Sub-zero temperature biosolvent-assisted liquid–liquid microextraction for quantifying propranolol and carvedilol in human urine using HPLC-UV: greenness profile evaluation

Herein, a biosolvent-based liquid-liquid microextraction method utilizing menthol as the extraction medium is proposed for the isolation and quantification of selected β-blockers (propranolol and carvedilol) in human urine. The method is based on the addition of 65 μL of molten menthol to aqueous urine samples, followed by sonication. Phase separation was achieved by centrifugation, and the menthol-rich phase was subsequently solidified using sub-zero cooling. The aqueous layer was removed <i>via</i> a syringe, and the solidified menthol layer was redissolved in methanol for HPLC analysis. The key experimental parameters influencing extraction performance were optimized through a two-step multivariate approach: Plackett-Burman design (PBD) for screening and face-centered central composite design (FC-CCD) for response surface optimization. Under optimized conditions, the method exhibited linearity in the range of 50-2000 ng mL^-1, with limits of detection of 11 ng mL^-1 for PROP and 17 ng mL^-1 for CARV. The method demonstrated intra-day and inter-day precision below 11%, and accuracy (% recovery) ranging from 87.2% to 110.2%. Robustness was evaluated separately for the LLME protocol and the HPLC method using Monte Carlo simulations and Plackett-Burman design, respectively. Environmental sustainability and practical applicability were assessed using the AGREEPrep, ComplexMoGAPI, Click Analytical Chemistry (CACI), and White Analytical Chemistry (WAC) metrics, confirming the method's greenness and suitability for routine analysis. Finally, the method was successfully applied to the analysis of authentic human urine samples, validating its effectiveness for real-world biological monitoring.