Highly Sensitive In-Capillary Derivatization and Field Amplified Sample Stacking to Analyze Narcotic Drugs in Human Serum by Capillary Zone Electrophoresis
Monika Malak, Hager Ebrahim, Heba Sonbol, Ahmed Ali, Yasmine Aboulella, Ghada M. Hadad, Samy Emara
Abstract
An in-capillary derivatization (in-CAP-D) integrated with field amplified sample stacking (FASS) has been developed for the determination of morphine (MOR) and its metabolite, morphine-6-glucuronide (MOR-6-G) in human serum using capillary zone electrophoresis (CZE) and fluorescence detection (in-Cap-D-FASS-CZE). Acetonitrile was employed in removing proteins and extracting MOR and MOR-6-G into the clear supernatant containing codeine (COD) as an internal standard (IS). The derivatization was achieved in an in-capillary mode by introducing the acetonitrile-treated samples into a running electrolyte containing an oxidizing agent of potassium ferricyanide, whereas MOR, MOR-6-G and COD were oxidized into dimer derivatives with highly fluorescent intensity. The effectiveness and sensitivity of the in-Cap-D-FASS-CZE method were affected by many parameters, and the following conditions were found to be optimal: 70 m Mdisodium tetraboratedecahydrate (pH, 10.5), 0.30 mM ferrricyanide and a separation voltage of 10 kV. In order to perform the FASS, samples were electrokinetically injected for 20 s at 20 kV into the capillary that was pre-field with a 4 s water plug. Analysis was performed at ambient temperature (22 ± 1 °C). The method’s validation revealed good linearity with respect to peak area ratios of MOR and MOR-6-G with the IS and the corresponding concentrations over the ranges of 1–2000 and 1.2 to 2000 ng/mL, respectively. Following one oral dose of controlled-release MOR sulphate tablet, the validated in-Cap-D-FASS-CZE method successfully enabled the determination of MOR and MOR-6-G in clinical serum samples.