Organic solvent-free micellar liquid chromatographic determination of amoxicillin, chlorzoxazone, and ibuprofen in pharmaceutical and biological matrices

Abstract

Athletes are frequently prescribed combination therapies to manage bacterial infections and musculoskeletal injuries, often involving amoxicillin (AMX), ibuprofen (IBU), and chlorzoxazone (CHL). However, their co-administration presents analytical challenges due to the structural diversity of these drugs within complex matrices. Simultaneous quantification of these agents is particularly demanding in the context of pharmacokinetic studies, therapeutic drug monitoring, and pharmaceutical quality control, where sensitivity, selectivity, and matrix compatibility are essential. To address this challenge, the present study introduces a rapid, robust micellar liquid chromatographic (MLC) method demonstrating an environmentally favorable profile relative to conventional acetonitrile-based RP-HPLC comparators, for the simultaneous determination of AMX, CHL, and IBU in their combined pharmaceutical dosage forms, and human plasma samples. The method utilizes a short RP-C18 column i.e., 75 mm, with UV detection at 220 nm, achieving complete separation of all three analytes in under 6 min using a low impact micellar mobile phase composed of sodium lauryl sulfate (SDS), Brij-35, and potassium dihydrogen phosphate at pH 3.0. Method validation was performed according to ICH guidelines, demonstrating linearity (R² > 0.999), accuracy (98.6-101.1%), adequate sensitivity (LOD: 0.44-0.74 µg/mL; LOQ: 1.32-2.25 µg/mL), and excellent baseline resolution between adjacent peaks (Rs > 4.5) across the concentration range of 2.5-25 µg/mL for all three analytes. Environmental sustainability was quantitatively assessed; the proposed method achieved an Eco-scale score of 87, compared to 68-78 for previously reported RP-HPLC methods, providing a quantitative basis for the green chemistry comparison. In alignment with green chemistry principles, the method's environmental sustainability was further evaluated using multiple metrics including the Analytical Eco-Scale, GAPI, AGREE, BAGI, and RGB12 tools. The collective results indicate that the proposed method's environmental profile compares favorably with previously reported RP-HPLC procedures, though it is acknowledged that the applied greenness metrics do not fully capture potential environmental persistence of non-ionic surfactants such as Brij-35 at high concentrations. Within the validated scope, the method demonstrates preliminary suitability for pharmaceutical quality control and therapeutic concentration monitoring; full bioanalytical validation per FDA/EMA guidance is recommended prior to pharmacokinetic applications. It is acknowledged that acetonitrile use in the plasma preparation step is included in the environmental assessments, and that long-term sustainability of the surfactant system at high concentrations warrants further evaluation.