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A suitable method has been developed and validated for simultaneously determining cocaine and its major metabolite, benzoylecgonine, 6-acetylmorphine, codeine, morphine and the methadone enantiomers in human hair samples by the in-line coupling between SPE and CyD-assisted CE with a previous sample pretreatment procedure based on pressurized liquid extraction. Optimal separation was achieved on a fused silica-capillary of 50 mu m i.d. and 80 cm total length using II mM alpha-CyD in an aqueous solution of 80 mM sodium phosphate at pH 2.5 as the separation medium and an applied voltage of 30 kV. The SPE-CE device consisted of a short length of capillary packed with Oasis HLB sorbent, which was inserted near to the inlet end of the CE capillary. Several parameters affecting the in-line preconcentration were evaluated. The LOQs reached for hair samples were in the range of 0.3-2.5 ng/mg with satisfactory analytical precision in both intraday and day-to-day experiments (RSDs <13%). Relative recoveries greater than 80% were obtained. The method has successfully been applied to the determination of these drugs of abuse in segmented hair from drug abusers who were undergoing methadone maintenance treatment. The results were consistent with the patients’ statements, indicating that the method established herein can be used for verifying a history of drug abuse. (C) 2016 Elsevier B.V. All rights reserved.