Analytical method development and validation for Simultaneous Estimation of Rifampicin and Levofloxacin in Bulk and Synthetic Mixtures Using Validated RP- HPLC Technique

Due in large part to the rise of Mycobacterium tuberculosis strains that are extensively drug-resistant (XDR) and multidrug-resistant (MDR), tuberculosis (TB) continues to be a significant worldwide health concern. Combination therapy is crucial for reducing treatment time, increasing effectiveness, and preventing the emergence of resistance. A validated reverse-phase high-performance liquid chromatography (RP-HPLC) approach was utilized to concurrently estimate levofloxacin (LEV), a fluoroquinolone used in cases of resistant tuberculosis, and rifampicin (RIF), a first-line antitubercular drug. Using a mobile phase of 0.03 M potassium dihydrogen phosphate buffer (pH 3.0): acetonitrile (55:45 v/v) at a flow rate of 0.8mL/min and a detection wavelength of 230nm, the chromatographic separation was accomplished on a Kinetics C18 column (250mm × 4.6mm, 5µm). With correlation values (r²) of 0.9985 and 0.9994, respectively, the technique demonstrated linearity over 1–5µg/mL for RIF and 2–10µg/mL for LEV. RIF and LEV had limits of detection (LOD) of 0.0921µg/mL and 0.0914µg/mL, respectively, and limits of quantitation (LOQ) of 0.2790µg/mL and 0.2771 µg/mL. Method reliability was confirmed by accuracy ranging from 98.14 to 101.32% and precision (%RSD) being less than 2%. A 2–¹ fractional factorial design was used to assess robustness, and the results showed that flow rate and acetonitrile concentration had a substantial impact on chromatographic parameters. A synthetic mixture including RIF and LEV with PLGA polymer was successfully analyzed using the described approach. All things considered, this validated RP-HPLC approach is straightforward, quick, precise, and appropriate for routine quality control applications as well as simultaneous estimation of levofloxacin and rifampicin in mixed dose forms.