Field amplified sample stacking and in-capillary derivatization for forensic analysis of morphine and morphine-6-glucuronide in human urine by capillary electrophoresis

Abstract A rapid, sensitive, selective and simple capillary zone electrophoresis (CZE) method, by integration of field amplified sample stacking (FASS) and in-capillary derivatization (in-CapD) coupled with fluorescence (FL) detection (CZE-FASS-in-CapD-FL), has been developed and validated for simultaneous determination of morphine and its metabolite, morphine-6-glucuronide (morphine-6-G)in human urine. The method was based on in-line derivatization by introducing the sample to running background electrolytes (BGE) of disodium tetraborate decahydrate and potassium ferricyanide, whereas analytes were oxidized into highly fluorescent dimer products. Various parameters influencing the sensitivity and efficiency of the proposed method were investigated, and the optimal conditions of the BGE were as follow: 60 mM disodium tetraborate decahydrate (pH 10.5), 0.25 mM potassium ferricyanide and separation voltage of 9 kV. For FASS, morphine and morphine-6-G were electrokinetically injected for 14 s at 16 kV into the capillary that was pre-injected with water plug for 4 s. The fluorescence signals were then detected at excitation and emission wavelengths of 340 and 450 nm, respectively. Analysis was performed at ambient temperature (22±1 °C). Validation of the method showed good linearity over morphine and morphine-6-G concentrations ranging from 2 to 2000 ng/mL and 2.5 to 2000 ng/mL, respectively. The mean recoveries were ranged from 97.72 to 87.26% and 97.14 to 85.43% for morphine and morphine-6-G, respectively. The validated CZE-FASS-in-CapD-FL method was effectively applied as quantitative forensic analysis of morphine and morphine-6-G in clinical urine samples.