Development of a certified reference material for the content of nitroimidazole parent drugs and hydroxy metabolites in pork meat

Nitroimidazoles have been applied in the past to poultry and pigs to treat protozoan diseases and to combat bacterial infections, but due to adverse health effects their use in food-producing animals has meanwhile been banned in the EU. The request for a certified reference material in a representative matrix was stipulated by the responsible Community Reference Laboratory and is underpinned by the need to improve the accuracy and comparability of measurement data and to establish metrological traceability of analytical results. The Institute for Reference Materials and Measurements (IRMM) has responded to this demand by developing and producing a new certified matrix reference material, ERM-BB124. This incurred lyophilised pork meat material was certified according to ISO guides 34 and 35 for the mass fractions of six nitroimidazole compounds. Processing of the frozen muscle tissue to the final material was accomplished by application of cutting, freeze-drying, mixing and milling techniques. Homogeneity and stability measurements were performed using liquid chromatography tandem mass spectrometry. The relative standard uncertainty due to possible heterogeneity showed to be below 1.8% for all analytes. Potential degradation during transport and storage was assessed by isochronous stability studies. No significant instability was detected at a storage temperature of −20 °C for a shelf-life of 2 years. The certified mass fraction values were assigned upon evaluation of the data acquired in an international laboratory inter-comparison involving 12 expert laboratories using different sample preparation procedures, but exclusively LC–MS/MS methods. Relative standard uncertainty contributions for the characterisation (between-lab variation of mean values) were found to be between 1.6 and 4.8%. Certified values for five analytes were in the range of 0.7 to 6.2 μg kg−1, with expanded relative uncertainties ranging between 7 and 14%. Dimetridazole could be certified as “<0.25 μg kg−1 with a probability of 95%”. All values are traceable to the International System of Units (SI). The material is intended to be used for method validation purposes (including trueness estimation) and for method performance assessment.