In the present study, a new generation of water-immiscible natural deep eutectic solvents (DESs) was synthesized using borneol as a hydrogen-bonding acceptor and decanoic acid, oleic acid, and thymol as a hydrogen-bonding donor in different molar ratios. These green hydrophobic solvents which are chemi- cally stable in aqueous solutions were used as extraction solvents for isolation and pre-concentration of warfarin in biological samples. In this method, fine droplets of DESs were dispersed into the sample so- lution by using the air-assisted liquid-liquid micro-extraction method to accelerate the cloudy emulsion system formation and increase the mass transfer of the analyte to the DES-rich phase. The borneol based deep eutectic solvent is a worthy generation of the extraction solvents in the ALLME method due to low- cost and less toxicity. A Plackett-Burman design was utilized for screening the experimental parameters. The effective parameters were then optimized by Box-Behnken design (BBD). Optimized extraction con- ditions were pH of sample solution of 3.9, number of aspiration/dispersion cycles of 15, the volume of DES of 60 μL, and rate and time of centrifuge of 60 0 0 rpm and 10 min, respectively. Under the opti- mized conditions, the developed NADES-ALLME method exhibited a wide linear range of 5–500 μg L −1 for plasma and urine samples with satisfactory recoveries above 88.80%. Limit of detections (LODs) and Limit of quantifications (LOQs) of warfarin were in the ranges of 0.5–2.7 and 1.65–8.91, respectively. The enrichment factors were obtained in the range of 148–164 and precisions were lower than 5.87%. Finally, the proposed method was successfully employed for the analysis of warfarin in human urine and plasma samples.
