Abstract

Simultaneous determination of sulfamethoxazole and trimethoprim in human plasma by RP-HPLC

Author(s): Syed N.Alvi, Muhammad M.Hammami

A simple and precise reversed-phase high performance liquid chromatography (HPLC) method for simultaneous determination of sulfamethoxazole (SUZ), and trimethoprim(TMP) in human plasma was developed and validated. Using cefmetazole sodium as an internal standard (IS), separation was achieved on Symmetry Shield C18 (4.6ï‚´150 mm, 4 m) column. The mobile phase, 30 mM sodium phosphate (pH 5.8), acetonitrile, and 0.05% triethylamine (83:17:0.05,) v/v,was delivered at a flowrate of 1.2ml/min. The eluentwasmonitored using spectrophotometric detection at 235 nm. Plasma samples were precipitated using acetonitrile, and extracts were evaporated and reconstituted in sodium phosphate buffer. No interference in blank plasma or of commonly used drugs was observed. The relationships between the concentrations of TMP and SUZ with their corresponding peak height ratios to the IS were linear over the range of 0.10-6.0 and 1.0-70 g/ ml, respectively. The intra-day and inter-day coefficients of variation were ï‚£5.2% and 8.7% and ï‚£7.0 and 11.3% for TMP and SUZ, respectively. The extraction recovery of TMP, SUZ, and the IS fromplasma samples were 95, 85, and 80%, respectively. The methodwas applied to assess the stability of TMP and SUZ under various conditions generally encountered in the clinical laboratory. TMP and SUZ in plasma were stable for at least 24 hr at RT, 8 weeks at -20ºC; and after three freeze-thaw cycles. TMP and SUZ in processed samples were stable at least 24 hr at RT, and 48 hr. at -20ºC. Stock solutions of TMP and SUZ in mobile phase were stable at 24 hr at RT, 8 weeks at -20ºC.