DEVELOPMENT & VALIDATION OF ANALYTICAL METHOD BY RP-HPLC FOR SIMULTANEOUSLY ESTIMATION OF ANTIBIOTICS IN A HOSPITAL ETP SLUDGE AND SOIL AS PER ICH GUIDELINE
DOI:
https://doi.org/10.22159/ijap.2026v18i3.57485Keywords:
Method development, Validation, Reverse-phase-high-performance-liquid-chromatography, Metronidazole, Mupirocin, Chloramphenicol, ICH-guidelines, Blue applicability grade index, Hospitals effluent treatment plant sludge and soilAbstract
Objective: The study aimed to develop and validate a simple, rapid, precise, and eco-friendly reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of metronidazole (MTZ), mupirocin (MPC), and chloramphenicol (CPL) in hospital effluent treatment plant (ETP) sludge and soil samples, with emphasis on compliance with green analytical chemistry (GAC) principles using the blue applicability grade index (BAGI) tool.
Methods: Chromatographic separation was achieved using a C18 column with a mobile phase comprising phosphate buffer and acetonitrile (72:28 v/v) at a flow rate of 1.5 mL/min and detection at 220 nm. A detection wavelength of 220 nm was selected after UV spectral evaluation and used consistently throughout method validation. The method was validated as per international-council-for-harmonisation (ICH Q2(R1) guidelines for parameters including system suitability, precision, accuracy, linearity, robustness, ruggedness, limit-of-quantitation (LOQ), limit-of-detection (LOD), solution stability, and forced degradation. Green assessment was conducted using the BAGI tool to evaluate solvent use, energy efficiency, waste generation, and sustainability.
Results: The method showed excellent linearity for all three antibiotics (R² > 0.999). The %RSD for precision and intermediate precision was below 2%, indicating high reproducibility. LOD and LOQ values confirmed the method’s sensitivity. Forced degradation studies revealed significant degradation under acidic and oxidative conditions, confirming its stability-indicating nature. BAGI assessment demonstrated complete compliance with green chemistry principles, indicating minimal solvent and energy consumption, reduced waste generation, and environmental safety.
Conclusion: The developed RP-HPLC method is accurate, precise, and environmentally sustainable. It can be effectively applied for routine analysis and monitoring of antibiotic contaminants in hospital ETP sludge and soil, supporting green analytical practices.
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