Nitroimidazoles analysis

Ellis JE, Yarlett N, Cole D, Humphreys MJ, Lloyd D (1994) Antioxidant defenses in the microaerophilic protozoan Trichomonas vaginalis—comparison of metronidazole-resistant and sensitive strains. Microbiol-SGM 140 2489-2494 Haggoud A, Reysset G, Azeddoug H, Sebald M (1994) Nucleotide sequence analysis of two 5-nitroimidazole resistance determinants from Bacterioides strains and of a new insertion sequence upstream of the two genes. Antimicrob Agents Chemother 38 1047-1051... 

It is noted [321] that alkylated 5-nitroimidazoles show more distinct maxima in a field of 220-260 nm than the corresponding 4-nitroisomers. This band is also slightly displaced to the long-wave area (7-13 nm) for 5-nitrosubstituted compounds in comparison with 4-nitroimidazoles. Thus, statistical analysis of the absorption spectra of 1-alkylnitroimidazoles allows establishing the nitro group position in the ring that is extremely valuable in the nitration chemistry of imidazoles. The absorption bands of... 

Differential UV spectrophotometry is also used for quantitative determination of compounds that increases the accuracy of the analysis of nitroimidazole-based pharmaceuticals [1131],... 

A detailed analysis of the mass spectra of methylated nitroimidazoles has been carried out [321, 1283, 1284, 1293-1296], These compounds are characterized by an intense molecular ion peak, the fragmentation pattern being the same as that of aromatic nitro compounds (Schemes 3.55 and 3.56) [1293],... 

By the negative chemical ionization (NCI) mass spectral method the negative ions of 2-nitroimidazole are obtained [1305], This method is much more sensitive than the positive chemical ionization one. The analytical potential of the NCI mass spectrometry for the analysis of different classes of nitro compounds has been discussed [1305], Mass spectra of 1,4-dinitro-, 2,4-dinitro-, and 4,5-dinitroimidazoles have been studied [1306], Comparisons of mass spectra (70 eV) of these compounds have shown that the fragment product ions have the same m/z values, but differ in their relative abundance and in the ion appearing the main peak. It seems that this difference is due to the nitramine functionality of 1,4-dinitroimidazole [1306]. 

The method of atomic adsorption analysis has been proposed for quantitative determination of mercury in the correspondent salt of 5-nitrotetrazole [1182], Chromatography is widely used for analysis and separation of nitroazoles. For example, thin-layer chromatography was used for separation of nitropyrazoles [1431, 1432], nitroimidazoles [1133, 1309, 1431], nitrobenzoxazole derivatives [1433], and 5-nitro-2,l,3-benzoselenadiazole [1434],... 

High-performance liquid chromatography (HPLC) has been used to analyze metronidazole [1435-1437], misonidazole [1309,1438], and other nitroimidazoles [1435, 1439] in body fluids or pharmaceutical dosage forms. HPLC analysis of effect of hypoxic-cell radiosensitizer misonidazole on the radiation-induced reduction of DNA bases (thymine, cytosine, and adenine) has been carried out [1440, 1441], HPLC was employed to characterize different nitroimidazoles [327, 366, 388,409, 450, 1442-1444], nitropyrazoles [246, 301], nitrothiazoles [366], l-aryl(hetaryl)-4-nitro-l,2,3-triazoles [601], nitrobenzimidazoles [707], nitrobenzofurazans [774, 1445-1449], nitrobenzotriazoles [1450],... 

The spectra of nitroimidazoles show features that are altered characteristically by substituents and by pH, and which are useful for the purposes of orientation. Chromatographic, spectrophotometric, and polarographic methods have been used ° in the simultaneous determination of N-substi-tuted and N-unsubstituted nitroimidazoles. The latter react with hydroxyl ions to form yellow nitroimidazole anions. The difficulty of reduction of these anions, together with the shift in the absorption maximum to longer wavelengths, makes the analysis possible. 

A 2008 paper has described for the first time a dilute and shoot strategy for the simultaneous extraction of wide variety of residues and contaminants (pesticides, myco-toxins, plant toxins, and veterinary drugs) from different foods (meat, milk, honey, and eggs) and feed matrices. Several antimicrobial classes were included (sulfonamides, quinolones, P-lactams, macrolides, ionophores, tetracyclines, and nitroimidazoles) in the analytical method. Sample extraction was performed with water/acetonitrile or acetone/1% formic acid, but instead of dilution of the extracts before analysis by UPLC-MS/MS, small extract volumes (typically 5 til) were injected to minimize matrix effects. Despite the absence of clean-up steps and the inherent complexity of the different sample matrices, adequate recoveries were obtained for the majority of the ana-lyte/matrix combinations (typical values for antimicrobials were in the range of 70-120%). In addition, the use of UPLC allows high-speed analysis, since all analytes eluted within 9 min. 

Several groups have adapted the method to analyze residues in a variety of matrices. Acetic acid (HQAc, 1%) and sodium acetate have been widely used to adjust and maintain pH and promote stability and recovery of base-sensitive residues. HOAc was used to adjust pH by Stubbings and Bigwood to determine residues [sulfonamides, quinolones, (fluoro)quinolones, ionophores, and nitroimidazoles] in chicken muscle. Buffering to acidic conditions improved the extraction efficiency of quinolones. Aeetonitrile extracts were subsequently purified by dSPE (see also Section 4.4.6.1) over Bondesil NH2 sorbent. An aliquot of the extracts was evaporated to dryness and re-dissolved in acetonitrile water (90 10, v/v) before LC-MS/MS analysis. Validation was performed on chicken muscle samples, and matrix-matched standards were used because suppression of the MS response was observed for many of the target analytes. 

In 2008, Carretero et al. described a multi-class method for the analysis of 31 antibacterials (including f)-lactams, macrolides, lincosamides, quinolones, sulfonamides, tetracyclines, nitroimidazoles, and trimethoprim) in meat samples by PLE-LC-MS/MS. Meat samples were homogenized and blended with EDTA-washed sand, then extracted with water by applying 1500 psi (Ib/in. ), at 70°C. One extraction cycle was 10 min. A drawback of the method is the large volumes of extracts (40 ml) obtained, which required evaporation to concentrate the extract volume prior to final analysis. This evaporation step considerably increases the time required for sample preparation. The proposed method has been applied to the analysis of 152 samples of cattle and pig tissues, with the presence of quinolones, tetracyclines, and sulfonamides detected in 15% of the samples, although at concentrations below the MRLs. 

ConnoUy L, Thompson CS, Haughey SA, Traynor IM, Tittlemeiser S, Elliott CT, The development of a mnlti-nitroimidazole residne analysis assay by optical biosensor via a proof of concept project to develop and assess a prototype test kiL Ana/. Chim. Acta 2007 598(1) 155-161. 

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... 

The nitroimidazoles, sulfonamides, and tetracyclines all present analytical challenges because of metabolism and/or chemical degradation. In the case of the nitroimidazoles, this is further complicated by the relatively low requirements for detection. Method development therefore has to take into account both metabolites as additional target compounds and low detection limits. Sulfonamide analysis has to take into account the potential for conversion of N -acetyl metabolites back to the parent compound. In contrast, in the analysis of honey, deconjugation is regarded as necessary to accurately determine sulfonamide concentrations. The facile, reversible formation of epimers is of particular concern in the analysis of those tetracyclines that can epimerize in the 4 position. Protein and metal binding are other issues that have to be overcome for successful tetracycline residue determination. 

Mottier P, Hure I, Gremaud E, Guy P, Analysis of four 5-nitroimidazoles and their corresponding hydroxylated metabolites in egg, processed egg, and chicken meat by isotope dilution liquid chromatography tandem mass spectrometry, J. Agric. Food Chem. 2006 54 2018-2026. 

Mohamed R, Mottier P, Treguier L, et al.. Use of molecularly imprinted solid-phase extraction sorbent for the determination of four 5-nitroimidazoles and three of their metabolites from egg-based samples before tandem LC-ESIMS/MS analysis, J. Agric. Food Chem. 2008 56 3500-3508. 

Ng CK, Sisanus AJ, Zaret BL, Soufer R. Kinetic analysis of technetium-99m-labeled nitroimidazole (BMS-181321) as a tracer of myocardial hypoxia. Circulation 1995 92 1261-1268. 

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