Methods for simultaneous determination and identification of MS-222 and metabolites in fish tissues

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MS-222 (methanesulfonate of meta-aminobenzoic acid ethyl ester) is a primary aromatic amine commonly used to anesthetize fish. Like all primary aromatic amines, its diazonium salt reacts with N-1-naphthylethylenediamine dihydrochloride to form a wine-red azo dye with a maximum absorbance at 545 nm. Basic carbon tetrachloride extraction separates the azo dyes of MS-222 and its acid metabolite, m-aminobenzoic acid, and quantitative determination of each compound is made colorimetrically. By this method, recoveries of 82to 110 percent for MS-222 and 84 to 117 percent for m-aminobenzoic acid were obtained from largemouth bass muscle and liver tissues spiked with 1 to 10 microgram/gram of each compound. Quantitative estimation and identification of each compound were possible from samples spiked with as little as 1 microgram/gram of MS-222 or m-aminobenzoic acid. Striped bass (Morone saxatilis) anesthetized in a 100-mg/1 solution of MS-222 at 17.5° C contained an average of 57.9 microgram/gram of MS-222 and 23.2 microgram/gram of m-aminobenzoic acid residues in the muscle tissue immediately after a 30-minute exposure to the drug (0-hour withdrawal samples). After this tissue was homogenized and stored in a refrigerator for 1 week at 1.7° C, residues were 100 percent m-aminobenzoic acid. Fish anesthetized with benzocaine and treated in like manner still contained residues of benzocaine and a small amount of p-aminobenzoic acid (3.4 percent) after storage. The ester and acid residues of both anesthetics decreased steadily with length of recovery time. The residues were measured by a modified Bratton-Marshall colorimetric method and confirmed by thin-layer chromatography.