Development and Validation of A RP-HPLC Method for the Simultaneous Determination of Twenty Related Substances of Sulfamethoxazole and Trimethoprim in Injection Dosage Form

A simple, robust and for the quantification of experimentally 20 related impurities of sulfamethoxazole (SM) and trimethoprim (TM) in liquid pharmaceutical dosage form, a novel RP-HPLC method was developed. Utilizing gradient elution on a Kromasil 100-5-C18; 5.0 m, (4.0x250) mm, column, chromatographic separation was accomplished. Solvent A (solution of 0.08% orthophosphoric acid in water) and Solvent B (mixture of acetonitrile and methanol, 80:20 v/v), given at a flow rate of 0.8 ml/min, respectively, make up the mobile phase. Using a photodiode array (PDA) detector, the analytes were identified and measured at 210 nm. Eight Sulfamethoxazole-related impurities and twelve Trimethoprim-related impurities can be quantified and evaluated using an organic impurities technique. According to the ingredients of the injection dosage form, the LOQ Standard solution concentration for Sulfamethoxazole Impurities is 0.0016 mg/mL and for Trimethoprim Impurities is 0.00032 mg/mL. For the OI process, the sample’s nominal concentrations of trimethoprim and sulfamethoxazole are 0.32 mg/mL and 1.6 mg/mL, respectively. The Validation range of the procedure was examined for test concentrations ranging from 0.1% to 1.0%. For all the stated contaminants, LOQ solution and LOQ spiked sample solution are stable for 24 hours at 5 °C sample temperature. At a sample temperature of 5°C, Trimethoprim EP Impurity-H is stable for up to 12 hours. According to ICH recommended guidelines, the method was validated, and it proved to be accurate and exact (repeatability and intermediate precision level) within the corresponding linear range of known SM and TM impurities.