A green UFLC approach for vancomycin therapeutic drug monitoring in human plasma: method development, validation, and clinical application in critically ill patients at a tertiary care hospital

Vancomycin remains a cornerstone for treating severe Gram-positive infections, yet its narrow therapeutic index and high inter-individual variability demand precise Therapeutic Drug Monitoring (TDM). Conventional HPLC-assays often require large plasma volumes, corrosive reagents, long analysis times, and rarely assess environmental sustainability. This study aims to develop, validate, and clinically apply a rapid, eco-efficient green UFLC-UV method for quantifying vancomycin in human plasma, integrating comprehensive greenness and sustainability metrics within the White Analytical Chemistry (WAC) framework. Chromatographic separation was achieved on a Shimadzu Solar C 18 (150 × 4.6 mm, 5 μm) column using 10 mM ammonium acetate buffer (pH -2.3), methanol (84:16, v /v) at 0.8 mL/min with UV detection at 240 nm. 100 μL of plasma was used with a single-step methanol protein-precipitation, followed by ICH-M10 validation. Sustainability was systematically assessed using AGREE, AGREEprep, ComplexGAPI, BAGI, and RAPI tools. Clinical applicability was demonstrated using plasma samples from seven critically ill patients receiving intravenous vancomycin. The method exhibited excellent linearity (0.5–256 μg/mL, r 2 = 0.9999), accuracy (95.28–108.08%), precision (RSD ≤ 5%), and recovery (100.83–101.30%). Vancomycin remained stable under all tested conditions (±15%). Greenness (AGREE_0.71), practicality (BAGI_77.5), and analytical performance (RAPI_90.0) confirmed a balanced “white” analytical profile. In clinical samples, five of seven patients showed sub-therapeutic trough concentrations (<15 μg/mL), highlighting the need for individualized TDM. Overall, This UFLC-UV assay represents a novel, low-volume, methanol-based, and ICH M10-compliant bioanalytical method for vancomycin, uniquely integrating multiple WAC sustainability tools. The method is rapid, robust, and environmentally conscious, providing a practical platform for patient-specific vancomycin monitoring in clinical settings. Novelty statement: This study presents the first rapid, low-volume UFLC-UV method for vancomycin quantification that integrates Green, Blue, and Red Analytical Chemistry principles under the White Analytical Chemistry (WAC) framework. Unlike previous HPLC or LC–MS/MS methods that emphasize speed or sensitivity alone, this approach simultaneously optimizes analytical performance, environmental sustainability, and clinical applicability. The method's validation across multiple greenness and sustainability tools (AGREE, BAGI, RAPI) establishes a comprehensive eco-efficiency benchmark. Its successful application in critically ill patients demonstrates real-world translational value for individualized vancomycin TDM. • First UFLC–UV method integrating Green, Blue, and Red Chemistry under WAC. • Requires only 100 μL plasma with minimal solvent for rapid quantification. • Evaluated using 5-WAC tools: AGREE, AGREEprep, BAGI, RAPI, ComplexGAPI. • Clinically validated for patient-specific vancomycin TDM in critical care.