Development of a microfluidic device with a screen printed electrode for studies on phase II metabolism

Vasiliadou, Rafaela

Abstract

Simulating human metabolism by electrochemistry (EC) and mass spectrometry (MS) provides an alternative approach to the existing in vitro and in vivo methodologies. Herein, screen printed electrodes (SPEs) were investigated as potent electrochemical tools with the aim of developing a microfluidic device with a SPE. The proposed chip could be used as a primary screening tool for phase II glutathione (GSH) adducts.

The reusability of SPEs was investigated with solvent treatment, mechanical polishing, and electrochemical activation. However, damages to the three-electrode configuration and lack of reproducibility prevented the effective removal of deposited material. Thus, SPEs were used as single-shot sensors for micking phase I and phase II metabolism.

The electrochemical reactions of dopamine, raloxifene, eugenol, and doxorubicin were examined initially on a bare SPE. Similar kinetics and reaction mechanisms were obtained as in previous studies with a glassy carbon electrode, confirming that SPEs can be used as cheaper alternatives. Controlled potential electrolysis (CPE) at the optimized potential was recorded for 30 min, in the presence of GSH, and subsequent offline MS permitted the detection of the corresponding GSH adducts of dopamine and raloxifene.

Dopaminoquinone and raloxifene di-quinone methide were generated via dehydrogenation and reacted covalently with GSH. However, the GSH adducts for eugenol and doxorubicin were not formed, leading to the generation of unconjugated metabolites. For example, eugenol was electrolysed via a single electron transfer with the addition of a proton to the corresponding phenoxyl radical. However, the oxidation reaction of GSH to form glutathione disulfide (GSSG) prevented further stabilisation to eugenol quinone methide and subsequent GSH adduct formation, mimicking only the catalytic pathway and polymerisation process. In the case of doxorubicin, the GSH adducts were probably formed at extremely low concentrations that were below the detection limits of the mass spectrometer or the expected doxorubicin semiquinone C7 free radical was generated but its trapping was unlikely, as proposed in previous in vitro and in vivo studies, owing to its quick oxidation back to the parent drug. Also, the possibility of generating non-reactive and highly unstable electrochemical products which were not capable of forming GSH adducts was considered. Thus, dopamine and raloxifene were selected for experiments in the microfluidic device. In addition, as the behaviour of acetaminophen on SPE is well-known, it was considered suitable for investigation with the proposed chip.

A polycarbonate disposable chip was developed, in which a 32 μL SPE- electrochemical cell was integrated with a serpentine channel. The chip was coupled online to an electrospray mass spectrometer for a semi-automated methodology. At an optimized flow rate of 5 μL min-1, 2.5x10-5 M solutions of dopamine and acetaminophen were allowed to electrolyse for 6.4 min in the SPE cell. The electrogenerated toxic intermediates dopaminoquinone and N-acetyl-p-benzoquinone imine reacted covalently with 5x10-5 M GSH and the resulting adducts were detected online by MS. Raloxifene failed to generate the GSH adduct in the microfluidic device owing to the requirement for lower flow rates that were incompatible with MS. In conclusion, a disposable and cost effective microfluidic device was developed for the simulation of phase II metabolism. This microdevice has the potential to reduce the expensive and time consuming use of the current in vitro and in vivo methodologies, in pharmaceutical industry and medical research.

Citation

Vasiliadou, R. (2015). Development of a microfluidic device with a screen printed electrode for studies on phase II metabolism. (Thesis). University of Hull. Retrieved from https://hull-repository.worktribe.com/output/4222259

• Thesis Type: Thesis
• Deposit Date: Mar 5, 2020
• Publicly Available Date: Feb 23, 2023
• Keywords: Chemistry
• Public URL: https://hull-repository.worktribe.com/output/4222259
• Additional Information: Department of Chemistry, The University of Hull
• Award Date: Oct 1, 2015