In today's world, antimicrobial agents play an extremely important role in the treatment of infectious diseases. Cotrimoxazole, a combination of trimethoprim and sulfamethoxazole, is commonly used in therapy. The active ingredients in combination act synergistically and are inhibitors of bacterial cell metabolism. To achieve the therapeutic effect or reduce the occurrence of adverse reactions, therapeutic monitoring of active ingredient concentrations is required in certain cases of cotrimoxazole use. The purpose of the master thesis was to develop and validate an analytical method for therapeutic monitoring of presumably low concentrations of both active ingredients in plasma samples.
The developed and optimized process of plasma sample preparation involved protein precipitation. 100 μL of plasma, 800 μL of acetonitrile as extraction solvent, and 100 μL of a standard addition solution of sulfamethoxazole and trimethoprim were used. The main challenge during the development of the sample preparation procedure was to reduce the matrix effect of trimethoprim, which was solved by choosing acetonitrile as the extraction solvent and modifying the chromatographic gradient program. The instrumental method was based on liquid chromatography coupled with tandem mass spectrometry using electrospray ionization in the positive mode. Separation of analytes was performed on a reversed-phase C18 column. Quantitative MRM transitions 291.2 ⒒ 230.2 (trimethoprim) and 254.1 ⒒ 65.2 (sulfamethoxazole) were determined based on the highest responses and the most suitable values of validation parameters. Isotopically labeled internal standards of both active ingredients were also included in the analysis, which proved to be a reasonable choice.
The validation of the method was performed according to guideline for the bioanalytical method validation, where the use of internal standards was found reasonable. The method meets the acceptance criteria of selectivity, accuracy, and precision. The linearity of the method was confirmed in the concentration range 1-200 μg/L and 21.4-4288 μg/L for trimethoprim and sulfamethoxazole, respectively. Different types of stability were evaluated and the active ingredients were found stable, a deviation was found only for freeze-thaw stability at the highest quality control sample. To extend the concentration range of the method, plasma samples were diluted and it was confirmed that the validation parameters were maintained within the specifications. The process efficiency, the extraction efficiency, and the absence of absolute and relative matrix effect were demonstrated. The applicability of the method for therapeutic drug monitoring will be confirmed on real samples.
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