Nitrofurans methods

Dicarbonyl compounds can be converted into furans by methods other than the classical Feist- Benary method, the essential feature of which is alkylation by a haloketone or similar species. A curious variation is provided by the use of trichloronitroethene, Cl2C=CCIN02, which condenses with two moles of a 1,3-dicarbonyl compound by Michael addition followed by elimination of two chloride ions the third chloride is lost at the aroma-tization step so that, for example, methyl 3-oxobenzenepropanoate is converted into the nitrofuran 38."... 

A variety of methods were developed for the identification and determination of the antimicrobial nitrofurans. They include LC, colorimetric and polarographic methods. Nitrofurans could be determined in animal tissues by extraction with acetonitrile, SPE and LC-UVD533. An LC-UVD method was statistically validated for the determination of nitrofuran drug residues in poultry534. 

Condensation of anthrandic acid (77-1) with an iminoether represents another method for preparing quinazolones. The reaction with the iminoether (77-2) from 2-cyano-5-nitrofuran and ethanohc acid can be visualized as proceeding through the formation of the amidine from addition-elimination of anthranilic acid cycliza-tion then affords the observed product (77-3). This is then converted to chloride (77-4) in the usual way. Displacement of the newly introduced chlorine with diethanolamine leads to the formation of nifurquinazol (77-5) [86], one of the antibacterial nitrofmans (see Chapter 8). 

Table 29.5 Physicochemical Methods for Nitrofuran Antibacterials in Edible Animal Products... 

In the liquid chromatographic methods, separation of nitrofurans is generally carried out on nonpolar reversed-phase columns, the preferred sorbent being octadecyl bonded silica (Tabic 29.5). Polar columns containing cyanopropyl-based sorbents (164, 165) have also been used for die isocratic separation of nitrofuran residues isolated from edible animal products. A literature survey shows that there exists a clear preference for acidic mobile phases containing acetonitrile as the organic modifier (Fig. 29.5.1). 

In liquid chromatographic analysis of nitrofuran antibacterials, the most popular detector is the ultraviolet visible (UV-vis) spectrophotometer. Nitrofurans exhibit strong absorption at wavelengths around 365 nm and are, therefore, ideal for direct determination (Table 29.5). Detection wavelengths of 275 nm (56, 57) and 400 nm (175) have also been suggested. Electrochemical detection is also frequently applied in liquid chromatographic methods for the determination of various nitrofuran antibacterials in edible animal products (172, 173, 179). 

Few simple amines are known in either the furan or the benzo[Z>]furan series. Simple nitrofurans on attempted reduction by mild chemical methods suffer what is probably a deaminative degradation. Similarly, attempted reduction of 2-nitrobenzo[6]furan affords not the amine but the product of its hydrolysis, benzofuran-2(3/f )-one. 

Both 2- and 3-nitrothiophenes are reduced by tin and hydrochloric acid to the corresponding aminothiophenes. Reduction of 2,5-dibromo-3,4-dinitrothiophene gives 3,4-diaminothiophene as a stable crystalline solid. 2-Acetamido-furans are prepared by the reduction of 2-nitrofurans in the presence of acetic anhydride. 2-Substituted 5-nitrofurans can be reduced to the S-aminofurans by an electrochemical method. Although catalytic reduction gives 2-aminofurans only in low yields, they can be trapped using ethyl ethoxymethylenecyanoacetate or ethoxymethylenemalononitrile. Benzofuranone 412 and not 2-aminobenzofuran is obtained from tin and hydrochloric acid treatment of 2-nitrobenzo[ ] furan 411. 

In order to determine nitrofuran activity in vitro, the minimum inhibitory concentration (MIC) in bacterial cultures must be found by using the serial two fold dilution technique with broth as a medium. Incubation time is 24 hours at 37°C. The cup method, which is usually employed for the assay... 

Reckendorf, Castringius and Spingler have utilized the colorimetric and spectrophotometric determination of nitrofurantoin in the urine and serum. Stone , and Puglisii have determined a trace of nitrofurantoin in milk by the colorimetric and spectrophotometric methods. Both procedures are based on the conversion of nitrofurantoin to 5-nitrofurfuraldehyde phenylhydrazone and are followed by the extraction and concentration on a chromatographic column. Final estimation depends upon development of a blue colour by the addition of hydramine base. Breinlich studied the titrimetric, spectrophotometric and chromatographic determinations of various nitrofurans and reported satisfactory results. 

As an aside, one might mention hair as a generally very stable matrix for most drugs and metabolites. These authors detected quantitatively sulfonamide and trimethoprim in horse tail hair 3 years after oral dosing in a horse, at a distance of 45-55 cm from the follicle. For the nitrofurans a MRPL of 1 p-g/kg has been set in the EU. Bound Residues and Nitrofuran Detection (FoodBRAND) comprises a rapid multi-residue screening test and also includes definitive multi-residue reference methods for protein-bound remnants of the nitrofurans. ... 

Methods for detecting residues of nitrofurans should not aim for the parent drugs because these are rapidly metabolized and do not persist in edible tissues. Nitrofurans form protein-bound metabolites that may persist in tissues for considerable periods after treatment. 

A common procedure for the analysis of nitrofuran metabolites involves hydrolysis of the protein-bound metabolites under acidic conditions followed by deriva-tization with 2-nitrobenzaldehyde (Eig. 7.5). After neutralization of the digest, solvent extraction is carried out with ethyl acetate. Residues are detected by LC-UV or LC-MS/MS In some cases an additional liquid-liquid extraction or solid-phase extraction step is applied to remove excessive matrix compounds. A broad overview of applied methods was published by Vass et al. in 2008. ... 

Because nitrofuran metabolites are very small molecules, the marker metabolites are not highly specific. This is especially the case for SEM. However, the presence of tissue-bound metabolites is more specific, because this indicates the administration of nitrofurans. Therefore, analytical methods are described focusing on bound residues. ... 

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... 

Bock C, Gowik P, Stachel C, Matrix-comprehensive in-house validation and robustness check of a confirmatory method for the determination of four nitrofuran metabolites in poultry muscle and shrimp by LC-MS/MS, J. Chromatogr. B 2007 856 178-189. 

Confirmation of quantity may also require the use of the method of standard addition or the inclusion of isotopi-cally labeled standards, although there are currently few such materials available for antimicrobial drugs, as noted in a review in 2009. Some examples of the application of isotopically labeled internal standards to methods for antimicrobial residues include the determination of chloramphenicol in meat, fish, and other biological matrices nitrofuran residues in milk and nitroimidazole residues in eggs and animal plasma. ... 

A comparison of mutagenic activity using normal and nitroreductase-deficient bacteria provides a method for detection and identification of nitroaromatic mutagens. But this idea is complicated by the existence of several bacterial nitroreductases with differing specificities (11). Three different nitroreductase-deficient derivatives of the normal tester strain TA98 were obtained from Dr H. S. Rosenkranz at Case Western University who has isolated and characterized these strains. Strain TA98NR was selected for its resistance to the nitrocompound niridazole and subsequently found to be resistant to the mutagenicity of niridazole, and also to that of the nitrofurans, nitronaphthalenes, and nitrofluorene (3). It is also resistant to 1-nitropyrene and a number of other nitro-PAH... 

Numerous analytical methods have been developed for the analysis of nitrofurans in various samples. These include microbiological, thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC) techniques. HPLC is the most widely used technique for determining nitrofurans of food sample origin, such as shrimps, fish, ° milk, egg and chicken... 

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