Enantioseparation of (±)-threo-methylphenidate in human plasma by cyclodextrin-modified sample stacking capillary electrophoresis

The (±)-threo-methylphenidate ((±)-threo-MP) is widely used for treatment of attention-deficit hyperactivity disorder (ADHD). According to clinical evidence, (+)-threo-MP possesses higher potency than (−)-threo-MP. Due to ppb level in plasma, till now, none of the capillary electrophoresis (CE) methods have been able to provide adequate sensitivity for therapeutic (±)-threo-MP monitoring. In this study, a cyclodextrin-modified field-amplified sample stacking CE method (CD-FASS-CE) for enantioseparation of (±)-threo-MP in human plasma was established for clinical applications. Phosphate buffer (50 mM, pH 3.0) was filled into uncoated fused silica capillary (40 cm, 50 μm I.D.), followed by a water plug (0.5 psi, 10 s). Electrokinetic injection (6 kV, 200 s) was used to load samples and to enhance sensitivity. Stacking and separation were performed at 20 kV and 200 nm using phosphate buffer (50 mM, pH 3.0) containing 20 mM HP-β-CD and 30 mM triethanolamine. Analytes were separated simultaneously by using CD-FASS-CE and had a lower detection limit of equal to the sub-ppb level. Linear calibration curves were obtained from 1 to 80 ng/mL (r = 0.998). The limit of detection for both isomers was 600 pg/mL. RSD and RE of precision and accuracy in intra- and inter-day assays were below 7.89%. This method was further applied to analyze (±)-threo-MP in four healthy Asian volunteers and that provided some relevant information for clinical treatments.