Online-coupled electromembrane extraction and HPLC as a highly efficient, selective and clean microextraction technique for determination of ultra-trace levels of tramadol in biological fluids

• Online HPLC coupling enables automated, selective tramadol detection. • Polypropylene SLM (1-octanol) ensures efficient analyte transfer. • Green approach: 15 µL solvent per extraction, showing eco-friendliness. • Fully automatable with excellent sample clean-up capability demonstrate the method’s value. • Validated in human urine/plasma with LOD 3 ng/mL was highly successful. A highly efficient microextraction method namely electromembrane extraction coupled online with HPLC apparatus is proposed for extremely selective and accurate measurement of tramadol drug in the human bio-fluid matrix. The method established utilizes a polypropylene sheet as a support for the liquid membrane (1-octanol, 15 µL), which is situated within a poly(methyl methacrylate) cell. 10 mL of donor solution (pH 13.0) is continuously circulated within the extraction cell; it is delineated from the acceptor medium (pH 1.0, 100 µL) through supported liquid membrane (SLM), facilitating selective extraction of target analyte from donor solution to acceptor solution through the membrane interface. This methodology is notably convenient and eco-friendly. It can easily be completely automated, and concurrently addressing all the sample pretreatment goals. This method utilizes an aqueous-phase extractant and enhances sample clean-up by blocking macromolecule extraction through the membrane. To attain the best optimal conditions, the factors influencing extraction efficiency of proposed method were investigated and optimized. Underneath ideal conditions, the proposed method provided an appropriate linearity in a span of 10-1000 ng mL -1 (R 2 = 0.998), good limit of detection (3.0 ng mL -1 ), and remarkable extraction repeatability (RSD, 3.9%). The applicability of the method was assessed through analyses of human urine and plasma fluids, and the results attained revealed the exceptional ability of method in the complicated matrices’ analysis.