Development of a rapid method for the determination and confirmation of nitroimidazoles in six matrices by fast liquid chromatography-tandem mass spectrometry.

A rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed to identify and to quantify nitroimidazoles, metronidazole (MNZ), ronidazole (RNZ) and dimetridazole (DMZ) and their corresponding hydroxy metabolites, MNZ-OH and 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMNNI) in plasma, milk, muscle, egg, honey and feed samples. The same sample clean-up procedure including a novel solid-phase extraction (SPE) on polymeric Strata-SDB cartridges was used for each matrix. The analytes were separated on Kinetex XB C-18 core-shell type HPLC column using isocratic elution mode with a mobile phase containing 0.1% formic acid in water/methanol (88/12, v/v, pH 2.6) at a flow rate of 0.7 ml/min. The main advantage of the developed method is that the analysis time of only 3 min, which is about three to ten times shorter than in other reported HPLC methods. The developed method was validated using a matrix-comprehensive in-house validation strategy. The matrix effect of LC-MS/MS analysis was also investigated. Results are presented from the successful application of the developed method to an incurred pork meat certified reference material and to incur porcine plasmas in a proficiency test in year 2011.

Chromatographic and spectroscopic characterization of sulphur-bound dimetridazole and ranidazole derivatives.

The 5-nitroimidazoles, dimetridazole and ronidazole, two important veterinary drugs, were reacted under reductive conditions with the sulfhydryl-containing substrates cysteine and glutathione to yield 5-amino-4-S-substituted imidazoles. After purification by reversed-phase liquid chromatography (RP-LC), the four adducts were characterized by RP-LC with photodiode array detection using conditions where their parent drugs were not eluted from the column. Structural identification was conducted by spectroscopic techniques, mainly 1-dimensional and 2-dimensional NMR. While the dimetridazole adducts were found to be monosubstituted at the C-4 position, the two ronidazole products contained two units of the sulfhydryl substrate, located at the C-4 and C-6 positions.

Chemical and biological properties of acetyl derivatives of the hydroxylamino reduction products of metronidazole and dimetridazole.

Metronidazole and related 5-nitroimidazoles undergo reduction of their nitro group apparently to produce such reactive species as 5-hydroxylaminoimidazoles. To define the role of these species we have sought ways to prepare them by the catalytic reduction of metronidazole, dimetridazole and flunidazole. Although their respective 5-hydroxylaminoimidazoles were too unstable to be isolated directly, their O,N-diacetyl derivatives were isolable. Of these, the diacetyl derivative of the hydroxylamine derived from dimetridazole, O,N-diacetyl-1,2-dimethyl-5-hydroxylaminoimidazole (DiacDMH), was used for further study. DiacDMH was converted to its monoacetyl derivative, N-acetyl-1,2-dimethyl-5-hydroxylaminoimidazole (AcDMH), by enzymatic deacylation. Both DiacDMH and AcDMH were examined for bactericidal activity against such strains as Bacteroides fragilis, Clostridium perfringens, and Escherichia coli strain SR58, which are known to be sensitive to dimetridazole, as well as a variety of other bacteria. No bactericidal activity was detected, even in the presence of deacetylating enzymes. As the 5-hydroxylaminoimidazole itself could not be shown to form in these bacterial incubations, it remains uncertain whether or not the hydroxylamino functionality of a 5-nitroimidazole has bactericidal activity.