) the results of the implementation, optimisation and validation of the method at the EURL-FA were presented to both networks of NRLs and discussion was launched on how to cope with this problem in the best way. As a conclusion, it was decided that the EURL-FA would organise a collaborative study among the NRLs in order to evaluate the fitness for purpose of the optimised method, which corrects the error found in the method description. This collaborative study was conducted in 2015 and the EURL-FA presented the results in the annual workshops of the EURL-FA Authorisation and EURL-FA Control respectively, in November 2015. The main aim of the collaborative study presented here was to assess the method performance characteristics of the corrected method based on high performance liquid chromatography coupled to spectrophotometric detection (LC-UV or LC-DAD) for the determination of diclazuril at additive level in feed. The required target limits of detection and quantification were respectively 0.1 mg kg-1 and 0.5 mg kg-1. According to the standard operational procedure (SOP) of the corrected method, the test portion, after addition of an internal standard, was extracted with acidified methanol and an aliquot of the extract was purified on a C18 solid phase extraction (SPE) cartridge, containing 5000 mg of the SPE sorbent. Diclazuril was eluted from the cartridge with a mixture of acidified methanol and water. After evaporation, the residue was dissolved in the mixture of N,N-dimethylformamide (DMFA) / water. The content of diclazuril was determined by ternary gradient reversed-phase high-performance liquid chromatography (HPLC) using a UV spectrophotometry detector measuring at 280 nm. In total 5 samples corresponding to 4 blind duplicates and 1 blank feed were sent to 15 laboratories and 14 laboratories reported results. Statistical evaluation revealed that the relative standard deviation for repeatability (RSDr) ranged from 4.5 % to 11.2 % and the relative standard deviation for reproducibility (RSDR) varied between 14.3 % and 18.1 %. The method showed acceptable within-laboratory and between-laboratory precision, since the calculated HORRAT values were in all cases below the critical value of 1.5. In addition, no false positive was found. Based on the data of the collaborative study and on the discussions, it was concluded during the workshops of 2015 that the EURL-FA will submit to the European Commission a recommendation for the revision of the current Community method. The recommendation will include a proposal for the final text of the method, drafted by the EURL-FA and consolidated after review by the NRLs (Authorisation and Control networks), for its inclusion in the revised Regulation replacing (EC) No 152/2009.JRC.F.5-Food and Feed Complianc