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Abstract

A method for the estimation of an anilide in which the anilide is first hydrolyzed enzymatically to an aniline and then the quantity of the aniline produced is estimated spectrophotometrically preferably colorimetrically. The hydrolysis of the anilide may be catalyzed by any enzyme of the type, EC 3.5.1.13, known as aryl acylamidases. Preferably enzymes isolated from the cells of Pseudomonas fluorescens ATCC 39005 or Pseudomonas putida ATCC 39004 are employed. The aniline may be analyzed, for example, by conversion to an indamine, an indophenol or an indoaniline, followed by colorimetric analysis of the colored quinone-type compound produced. This conversion may take place in the presence of an oxidizing agent, such as a copper (II) salt, and/or a base, such as ammonia. A diagnostic kit to allow the routine use of the above method is also provided. In one embodiment of the kit, it comprises, (a) an aryl acylamidase, in solution or on a solid support, (b) an organic compound suitable for the conversion of the aniline to an indamine, an indophenol or an indoaniline, preferably in solution, and (c) a base, preferably in solution. The kit may further comprise an oxidizing agent, again preferably in solution. The above method of analysis and diagnostic kit may be particularly useful in the estimation of anilide drugs, such as paracetamol, dissolved in biological fluids.

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