Biosolvent-based liquid liquid microextraction at sub-zero temperature for the determination of amphotericin B in human urine followed by HPLC-UV

• Menthol used as biosolvent for the extraction of amphotericin B. • No organic solvent/disperser is required for the extraction of the analyte. • The analytical protocol is rapid using environmentally friendly solvents. • The LOD achieved was 50 ng/mL and the method is linear in the range of 200–2000 ng/mL. • High WAC and CACI scores indicate that the method is both cost- and time-effective. Herein, a biosolvent-based LLME using menthol as extraction media has been proposed for the isolation of ATB in human urine. The principle of the method is based on the mixing of aqueous sample with 50 μL of menthol followed by sonication. Phase separation was achieved by centrifugation and the menthol layer was solidified under sub-zero conditions. Then, the aqueous phase was retrieved by a syringe and the molten menthol was redissolved and analyzed. The main experimental parameters affecting the extraction performance of the analyte were investigated using a multivariate analysis consisting in two steps: a Plackett-Burman design followed by a face-centered central composite design. Under optimized conditions the method is linear in the range of 200–2000 ng/mL with LOD and LLOQ of 50 ng/mL and 200 ng/mL, respectively. The precision of the method (intra-day and inter-day) was less than 14.9 % while the accuracy (expressed as relative recoveries) was between 86.7 and 118.5 %. The robustness of the LLME conditions was evaluated using Monte-Carlo simulations experiments, resulting in a CpK value of 2.091 with a standard deviation of 0.2 of both tested parameters. The greenness and the applicability of the method were assessed through the “White Analytical Chemistry and “Click Analytical Chemistry” indexes. The applicability of the developed analytical scheme was demonstrated by analyzing real urine samples.