Eco-friendly simultaneous determination of oxytetracycline HCl, sulfadimethoxine sodium, and trimethoprim glutamate in pharmaceutical formulations using HPLC

• First simultaneous HPLC analysis of OTC, sulfadimethoxine sodium, and trimethoprim glutamate . • Eco-friendly method with AMVI = 4.67 and AGREE score = 0.82 aligns with green chemistry. • Validated per ICH: LODs 1.05–8.33 µg/mL, recoveries 98.4–102.1 %, RSD < 2 %. • Applied successfully to veterinary formulation with 99.5–100.6% label claim recovery. • High blueness (BAGI = 87.5) ensures cost-effective, industry-friendly implementation. The development of environmentally sustainable analytical methods for simultaneous determination of multiple active pharmaceutical ingredients (APIs) is critical for advancing pharmaceutical quality control. This study introduces a novel, green reversed-phase high-performance liquid chromatography (RP-HPLC) method for the concurrent quantification of oxytetracycline (OTC), sulfadimethoxine sodium (SFM), and trimethoprim glutamate (TMG) in veterinary formulations. Using a Hypersil BDS C18 column (250 mm × 4.6 mm, 5 µm) with an isocratic mobile phase (phosphate buffer pH 7: methanol, 55:45 v/v) at 1.0 mL/min and detection at 280 nm, the method achieved baseline separation of all analytes within 6 min. Validation per ICH guidelines demonstrated excellent linearity (r2 ≥ 0.9994) across ranges of 10–200 µg/mL (SFM), 30–300 µg/mL (OTC), and 6–40 µg/mL (TMG). Sensitivity was confirmed with limits of detection (LOD) of 2.34, 8.33, and 1.05 µg/mL and limits of quantification (LOQ) of 7.09, 25.24, and 3.20 µg/mL for SFM, OTC, and TMG, respectively. Accuracy (98.40–102.05 % recovery) and precision (%RSD < 2 %) met regulatory standards. The method’s environmental sustainability was rigorously evaluated using AGREE (score: 0.82), NEMI, ComplexGAPI, and AMVI tools, highlighting reduced solvent consumption (AMVI = 4.67 vs. 350 for conventional methods) and compliance with green chemistry principles. Additionally, the BAGI tool affirmed its practicality (score: 87.5/100), underscoring cost-effectiveness and ease of implementation. Successful application to a commercial veterinary powder, containing oxytetracycline HCl, sulfadimethoxine sodium, and trimethoprim glutamate, confirmed label claims (99.54–100.59 % recovery) with no matrix interference. This method represents the first simultaneous analysis of OTC, SFM, and TMG, offering a robust, eco-friendly alternative to pharmacopeial methods while enhancing efficiency in pharmaceutical quality control.