Deliverables
The screening and confirmatory methods for coccidiostats will eventually be implemented in laboratories involved in regulatory control, in order to improve the ability of those laboratories to enforce EU regulations. Prior to methodology transfer and as a part of method validation, the methods were checked in a realistic experimental set-up.
For this purpose the availability of samples including blank, spiked and incurred sample material was considered essential. Therefore, the preparation of blank, spiked and incurred materials from poultry (egg and liver) was incorporated in the project.
The availability of these materials served two distinct purposes:
- To verify the analytical methodology for screening and confirmatory analysis.
- Samples could be used in the organisation of international ring tests, aimed at methodology transfer and dissemination of project results, targeted at the CRL-NRL network.
With a serial kill procedure such sample material was obtained that it could be used either directly or after dilution. In this way, a realistic ratio between metabolites and parent compound and a low risk of failure with respect to too low concentration levels at the time of slaughter could be obtained.
Compounds halofuginone, nicarbazin, nitroimidazoles and toltrazuril were selected as the priority coccidiostat drugs for assay development. The first phase was to produce a panel of high quality antibodies capable of binding these drugs in a specific manner. To accomplish this the small molecular weight drugs had to be coupled to larger carrier proteins to render the conjugate immunogenic. The coccidiostat compounds were conjugated to antigenic proteins such as albumen, haemocyanin and thyroglobulin using well-documented procedures such as carbodiimide, mixed anhydride reactions and NHS ester cross-linker. Following the completion of the immungen preparation the resultant conjugates were used in a range of experimental animals to illicit humoral immune responses. The obtained polyclonal antibodies were fully characterised with regard to titre, sensitivity, and cross reactivity. Dose response curves were generated against each of the coccidiostats included in the project as well as a range of chemically and structurally related compounds to determine the sensitivity and cross reactivity of each reagent produced. In conclusion, highly useful antibodies were produced to all the selected compounds. In a number of cases, most notably nicarbazin, nitroimidazoles and toltrazuril, there have been no reported successes by any research group in the world developing immunoreagents of this quality.
High quality test kits were produced for coccidiostats halofuginone, nicarbazin and nitroimidazoles. The all-in-one dry chemistry microtiter wells were coated with anti-rabbit IgG and then the analyte specific antibody was immobilised to the well. The insulation layer was dispensed into the wells and dried. The label was added in a small volume on top of the layer and dried. A custom-built small-volume dispensing machine was used for label dispensing during the assay development stage, when only few test kits were produced at a time.
However, the scaling up of the process proved to be difficult. Lot of effort was put into the tuning of the system, but the results were not satisfactory. Therefore, a completely new system for small volume label dispensing was constructed based on different kind of dispensing technique. However, the system could not be completed in time. Consequently, in the production of the test kits the label was dispensed manually to the microtiter wells. The stability and accuracy of the test kits were monitored during a one-year period. In addition, accelerated stability tests were conducted and the results were in accordance with the real time stability tests indicating that the kits were stable at least a year.
The confirmatory tests were based on quantitative and specific LC-MS-MS techniques. The selected coccidiostats for method development were halofuginone, nicarbazin, toltrazuril and nitroimidazoles. The sample matrix was poultry eggs and liver. Extraction protocols were developed or modified in order to save time and limit the usage of hazardous solvents. The aim was to develop confirmatory methods with sufficient sensitivity, that is to say to obtain a detection capability lower or equal to the minimum required performance limit, i.e. CCb< MRPL (b=5%). Particular emphasis was placed on the detection and elimination of potential problems associated with ionisation suppression, a phenomenon that frequently occurs in LC-MS analysis. If possible, deuterium labelled internal standards were used to facilitate the development of more robust analytical methods. All analysis methods were validated according to criteria laid down in the Annex to EC directive 96/23/EEC, which expresses a preference for the use of mass spectrometric methods in the confirmation of compounds with an MRL and an obligation to use of mass spectrometric methods in the reference analysis of compounds listed in Annex IV of Council Regulation 2377/90.
The ring test for each analyte/group of analytes was conducted amongst a number of NRLs, who were not involved in method development. The preparation of the test samples and the coordination with respect of the confirmatory assays was carried out under the supervision of RIKILT, while the coordination of the screening assays was in the hands of the University of Turku. Batches incurred material with residue concentrations close to the target concentrations were prepared from the materials obtained from the animal studies and spiked batches were prepared by fortification of blank material. The homogeneity of the incurred and spiked ring test batches was verified. Each laboratory tested 14 unknown samples per matrix and reported the results to RIKILT or University of Turku. The results were analysed qualitatively.
The screening tests for halofuginone and nicarbazin performed satisfactorily. The confirmatory methods on halofuginone and dimetridazole were evaluated to perform according to expectation, while the method on nicarbazin in one instance performed less satisfactory. Data on the method for toltrazuril were incomplete, but most likely this method performed satisfactorily as well. A Technology Transfer Event was arranged in BVL Berlin to introduce nicarbazin screening and confirmatory methods to several NRLs.
Council Directive 96/23/EC requires residue monitoring from the NRLs. There is strong evidence that coccidiostat residues can occur in poultry products, and consumers may be exposed to harmful residue concentrations. However, the lack of suitable methods has complicated the mandatory residue control.
To address this issue, rapid immunoassays were developed for the screening of three coccidiostats, halofuginone, nicarbazin and nitroimidazoles. The assays were based on the use of highly specific polyclonal antibodies, time-resolved fluorometry and a novel all-in-one dry chemistry assay concept. The dry chemistry concept enabled a one-step assay procedure, which could be fully automated with the help of an immunoanalyser.
All the assay specific reagents were predispensed into a single microtiter wells in advance and dried. Only the addition of a sample was required to start the assay, and the total assay time was 18 minutes. The sample material consisted of poultry eggs and liver. Prior to applying the samples to immunoassays, the samples were manually extracted according to a simple protocol. All the screening assays were validated according to Commission Decision 2002/657/EC.
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