Nitroimidazoles and Their Metabolites

Nitrofurans are banned substances within the EU and in some other countries because of their mutagenic and geno-toxic characteristics. Nitrofuran metabolites are still found, primarily in aquaculture products originating from Southeast Asia, with SEM (the metabolite of nitrofurazone) having the highest incidence. Methods for detecting residues of nitrofurans aim for protein-bound metabolites that may persist in tissues for considerable periods after treatment. Methods are reported for the detection and identification of nitrofuran metabolites in many different food products. The main difficulty in nitrofuran metabolite analysis is the low selectivity of SEM as a marker metabolite of nitrofurazone. Several other possible sources of SEM have been identified and investigated, the most important of which is the use of 

The most commonly reported nitroimidazoles in the literature include dimetridazole (DMZ), metronidazole (MNZ), ronidazole (RNZ), and ipronidazole (IPZ) (Fig. 7.7). More recently, additional members of this class such as carnidazole (CNZ), ornidazole (ONZ), tinidazole (TNZ), and ternidazole (TRZ) have also been incorporated into analytical methods. Extensive studies on the metabolism of nitroimidazoles in poultry, beef, swine, and fish have shown that the hydroxy metabolites of the parent compounds, formed by oxidation of the side-chain in the C2 position of the imidazole ring, are of additional concern. These compounds have been shown to have toxicity equal to that of their parent forms and are often rapidly formed by metabolism within the animal. Studies in both poultry and rainbow trout have shown that the distribution of parents to metabolites is analyte- and species-specific. As a result, it has been suggested that it is imperative that a residue control program considers both the parent and the metabolites. The metabolites indicated in the literature include MNZ-OH, IPZ-OH, and HMMNI (2-hydroxymethyl-l-methyl-5-nitroimidazole) as the hydroxy metabolites of MNZ, IPZ, and DMZ, respectively. HMMNI has also been identified as a metabolite of RNZ, but through a different pathway. HMMNI is not a major metabolite of RNZ, however, and is therefore not suitable on its own for use as a marker of RNZ use. 

In terms of instrumentation, earlier quantitative approaches concentrated on HPLC with UV detection. Early confirmatory approaches utilizing both GC and GC-MS for quantification and confirmation were abandoned because of the need for a derivatization step that made data interpretation challenging because the same end product was created for RNZ and the metabolite of DMZ. Most current methods reported in the literature are LC-MS/MS-based, due to its selectivity and confirmatory capability in the 1 txg/kg range. A summary of current quantitative and confirmatory methodology has been provided in Table 7.3. In addition. Table 7.6 at the end of this chapter outlines the common MS/MS transitions currently reported in the literature. 

TABLE 7.3 A Summary of Current Quantitative and Confirmatory Methods for Analysis of Nitroimidazoles and Their Related Hydroxy Metabolites 

Most nitroimidazole methods also incorporate further clean-up steps such as solid phase extraction (SPE). Some common phases used include Oasis Oasis MCX, Chromabond XTR, SCX, Bakerbond Silica, and MIP-SPE (molecular imprinted polymer). In addition, most methods incorporate a solvent washing step with solvents such as hexane prior to instrumental analysis for removal of non-polar material. More recent work bypassed extensive clean-up techniques such as SPE as it was felt that recent advances in MS/MS technologies and 

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