SIMULTANEOUS DETERMINATION OF SULFAMETHOXAZOLE AND TRIMETHOPRIM IN RAT PLASMA BY LC-ESI-MS AND ITS APPLICATION TO A PHARMACOKINETIC STUDY

A sensitive and selective liquid chromatography-mass spectrometry (LC–MS) method for determination of sulfamethoxazole and trimethoprim in rat plasma was developed and validated. The analytes and internal standard (IS) were extracted from plasma by liquid–liquid extraction using ethyl acetate, and chromatography involved Agilent Extend-C18 column (2.1 mm x 50 mm, 3.5 um) using 0.1 formic acid in water, and acetonitrile with methanol (1:1, v/v) as a mobile phase with gradient elution. Detection involved positive ion mode electrospray ionization (ESI), and selective ion monitoring (SIM) mode was used for quantification of target fragment ions m/z 253.8 for sulfamethoxazole, m/z 290.9 for trimethoprim and m/z 294.9 for IS. The assay was linear over the range of 20–40000 ng/mL for sulfamethoxazole and 10–1000 ng/mL for trimethoprim, with a lower limit of quantitation (LLOQ) of 20 ng/mL for sulfamethoxazole and 10 ng/mL for trimethoprim. Intra- and inter-day precisions were less than 15% and the accuracies were in the range of 92.5%–109.3% for both sulfamethoxazole and trimethoprim. This developed method was successfully used for the determination of sulfamethoxazole and trimethoprim in rat plasma for pharmacokinetic study.