Evaluation and further development of methods

In the past few years numerous methods for the detection of natural and synthetic hormones, of antibiotic substances (including sulfonamides, tetracyclines, quinolones) and of anthelmintics have been worked on. With the cooperation of the working group , this methodical work has found its way into official investigation procedures. The working group regularly sifts through new, available analytical methods for the detection of pharmacologically active substances, the process characteristics of which make an application in testing laboratories appear practicable in compliance with the EU validation regulations. As a rule, checks for selectivity, sensitivity, precision and recovery are first carried out in at least three laboratories from the group of working groups on the basis of grown or spiked samples. The results are presented in the working group and discussed in detail. In the event of a successful pre-validation, the second step is to check the corresponding method with a larger number of participants from the group of active and corresponding members. The methods currently being worked on include the determination of:

 

• Coccidiostats
  In comparative studies by different laboratories, experiences in the analysis of different matrices and modifications of the generally recognized method for the determination of coccidiostats with LC-MS-MS are evaluated.
• ß-lactam antibiotics
  The active ingredients from the group of ß-lactam antibiotics can be determined very sensitively using the LC-MS-MS method. A method that has been extensively tested in the context of a dissertation (link) is tested with spiked samples with regard to its robustness for use in routine laboratories and its suitability for checking the maximum levels stipulated in food.
• Aminoglycosides
  For the determination of aminoglycosides in biological matrices, the coupling of HPLC with a mass-selective detector (tandem MS) appears to be well suited. The comparative investigation of published analytical methods in several laboratories is currently being carried out.
• Antibiotics in honey
  For the determination of antibiotics in honey using LC-MS-MS, various test methods were compared with regard to sample preparation. The following working hypotheses were formulated from the results of the internal laboratory comparison test "Veterinary medicinal products in honey":
  1.) When honey is normally stored at room temperature, anhydroerythromycin is formed from erythromycin through elimination of water.
  2.) A work-up process that does not contain an acidic hydrolysis step leads to sub-findings for sulfonamides and false negative results for Dapsone.
  3.) Macrolides are unstable in acidic solution.
  4.) An acidic environment promotes the epimerization of tetracyclines.
  5.) Influences of the pH value of the extraction agent used on the results of trimethoprim, nitroimidazoles, tylosin A, tylosin B, and chloramphenicol are not discernible.

screening test systems

The working group has set itself the goal of following the new developments in rapid test systems in addition to checking physical-chemical measurement methods such as LC-MS.

After an extensive comparison of the commercially available ELISA test kits, the Premitest as a rapid antibiotic test in organ material was compared in several laboratories with the results of the three-plate inhibitor test and chemical analysis methods.

Validation of methods

The aim of validating test methods includes ensuring a scientifically based and legally secured comparability of analysis results. Validation is part of quality assurance systems according to DIN EN ISO / IEC 17025.

Analysis results from different laboratories within the EU and beyond must be comparable accurate, correct and precise. For test methods validated before June 10th, 2021 as part of the official residue controls, the decision 2002/657/EG is still valid in part until June 10th, 2026 (Reference 1). Since June 10, 2021, the requirements of the Implementing Regulation (EU) 2021/808 must be observed for new or revalidations (Reference 4).

A sub-working group of the AG has developed a procedural proposal for the practical implementation of validation in routine laboratories in accordance with the above-mentioned EU decision. This was published after discussion in the working group (Reference 2). In addition, a comparative analysis of different validation methods was carried out.

The results of these discussions have found their way into the validation procedure recommended by the BVL using the "InterVal" software from quo data GmbH.

links and literature

Left

BVL

https://www.bvl.bund.de/DE/Arbeitsbereiche/05_Tierarzneimittel/tierarzneimittel_node.html

https://www.bfr.bund.de/de/lebensmittel-391.html

https://food.ec.europa.eu/safety/chemical-safety/residues-veterinary-medicinal-products_en

literature

1) Commission decision of August 12, 2002 implementing Council Directive 96/23/EC on the performance of analytical methods and the interpretation of results
(2002/657/EC)
2) Validation of procedures for testing for pharmacologically active substances in routine laboratories
food chemistry 57, 99-102 (2003)
3) Discussion on method validation of procedures for testing for pharmacologically active substances
food chemistry 58, 56-57 (2004)
4) COMMISSION IMPLEMENTING REGULATION (EU) 2021/808 of March 22, 2021 on performance criteria for analytical methods for residues of pharmacologically active substances in the
animals used for food production and on the interpretation of results, as well as on the methods to be used for sampling and the overturning of the decisions
2002/657/EC and 98/179/EC