Validated Method for Determination of Eight Banned Nitroimidazole Residues in Natural Casings by LC/MS/MS with Solid-Phase Extraction

A sensitive method based on solid-phase extraction-liquid chromatography-tandem mass spectrometry interfaced with electrospray ionization (SPE-LC-MS/MS-ESI) was developed for the simultaneous determination of 8 banned nitroimidazole (NOZ) drugs including metronidazole (MNZ), ronidazole (RNZ), dimetridazole (DMZ), tinidazole, ornidazole, secnidazole, metronidazole-OH (MNZOH, the metabolite of MNZ), and 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI, the metabolite of RNZ and DMZ) in natural casings. After extraction with ethyl acetate and evaporation, the NOZs were reconstituted in ethyl acetate and purified on a strong cation-exchange SPE column, and then LC/MS/MS analysis was performed by positive ESI applying multiple reaction monitoring of 2 transition reactions for each compound. The method was validated according to the European Union requirements (Commission Decision 2002/657/EC). Specificity, linearity, decision limit (CC alpha), detection capability (CC beta), accuracy, and precision were determined. Average recoveries of the 8 NOZs from natural animal casing fortified at 3 levels (0.1, 0.5, and 1 mu g/kg) ranged from 87.3 to 116.5%. The calculated CC alpha for NOZs ranged from 0.029 to 0.049 mu g/kg, and CC beta ranged from 0.049 to 0.083 mu g/kg. Repeatability was in the range of 3.35-10.1%, and within-laboratory reproducibility was <10.3%.