Validation of a liquid chromatography–tandem mass spectrometry method for the identification and quantification of 5-nitroimidazole drugs and their corresponding hydroxy metabolites in lyophilised pork meat

A liquid chromatography–electrospray ionisation tandem mass spectrometry method for the simultaneous detection and quantitation of 5-nitroimidazole veterinary drugs in lyophilised pork meat, the chosen format of a candidate certified reference material, has been developed and validated. Six analytes have been included in the scope of validation, i.e. dimetridazole (DMZ), metronidazole (MNZ), ronidazole (RNZ), hydroxymetronidazole (MNZOH), hydroxyipronidazole (IPZOH), and 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI). The analytes were extracted from the sample with ethyl acetate, chromatographically separated on a C18 column, and finally identified and quantified by tandem mass spectrometry in the multiple reaction monitoring mode (MRM) using matrix-matched calibration and 2H3-labelled analogues of the analytes (except for MNZOH, where [2H3]MNZ was used). The method was validated in accordance with Commission Decision 2002/657/EC, by determining selectivity, linearity, matrix effect, apparent recovery, repeatability and intermediate precision, decision limits and detection capabilities, robustness of sample preparation method, and stability of extracts. Recovery at 1 μg/kg level was at 100% (estimates in the range of 101–107%) for all analytes, repeatabilities and intermediate precisions at this level were in the range of 4–12% and 2–9%, respectively. Linearity of calibration curves in the working range 0.5–10 μg/kg was confirmed, with r values typically >0.99. Decision limits (CCα) and detection capabilities (CCβ) according to ISO 11843-2 (calibration curve approach) were 0.29–0.44 and 0.36–0.54 μg/kg, respectively. The method reliably identifies and quantifies the selected nitroimidazoles in the reconstituted pork meat in the low and sub-μg/kg range and will be applied in an interlaboratory comparison for determining the mass fraction of the selected nitroimidazoles in the candidate reference material currently developed at IRMM.