In this study, we are used Liquid–liquid–liquid microextraction (LLLME) with directly suspended droplet using uvvisible spectroscopy for extraction and determination of methadone in hair. The target compound was extracted from the aqueous sample solution (donor phase, pH 9) into an organic phase and then was back-extracted into a directly suspended droplet of an acidic aqueous solution (acceptor phase, pH 1). In this method, without using a microsyringe as supporting device, an aqueous large droplet is freely suspended at the top-center position of an immiscible organic solvent, which is laid over the aqueous sample solution while being agitated. Then, the droplet was withdrawn into the microsyringe and determined with UV detection at λ=205 nm. 1-octhanol (350μL) used as organic phase. The results show extraction time: 15 min, back-extraction time: 5 min and stirring rate: 600 rpm. Also, these conditions used for determination methadone in hair samples of abusers were collected from 13 men ranging from 16 to 45 years old. The calibration graph was linear in the range of 0.2 -10 μg mL-1 with r = 0.995(N=5). The limit of detection and the enrichment factor were 1.7 ng mL-1 and 159.92, respectively. The relative recovery was in the range of 82–98% with an average of 91.50%. Also the relative standard deviation (RSD, n = 5) obtained 4.3%. All experiments were carried out at room temperature (25±0.5 °C).
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