Electrochemistry–Mass Spectrometry for Generation and Identification of Metabolites of Selected Drugs from Different Therapeutic Groups in Comparison with In Vitro and In Vivo Approaches

At a Glance

Metadata	Details
Publication Date	2025-09-05
Journal	Separations
Authors	Małgorzata Szultka‐Młyńska
Institutions	Nicolaus Copernicus University
Analysis	Full AI Review Included

Technical Documentation & Analysis: MPCVD Diamond for Advanced Electrochemistry-Mass Spectrometry (EC-MS)

Executive Summary

This research successfully validates the use of electrochemical (EC) simulation coupled with mass spectrometry (MS) as a reliable, high-throughput alternative to traditional in vitro methods for predicting drug metabolism. The key findings directly underscore the necessity and superior performance of 6CCVD’s specialized diamond materials:

Superior Electrode Performance: The Magic Diamond^TM (MD) electrode—an ultra-thin crystalline diamond layer on silicon—demonstrated the highest efficiency and generated the most significant number of metabolite signals compared to Glassy Carbon (GC), Gold (Au), and Platinum (Pt).
Wide Potential Window: The MD electrode enabled a significantly broader potential range (up to 3000 mV) necessary to mimic complex oxidative Phase I and II metabolic reactions, a critical advantage over conventional electrodes (limited to 2000 mV).
Material Requirement Match: The MD electrode’s characteristics (wide potential window, low background current, corrosion resistance) are intrinsic properties of Boron-Doped Diamond (BDD), a core product offered by 6CCVD.
Application Validation: The EC-MS method successfully generated and identified pharmacologically active metabolites (e.g., enalaprilat, hydroxymidazolam) that correlated strongly with results from liver microsome incubation and real patient samples.
Core Value Proposition: 6CCVD provides the high-purity, custom-doped MPCVD diamond required to replicate and advance this highly efficient drug screening and metabolite identification technique.

Technical Specifications

The following parameters highlight the critical role of the diamond working electrode in achieving high-fidelity metabolic simulation:

Parameter	Value	Unit	Context
Working Electrode Material	Magic Diamond^TM (MD)	N/A	Ultra-thin crystalline diamond layer on Si substrate.
MD Potential Range	0 to 3000	mV	Required for broad Phase I/II oxidation simulation.
Conventional Electrode Range	0 to 2000	mV	Limitation of GC, Au, and Pt electrodes.
Electrode Accessible Area	15	mm²	Area used within the thin-layer ReactorCell^TM.
Cell Volume	0.7	µL	Thin-layer cell design for efficient reaction.
Cell Temperature	36.7	°C	Maintained to simulate physiological conditions.
Potential Scan Rate	10	mV/s	Used for recording mass voltammograms.
Mobile Phase Flow Rate	10	µL/min	Low flow rate for EC simulation.
Drying Gas Temperature (X2)	290-350	°C	Optimized MS parameter for desolvation.
Fragmentor Voltage (X1)	70-150	V	Optimized MS parameter for fragmentation control.

Key Methodologies

The experimental protocol relied on precise control of the electrochemical environment, enabled by the specialized working electrode:

EC System Setup: Experiments utilized the ROXY^TM system (Antec) with a potentiostat and a ReactorCell^TM, employing a classical three-electrode arrangement (working, counter, and reference electrodes).
Working Electrode Selection: Four materials were tested: Platinum (Pt), Gold (Au), Glassy Carbon (GC), and Magic Diamond^TM (MD). The MD electrode, consisting of an ultra-thin crystalline diamond layer on a silicon substrate, was selected for its superior performance.
Phase I Simulation: A 5 µM drug solution was introduced into the reaction chamber (maintained at 36.7 °C) at a flow rate of 10 µL/min.
Oxidation Potential Application: The working electrode potential was linearly increased at 10 mV/s, reaching up to 2000 mV for conventional electrodes and up to 3000 mV for the MD electrode.
Phase II Simulation: Phase I products were mixed with glucuronic acid solution and UDPGA (uridine 5′-diphosphoglucuronic acid triammonium salt) via a 100 µL reaction coil to simulate glucuronide conjugation.
Detection: Products were detected directly online using a Triple Quad MS system in Multiple Reaction Monitoring (MRM) mode, allowing for simultaneous quantification and confirmation of metabolites.

6CCVD Solutions & Capabilities: Driving EC-MS Innovation

The research highlights that the success of EC-MS for drug metabolism studies hinges entirely on the performance of the working electrode, specifically the wide potential window and stability offered by diamond. 6CCVD is the ideal partner to supply the necessary materials for replicating and scaling this advanced analytical technique.

Applicable Materials

The “Magic Diamond^TM” electrode described is functionally equivalent to 6CCVD’s Boron-Doped Diamond (BDD), grown via Microwave Plasma Chemical Vapor Deposition (MPCVD) on a silicon substrate.

Material Recommendation: Heavy Boron-Doped SCD or PCD on Silicon Substrates.
- SCD (Single Crystal Diamond): Recommended for ultra-low background current (Ra < 1 nm polishing available) and maximum stability in harsh chemical environments.
- PCD (Polycrystalline Diamond): Recommended for large-area EC cells, offering plates/wafers up to 125 mm in diameter, ideal for scaling up high-throughput screening.
Thickness Control: 6CCVD can precisely control the ultra-thin crystalline layer thickness, offering BDD films from 0.1 µm up to 500 µm, ensuring optimal electrochemical properties and adhesion to the silicon substrate.

Customization Potential

The experimental setup used a specific 15 mm² electrode area. 6CCVD specializes in providing custom geometries and integration features essential for advanced EC systems:

Custom Dimensions and Shaping: We provide diamond plates and wafers in custom shapes and sizes, including laser cutting services to match the exact dimensions (e.g., 15 mm²) required for thin-layer ReactorCell^TM designs.
Integrated Metalization: The EC system requires robust electrical connections. 6CCVD offers in-house metalization services, including deposition of Ti, Pt, Au, Pd, W, and Cu contacts, ensuring low-resistance ohmic contacts necessary for stable high-voltage operation (up to 3000 mV).
Surface Finish: For reproducible results and minimal fouling, 6CCVD guarantees ultra-smooth surfaces: Ra < 1 nm for SCD and Ra < 5 nm for inch-size PCD.

Engineering Support

6CCVD’s in-house team of PhD material scientists and engineers are experts in optimizing BDD properties for electrochemical applications.

Material Selection Consultation: We assist researchers in selecting the optimal boron doping level, crystal orientation, and substrate type (SCD vs. PCD on Si) to maximize the potential window and signal intensity for specific drug metabolism simulation or redox reaction projects.
Global Supply Chain: We offer reliable global shipping (DDU default, DDP available) to ensure timely delivery of critical materials for international research and development programs.

For custom specifications or material consultation, visit 6ccvd.com or contact our engineering team directly.

View Original Abstract

The metabolism of antibiotics, antidepressants, and cardiovascular drugs has been investigated widely over the last few decades. The aim of this study was to develop an efficient analytical protocol based on the combination of electrochemistry (EC) and mass spectrometry for the identification of electrochemical products (potential pharmacologically active metabolites) of selected drugs (enalapril, metronidazole, midazolam, propranolol, venlafaxine). The electrochemical mimicry of the oxidative phase I and II metabolism was achieved in a thin-layer cell equipped with different working electrodes (magic diamond (MD), glassy carbon (GC), gold (Au), platinum (Pt)). The structures of the electrochemically generated metabolites were elucidated based on accurate mass ion data and tandem mass spectrometry (MS/MS) experiments. The in silico prediction of the main sites of selected drugs’ metabolism was performed using Biotransformer 3.0, GLORYx, and Xenosite software. Moreover, incubation with liver microsomes (LMs) was performed to examine the proposed metabolic pathways of target compounds. The data from in vitro experiments agreed with the data from electrochemical oxidation, which predicted some potential metabolites found in the real samples from patients. For enzymatic incubation, N-dealkylation, O-demethylation, and hydroxylation were the metabolic pathways involved mainly in their metabolism. Their in vitro phase II metabolites were identified as glucuronic acid conjugates. Finally, different in vivo phase I and II metabolites were identified for the studied drugs, including metabolic pathways for in vivo phase I N-demethylation, N-dealkylation, O-demethylation, and hydroxylation, while the metabolic pathways for in vivo phase II metabolites were identified as glucuronic acid conjugates.

Tech Support

Original Source

DOI: https://doi.org/10.3390/separations12090243

References

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