Rapid assay for determination of trimethoprim and sulfamethoxazole levels in serum by spectrofluorometry.

A rapid spectrofluorometric method for determining the levels of both trimethoprim and sulfamethoxazole from the same specimen of serum is described. The method involves stepwise extraction of the specimen first with chloroform at an alkaline pH (pH 9.0) for trimethoprim followed by n-butyl chloride at an acidic pH (pH 2.0) for sulfamethoxazole. To quantitate trimethoprim, the chloroform layer was subjected to fluorometry by exciting the specimen at 295 nm and measuring the relative intensity at 330 nm. To determine sulfamethoxazole levels, the n-butyl chloride layer was subjected to fluorometry by exciting the specimen at 285 nm and measuring the relative intensity at 330 nm. Relative intensities were linear (r greater than 0.99) over the concentration ranges of 0.5 to 40 microgram/ml for trimethoprim and 1 to 400 microgram/ml for sulfamethoxazole. Values obtained by this spectrofluorometric procedure were in excellent agreement with those obtained by a conventional fluorometric assay for trimethoprim and a colorimetric assay for sulfamethoxazole. Elevated levels of endogenous metabolic products and numerous other drugs, including a number of antimicrobial agents, did not interfere with the method. Although salicylates interfere with the determination of sulfamethoxazole, an appropriate correction can be made. This method can also be used to determine the drug levels in cerebrospinal fluid.