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.