Study of aldehyde oxidase with phthalazine as substrate using both off-line and on-line capillary electrophoresis.

An optimized and economical capillary electrophoretic method for both off-line and on-line study of the enzyme aldehyde oxidase and its substrate phthalazine was developed. The separation of the substrate phthalazine and its metabolite 1-phthalazinone was achieved using micellar electrokinetic chromatography (MEKC) with sodium dodecyl sulphate in the background electrolyte (BGE). The BGE consists of 25 mM sodium phosphate buffer containing 50 mM sodium dodecyl sulphate at pH 7.4. A bare-fused-silica capillary with a capillary length of 40 cm, 50 μm ID and effective length of 30 cm was used to develop the capillary electrophoresis method. Improved separation conditions were elaborated and the separation method was validated based on the ICH and EMA guidelines. The limit of detection for phthalazine and 1-phthalazinone was 8 μM and 3 μM, respectively. The limit of quantification was 25 μM for phthalazine and 10 μM for 1-phthalazinone. The linearity of the detector response was checked for 1-phthalazinone at nine different concentrations in the range 10-500 μM and the determination coefficient was 0.9994. Accuracy was tested by comparing the corrected peak area of 1-phthalazinone reference solution at 20 μM and 50 μM with the corrected peak area of 20 μM and 50 μM 1-phthalazinone in the presence of human liver cytosol (HLC). Accuracy values of +5.3% and -2.5% were obtained at 20 μM and 50 μM, respectively. The on-line enzymatic reaction was successful with the application of the method of transverse diffusion of laminar flow profiles (TDLFP), which enables the mixing as well as separation of the enzyme and substrate inside the nanoliter-scale capillary. TDLFP is examined to be precise when performing 5 consecutive injections, with a relative standard deviation of 7.16% which is within the limitation of EMA standards. This miniaturized and low-cost incubation and separation method could be further introduced into industry and extended to other substrates.