Screening and Identification Methods for official control of Banned Antibiotics and Growth promoters in Feedingstuffs
||Type of the activity: research projectFinancing: European Community under the ‘Competitive and sustainable growth’
Programme (FP5). Project G6RD-CT-2000-00413
Duration of the project: 03/2001 – 07/2005
The authorisation of several antibiotics (avoparcin, zinc-bacitracin, spiramycin, tylosin and
virginiamycin) and growth promoters (carbadox and olaquindox) as feed additives has been withdrawn for reasons of human health concerning resistance of pathogenic bacteria (antibiotics) and toxicity (growth promoters). Available methods of analysis are not suited for an efficient and effective control of possible illegal use of these substances. There was a need to develop adequate methods.
SIMBAG-FEED project, Screening and identification methods for official control of possible illegal use of banned antibiotics and growth promoters in feedingstuffs“, was proposed within the framework of the specific programme “Competitive and Sustainable Growth”, Generic Activity “Measurements & Testing”, Dedicated Call 10/99, Topic II.15.
The main objective of SIMBAG-FEED was to develop, improve and validate multi-screening and multi-identification methods for the official control of the possible illegal use of antibiotics and growth promoters in feed. The methods will be integrated in a control strategy, which allows the efficient and effective control of the ban throughout the European Union.
Several types of multi-methods have been considered for inclusion in the control strategy, ranging from cheap and fast screening methods to confirmatory methods, applying sophisticated instrumentation. The following multi-methods have been studied in SIMBAG-FEED: – Microbiological plate test (MICRO) – High voltage electrophoresis (HVE) – Thin layer chromatography (TLC) – Liquid chromatography (LC) – Liquid chromatography-mass spectrometry (LC-MS).
The first three types of methods may be regarded as screening or post-screening methods. For the detection of banned substances, LC with single wavelength UV detection should also be regarded as a screening technique. LC with diode array detection (DAD) and especially LC-MS are powerful techniques for confirmatory analysis.
The second objective of SIMBAG-FEED was the set-up of a reference bank for storage of the banned substances. The reference bank contains pure reference standards with guaranteed stability during a period of at least 5 and probably 10 year. The amounts of material should be sufficient to provide the official control laboratories in the EU with reference material for a period of at least 10 year. The reference bank is an important requisite for the official control in the long run.
Antibiotics: For the antibiotics tylosin (TYL), spiramycin (SPIR), virginiamycin (VIRG), zinc bacitracin (BAC) and avoparcin (AVO) the target was achieved to develop multi-methods both for screening, post-screening and confirmation. For the antibiotics a 3- or 2-step control strategy has been established for the efficient and effective control of illegal use of antibiotics in feed. The initial screening is done with the microbiological plate test, the post-screening procedure with TLC (for TYL, SPIR and VIRG) and/or HVE (all 5 compounds) and finally confirmation with LC-MS/MS. Eventually, the post-screening step may be omitted. However, this may lead to a larger number of negative results in the LC-MS/MS confirmation.
The number of samples per day is in the range of 50-75 for microbiological screening, 20-25 for TLC, 20-25 for HVE and 10 for LC-MS/MS. For TYL, SPIR and VIRG this control strategy is supported by fully validated methods, incl. collaborative studies. For BAC, the LOD’s for the screening and postscreening step are somewhat higher. For AVO and BAC, the HVE post-screening step is only partly validated by means of collaborative studies and at present the LC-MS/MS confirmation could not be implemented by all laboratories.
Growth promoters: For the growth promoters olaquindox (OLA) and carbadox (CAR) multi-methods have been developed and validated through within- and between-lab validation and tested in collaborative studies. The control strategy consists of a 2-step approach, starting with LC-UV screening and quantification, followed by confirmation with LC-DAD or LC-MS/MS. If LC-DAD is applied directly, the control strategy can be reduced to a 1-step approach. The number of samples per day is in the range of 20-25 for LC-UV/DAD and 10 for LC-MS/MS. The control strategy is supported by fully validated methods, incl. collaborative studies.
Reference standards bank: The antibiotics zinc bacitracin, spiramycin, tylosin tartrate and growth promoters carbadox and olaquindox could be purchased and bottled according to the project objectives. The identity, content and purity was assessed by means of NMR, LC-MS/MS, continuous infusion MS/MS, LC-UV and microbiological inhibition methods. The homogeneity, short-term and long-term stability was tested. The materials were found to be homogeneous. Long-term stability was tested during a period of 12 months. No significant degradation could be observed under all tested conditions. Although it is not possible to guarantee the stability for a period of 10 years, from experience of many official control laboratories it is most likely that the reference standards are stable during this period under appropriate storage conditions.
The reference standards zinc bacitracin, spiramycin, tylosin tartrate, carbadox and olaquindox are available in sufficient amounts to supply the official control laboratories in the European Community for a period of 10 years.
Two workpackages cover final validations by means of collaborative studies and the establishment of a bank of reference standards.
Dissemination and use
The method protocols including the results of the collaborative studies and the concepts of the control strategy have been made available to the official feed control laboratories in the European Community. Furthermore, the method protocols of the fully validated methods, incl. collaborative studies, will be submitted to CEN for international standardisation.
The results of method development and validation, the results of the collaborative studies and the concepts of the control strategy have been or will be published in international scientific journals.
The SIMBAG-FEED project includes 10 partners (Official laboratories and institutes, Research centres, Universities) from 8 European countries involved in the feedingstuffs control. The project is co-ordinated by the Institute of Food Safety of Wageningen (RIKILT) in the Netherlands.
Dr Jacob De Jong
RIKILT – Institute of Food Safety
Bornsesteeg 45 PO Box 230
Tel : +31-317-475581
Email : Jacob.firstname.lastname@example.org