Simultaneous determination of Pyridoxine HCl, Isoniazid, Trimethoprim, and Sulfamethoxazole by Reverse Phase Liquid Chromatographic Method

Tuberculosis may be considered as a grave and persistent threat to global health.One of the major challenges in the treatment of tuberculosis is drug resistance, or sometimes, multiple drug resistance.The combination of Pyridoxine HCl, Isoniazid, Trimethoprim, and Sulfamethoxazole has recently been approved by a regulatory agency as a potent treatment for multiple drug-resistant tuberculosis.Hence a successful attempt was made to develop and validate a novel reverse phase high performance liquid chromatographic method for simultaneous determination of Pyridoxine HCl, Isoniazid, Trimethoprim, and Sulfamethoxazole.A mixture of acetonitrile and phosphate buffer in a ratio of 10:90 was employed as the mobile phase for the first 4 minutes in gradient programming, followed by adjusting the ratio of acetonitrile and phosphate Buffer to 25:75 until 8 minutes while maintaining the pH of the mobile phase at 4 throughout the measurement.With this developed method, the retention times of Pyridoxine HCl, Isoniazid, Trimethoprim, and Sulfamethoxazole were found to be 3.19± 0.0574, 3.65±0.0374, 4.88±0.0167, and 7.96±0.0989, respectively. The range of values was found to be 5µg/ml to 30µg/ml, 10µg/ml to 30µg/ml, 12µg/ml to 36µg/ml, and 10µg/ml to 50µg/ml for Pyridoxine HCl, Isoniazid, Trimethoprim, and Sulfamethoxazole, respectively. All the system suitability parameters and validation parameters results were within the acceptance criteria, in accordance with ICH guidelines for analytical method validation.