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

Executive Summary

This research validates an advanced Electrochemistry-Mass Spectrometry (EC-MS) protocol as a rapid, reliable alternative for simulating and identifying drug metabolites, correlating results across in silico, in vitro, and in vivo models.

Core Achievement: Developed an efficient EC-MS method to mimic oxidative Phase I (N-dealkylation, hydroxylation) and Phase II (glucuronidation) metabolism for five key pharmaceuticals (enalapril, metronidazole, midazolam, propranolol, venlafaxine).
Electrode Superiority: The Magic Diamond (MD) electrode demonstrated superior performance, enabling electrochemical processes across a wider potential range (up to 3000 mV) and yielding the most significant number and intensity of metabolite signals compared to Glassy Carbon (GC), Gold (Au), and Platinum (Pt).
Validation Success: Metabolite profiles generated by the EC-MS system showed remarkable similarity to those obtained from conventional in vitro liver microsome (LM) incubation and were confirmed in real biological samples from patients.
Method Optimization: Mass Spectrometry (MS) parameters were successfully optimized using a Central Composition Plan (CCD), focusing on fragmentor voltage (70-150 V) and drying gas temperature (290-350 °C) for simultaneous drug and metabolite detection.
Future Utility: The EC-MS approach is positioned as a powerful screening tool to predict potential pharmacologically active or reactive metabolites early in drug development, supporting individualized patient treatment.

Technical Specifications

Parameter	Value	Unit	Context
Working Electrode Materials	MD, GC, Au, Pt	N/A	Tested for EC simulation efficiency.
MD Electrode Potential Range	0 to 3000	mV	Used for simulating Phase I metabolism (oxidation).
GC, Au, Pt Electrode Potential Range	0 to 2000	mV	Standard potential range for comparison.
Working Electrode Area	15	mm²	Accessible area in the ReactorCell^TM.
Cell Volume (Spacer)	0.7	µL	Volume separated by the 50 µm spacer.
Potential Scan Rate	10	mV/s	Used for recording mass voltammograms.
Mobile Phase Flow Rate (EC)	10	µL/min	Infusion rate into the reaction chamber.
LC Column	ACE5 C18 300 (150 x 4.6)	mm	Used for chromatographic separation.
LC Mobile Phase Flow Rate	0.4	mL/min	Standard flow rate for LC separation.
MS Ionization Mode	ESI(+)	N/A	Selected for higher signal intensity.
Drying Gas Temperature Range (Optimized)	290-350	°C	Optimized using CCD for efficient desolvation.
Fragmentor Voltage Range (Optimized)	70-150	V	Optimized for structural fragmentation (MS/MS).
Physiological pH Simulation	7.4	N/A	pH of buffer used for EC simulation.
Microsome Incubation Temperature	37	°C	Standard temperature for in vitro enzymatic studies.
Microsome Concentration (MLMs)	10	mg/vial	Concentration used for enzymatic studies.

Key Methodologies

The study utilized a multi-pronged approach combining computational prediction, electrochemical simulation, in vitro enzymatic assays, and clinical sample analysis.

1. Electrochemical (EC) Simulation (ROXY^TM System)

Phase I Simulation: A 5 µM drug solution was introduced into the ReactorCell^TM (kept at 36.7 °C) via an infusion pump (10 µL/min). Potential was linearly increased (10 mV/s) from 0 to 2000 mV (GC, Pt, Au) or 0 to 3000 mV (MD).
Electrode Selection: The Magic Diamond (MD) electrode was chosen as the optimal working electrode due to its wide potential window, low background currents, and high corrosion resistance, allowing for the study of a broader range of oxidative reactions.
Phase II Simulation: Phase I products leaving the chamber were mixed with 25 µM glucuronic acid solution and passed through a 100 µL reaction coil to achieve spontaneous enzymatic catalysis (glucuronidation).
Optimization: A Central Composition Plan (CCD) was used to optimize MS parameters, specifically the fragmentor voltage (X1) and drying gas temperature (X2), to maximize the Multiple Reaction Monitoring (MRM) signal intensity (PP_MRM).

2. In Vitro Enzymatic Incubation

Enzyme Source: Microsomes derived from mouse liver cells (MLMs) were used.
Reaction Setup: Drugs were incubated with MLMs (2 mg/mL) in a sodium phosphate buffer (pH 7.4) containing cofactors (NADPH/UDPGA) at 37 °C for up to 120 minutes.
Analysis: Reactions were stopped by cooling and adding acetonitrile, followed by centrifugation and analysis using RP-HPLC-ESI-MS/MS.

3. In Silico Prediction

Software Used: GLORYx, Biotransformer 3.0, and XenoSite predictor (FAME 3 models).
Purpose: Predicted putative metabolites, classified by probability (probable, minor, less probable), and identified the most vulnerable Sites of Metabolism (SOMs) in the drug structures.

4. Real Sample Analysis (In Vivo)

Sample Preparation: Urine and plasma samples from patients were subjected to Microextraction in Packed Syringe (MEPS) using C18 sorbent for efficient cleanup and preconcentration.
Detection: LC-ESI-MS/MS was performed in MRM mode using the optimized parameters to confirm the presence of metabolites identified in the EC and in vitro studies.

Commercial Applications

The methodologies and materials validated in this study have direct relevance across several high-tech engineering and commercial sectors:

Drug Discovery and Development: EC-MS provides a fast, cost-effective screening platform for predicting Phase I and II metabolism, significantly accelerating the identification of active or toxic metabolites compared to traditional in vitro methods.
Boron-Doped Diamond (BDD) Electrode Manufacturing: The superior performance of the Magic Diamond (MD) electrode (a type of BDD) validates its use in advanced electrochemical reactors, driving demand for ultra-thin crystalline diamond layers deposited on silicon substrates.
Advanced Analytical Instrumentation: The integration of electrochemistry with mass spectrometry (EC-MS) is critical for developing next-generation online analytical systems used in process monitoring, quality control, and environmental analysis.
Environmental Remediation and Sensing: BDD/MD electrodes, due to their wide potential window and stability, are ideal for electrochemical degradation studies of persistent organic pollutants (POPs) and pharmaceuticals in wastewater treatment, simulating environmental redox processes.
Chemical Synthesis: The EC reactor can be used for controlled, clean electrochemical synthesis of specific oxidized or reduced chemical intermediates, which are often difficult or expensive to produce via conventional chemical routes.

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

2015 - Electrochemistry-mass spectrometry to study reactive drug metabolites and CYP450 simulations [Crossref]
2004 - Electrochemistry/Mass Spectrometry (EC/MS)-A New Tool To Study Drug Metabolism and Reaction Mechanisms [Crossref]
2010 - Electrochemistry and LC-MS for Metabolite Generation and Identification: Tools, Technologies and Trends
2008 - Electrochemistry-mass spectrometry in drug metabolism and protein research [Crossref]
2016 - Electrochemistry-coupled to mass spectrometry in simulation of metabolic oxidation of methimazole: Identification and characterization of metabolites [Crossref]
2001 - Enalapril: Pharmacokinetic/dynamic inferences for comparative developmental toxicity. A review
2004 - Simultaneous determination of enalapril and enalaprilat in human plasma by liquid chromatography-tandem mass spectrometry [Crossref]
2018 - Development of a LC-MS method for simultaneous determination of amoxicillin and metronidazole in human serum using hydrophilic interaction chromatography (HILIC) [Crossref]
1999 - Sensitive and specific determination of midazolam and 1-hydroxymidazolam in human serum by liquid chromatography-electrospray mass spectrometry [Crossref]