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A normal-phase high-performance liquid chromatographic method was developed to determine therapeutic concentrations of trimethoprim, sulphamethoxazole, and its N4-acetyl derivative in biological fluids. The compounds are extracted at pH 6.2 using ethyl acetate--chloroform in a single extraction. The detection limit is 15 ng/ml for trimethoprim, 20 ng/ml for sulphamethoxazole, and 10 ng/ml for its N4-acetyl metabolite. The method is rapid, sensitive, precise, and well suited to clinical pharmacokinetic investigations.
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