Sulfonamide detection methods

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. 

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. 

Seven replicate recoveries of flucarbazone-sodium, sulfonic acid, sulfonamide and NODT from well water fortified af 50 ng L averaged 106,100,89 and 106%, respec-fively. Therefore, the LOQ is 50 ng L for each analyte. The method detection limits for flucarbazone-sodium, sulfonic acid, sulfonamide and NODT, as determined by the United States Environmental Protection Agency (USEPA) recommended technique, are 5, 11, 20 and 19ngL, respectively. 

Rapid progress has been reported in the development of methods for sulfonamide residues in tissues, milk, and eggs since the subject was reviewed by Horwitz ( ) in 1981, The colorimetric method of Tishler et al (j ) has in the past been used to detect violative levels of sulfonamide residues in animal tissues. The lack of specificity and the variable background levels produced by this method have been discussed by Horwitz ( ), Matusik et al (15), and Lloyd et al (16), Recently, a number of specific chromatograpiic methods have been described for determination of residues of a variety of sulfonamides, These are summarized in Table 1 and suggest that HPLC is emerging as the method of choice followed by GLC and TLC methods. 

If tlie identity of the detected drug residue is still unknown after these tests have been performed, a TLC-bioautography procedure is then applied to isolate and tentatively identify the residues (64, 72). This method is not quantitative and only gives direction to the analyst as to what determinative/confirmatory method of analysis should be used. Following this tentative identification, presumptive positives are then quantified and confirmed by validated physicochemical methods. A TLC method (73) is applied for analyzing presumptive sulfonamide residues, a GC-ECD method (74) followed by a GC-MS NCI method (75) for analyzing chloramphenicol, and LC/UV methods (76,77) for analyzing -lactams and tetracyclines. 

Most immunochemical methods published for the determination of sulfonamides in edible animal products, serum, and urine concern sulfamethazine analysis (Table 28.3). Early methods for screening sulfamethazine in swine blood (54) necessitated extraction of the antibiotic from the sample and application of long assay protocols that rendered them impractical for routine analysis in hog slaughterhouses. Later methods developed for the detection of sulfamethazine residues in swine serum (55), urine and muscle (8), and in milk (9) addressed the extraction and assay problems of previous methods. 

Following their extraction and cleanup, residues of sulfonamides and diaminopyrimidine potentiators in sample extracts can be detected by direct nonchro-matographic methods, or thin-layer, gas, liquid, or supercritical fluid chromatographic methods (Table 29.7). 

In thin-layer chromatographic methods, sulfonamide residues were first separated on commercially available silica gel plates using various solvent mixtures as mobile phase, and subsequently detected by fluorometry after spraying... 

A multiresidue preparation technique—MSPD—has also been applied to the analysis of CAP residues in meat samples. Two fractions were collected by elution with methylene chloride and ethyl acetate. No additional purification was necessary. Diode assay detection and fluorescence detectors were recommended for the multiresidue analysis of sulfonamides, benzimidazoles, nicarbazin, furazolidone, and CAP. The percentage recoveries and linearity of the method were evaluated. The method was linear from 50 to 250 /tg/kg of CAP. Not only do the authors recommend the MSPD multiresidue procedure for HPLC analysis, but it could be associated with several detection modes, such as immuno- or receptor assays. The MSPD technique represents a new approach in the field of biological-matrix extraction and provides a great possibility for the analysis of a wide range of compounds (20). 

The method detection limit in the SIM quantitation procednre was between 0.2 and 0.5 pg/kg for most compounds in milk. The results of the pre-screening and the quantitation of 5 sulfonamides, i.e., sulfadiazine, sulfapyri ne, sidfamethazine, sulfisoxasole, and snlfaquinoxaline, spiked at 1 ppb, are shown in Figure 14.7. 

Liquid chromatography-ionspray-mass spectrometry has been shown to be an attractive approach for the determination of semduramicin in chicken liver. Tandem MS using the CID of the molecular ions further enhanced the specificity providing strucmre elucidation and selective detection down to 30 ppb. Liquid chromatography-ionspray-mass spectrometry has also been successfully applied for the assay of 21 sulfonamides in salmon flesh. Coupling of LC with either ISP-MS or ISP-MS-MS has also been investigated as an attractive alternative for the determination of erythromycin A and its metabolites in salmon tissue. The combination of these methods permitted the identification of a number of degradation products and metabolites of erythromycin at the 10-50 ppb level. Tandem MS with CID has also been... 

Another current trend that is well underway is the use of more specific analytical instrumentation that allows less extensive sample preparation. The development of mass spectrometric techniques, particularly tandem MS linked to a HPLC or flow injection system, has allowed the specific and sensitive analysis of simple extracts of biological samples (68,70-72). A similar HPLC with UV detection would require significantly more extensive sample preparation effort and, importantly, more method development time. Currently, the bulk of the HPLC-MS efforts have been applied to the analysis of drugs and metabolites in biological samples. Kristiansen et al. (73) have also applied flow-injection tandem mass spectrometry to measure sulfonamide antibiotics in meat and blood using a very simple ethyl acetate extraction step. This important technique will surely find many more applications in the future. 

Tsai, C.-E. Kondo, F. A sensitive high-performance liquid chromatographic method for detecting sulfonamide residues in swine serum and tissues after fluorescamine derivatization. J.Liq.Chromatogr,... 

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. ... 

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