Rapid multi-class multi-residue method for the confirmation of chloramphenicol and eleven nitroimidazoles in milk and honey by liquid chromatography-tandem mass spectrometry (LC-MS)

Abstract

A confirmatory method was developed to allow for the analysis of eleven nitroimidazoles and also chloramphenicol in milk and honey samples. These compounds are classified as A6 compounds in Annex IV of Council Regulation 2377/90 (European Commission 1990) and therefore prohibited for use in animal husbandry. Milk samples were extracted by acetonitrile with the addition of NaCl; honey samples were first dissolved in water before a similar extraction. Honey extracts underwent a hexane wash to remove impurities. Both milk and honey extracts were evaporated to dryness and reconstituted in initial mobile phase. These were then injected onto a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system and analysed in less than 9 min. The MS/MS was operated in multiple reaction monitoring (MRM) mode with positive and negative electrospray ionization. The method was validated in accordance with Commission Decision 2002/657/EC and is capable of analysing metronidazole, dimetridazole, ronidazole, ipronidazole and there hydroxy metabolites hydroxymetronidazole, 2-hydroxymethyl-1-methyl-5-nitroimidazole, and hydroxyipronidazole. The method can also analyse for carnidazole, ornidazole, ternidazole, tinidazole, and chloramphenicol. A recommended level of 3 µg l⁻¹/µg kg⁻¹ for methods for metronidazole, dimetridazole, and ronidazole has been recommended by the Community Reference Laboratory (CRL) responsible for this substance group, and this method can easily detect all nitroimidazoles at this level. A minimum required performance level of 0.3 µg l⁻¹/µg kg⁻¹ is in place for chloramphenicol which the method can also easily detect. For nitroimidazoles, the decision limits (CCα) and detection capabilities (CCβ) ranged from 0.41 to 1.55 µg l⁻¹ and from 0.70 to 2.64 µg l⁻¹, respectively, in milk; and from 0.38 to 1.16 µg kg⁻¹ and from 0.66 to 1.98 µg kg⁻¹, respectively, in honey. For chloramphenicol, the values are 0.07 and 0.11 µg l⁻¹ in milk and 0.08 and 0.13 µg kg⁻¹ in honey. Validation criteria of accuracy, precision, repeatability, and reproducibility along with measurement uncertainty were calculated for all analytes in both matrices.

Keywords:

• liquid chromatography/mass spectrometry (LC/MS)
• extraction
• clean-up
• liquid chromatography/mass spectrometry (LC/MS)
• veterinary drug residues
• chloramphenicol
• milk
• honey