Abstract
Sulfonamides are an important class of antibacterial compounds used in veterinary practice and animal production. A number of specific compounds are approved for use in certain species under limitations requiring withdrawal long enough to eliminate the residues from edible tissues. In most cases, the legal tolerance is 0.1 ppm. This residue is measured in regulatory laboratories by a modified Bratton-Marshall procedure of solvent extraction, transfer to an acid solution, removal of potentially interfering amines by extraction from basic solution, diazotization of the sulfonamide in a reacidified solution, and spectrophotometric measurement of the developed color. The tissue metabolites of the sulfonamides are usually the parent compound and the N4-acetyl derivative, which is hydrolyzed to the parent compound by evaporation of the extracting solvent in the presence of acid.
The only collaborative studies which have been performed on methods for veterinary sulfonamides involve their determination as additives to feeds at levels of the order of magnitude of 100 ppm. Recoveries (94-103%) are satisfactory for the collaborative studies available. The reproducibility coefficient of variation of 5% is satisfactory for sulfaquinoxaline, sulfaguanidine, sulfanitran, and sulfamethazine; the 13% coefficient of variation for sulfadimethoxine is not satisfactory, indicating that the method for this compound needs improvement.
No collaborative studies have been performed on tissue residue methods. Therefore the between-laboratory variability and the susceptibility to outliers of the method used to enforce residue tolerances are unknown. Based upon within-laboratory studies, however, it appears that all of the diazotization-based methods are being operated close to their limit of reliable determination, a poor analytical practice.
Current methodological approaches utilize gas chromatography, high pressure liquid chromatography, and even gas chromatography-mass spectrometry with an isotopically labeled internal standard. None of these proposed methods have yet reached the stage of collaborative studies for either feeds or tissues.
In this discussion of analytical methods for sulfonamides, an introductory section covering the regulatory aspects and chemical properties of sulfonamides is followed by three main sections: first, general problems, including sampling and sample preparation, extraction, protein precipitation, cleanup, emulsions, color reactions, confirmation of identity, interferences, and sulfonamide metabolites; second, collaboratively studied methods of analysis, including methods for feeds, concentrates and premixes, and tissues, and statistical parameters; and last, other available analytical methods, including methods in the Food Additives Analytical Manual, validation studies for New Animal Drug Applications, the Tishler method, methods for sulfonamides as contaminants in feeds, methods involving chromatography, polarography, and automation, and prospective methods.
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