Simultaneous determination of sulfamethoxazole and trimethoprim in microgram quantities from low plasma volume by liquid chromatography–tandem mass spectrometry

A highly sensitive, selective and high-throughput liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous quantification of sulfamethoxazole (SMZ) and trimethoprim (TMP) has been developed and validated using imipramine as an internal standard. The analytes were extracted from 50 µL human plasma using solid phase extraction (SPE) and separated on Thermo Hypersil Gold C18 (50 mm × 4.6 mm, 5 μm) column under isocratic conditions in a run time of 2.5 min. Detection was carried out by tandem mass spectrometer, interfaced with electro spray ionization and operating in positive ionization mode. The calibration curves were linear over the concentration range of 0.88–80 µg/mL for SMZ and 0.03–30 µg/mL for TMP. The intra- and inter-day accuracy and precision (% CV) evaluated at four quality control levels were within 93.5–105.0% and 1.3–7.2% respectively. The absolute recovery was greater than 81% for both the analytes at two concentration levels. Stability of SMZ and TMP was assessed under different storage conditions. The validated method was successfully applied for a bioavailability study in 12 healthy volunteers after oral administration of 800 mg of SMZ and 160 mg of TMP succinate tablet formulations under fasting condition.