| Metadata | Details |
|---|
| Publication Date | 2025-08-28 |
| Journal | Frontiers in Pharmacology |
| Authors | Paulina JerszyĆska, MaĆgorzata SzultkaâMĆyĆska |
| Institutions | Nicolaus Copernicus University |
| Citations | 1 |
| Analysis | Full AI Review Included |
- Method Validation: Electrochemistry coupled with Mass Spectrometry (EC-MS) was successfully validated as a rapid, ethical, and complementary instrumental technique for simulating oxidative Phase I and Phase II drug metabolism.
- Performance Comparison: EC-MS results showed strong agreement with metabolites identified in Human Liver Microsome (HLM) incubations and real patient plasma samples for five selected psychotropic drugs (Quetiapine, Clozapine, Aripiprazole, Venlafaxine, Vortioxetine).
- Optimal Hardware: The Boron-Doped Diamond (BDD) working electrode proved optimal, allowing electrochemical processes to be conducted across a wide potential range (0-3000 mV) necessary for efficient oxidative mimicry.
- Metabolic Mimicry: EC-MS effectively simulated key Phase I reactions, including N-dealkylation, S-oxidation, P-oxidation, hydroxylation of aromatic systems, and dehydrogenation, primarily occurring in the 1800 to 2200 mV potential window.
- Sample Preparation Efficiency: Biological sample preparation utilized Microextraction by Packed Sorbent (MEPS) with a C18 sorbent, achieving high average recoveries (98.16% ± 1.75%) and excellent repeatability for isolating drugs and metabolites from plasma.
- Future Utility: The study confirms EC-MS as a viable future alternative in vitro method for studying oxidation-reduction reactions, reducing the reliance on animal experiments and complex enzymatic systems.
| Parameter | Value | Unit | Context |
|---|
| EC Working Electrode | Boron-Doped Diamond (BDD) | N/A | Optimal electrode material tested (vs. Pt, Au, GC). |
| EC Potential Range (BDD) | 0 to 3000 | mV | Total linear potential sweep range. |
| Optimal EC Potential Range | 1800 to 2200 | mV | Range for most efficient transformation product (TP) formation. |
| EC Potential Sweep Rate | 10 | mV/s | Rate of linear potential increase. |
| EC Cell Temperature | 36.7 | °C | Controlled temperature for electrochemical transformation. |
| EC Flow Rate | 10 | ”L/min | Xenobiotic solution flow through the ReactorCellTM. |
| LC Column Specification | ACE C18 (150 mm x 4.6 mm) | N/A | Column used for chromatographic separation. |
| LC Flow Rate | 0.4 | mL/min | Mobile phase flow rate. |
| MS Ionization Mode | ESI(+) | N/A | Electrospray Ionization (Positive mode). |
| MS Spray Voltage | 4000 | V | ESI operation parameter. |
| MS Drying Gas (N2) Flow | 6.0 | L/min | Nitrogen flow rate. |
| MEPS Recovery (C18) | 98.16 ± 1.75 | % | Average recovery of analytes from plasma using optimal C18 sorbent. |
| HLM Incubation Temperature | 37 | °C | In vitro enzymatic reaction temperature. |
- In Silico Metabolite Prediction: Putative metabolite structures were predicted using GLORYx and Biotransformer 3.0 software based on the compoundsâ SMILES strings, integrating machine learning for Site of Metabolism (SoM) prediction.
- EC Simulation (Phase I Oxidation):
- Drug solutions (5 or 10 ”M) were prepared in optimized mobile phase (ammonium acetate buffer, pH 5-7).
- Solutions were pumped through the ReactorCellTM equipped with a BDD electrode at 10 ”L/min.
- The electrode potential was linearly increased (0 to 3000 mV) to induce oxidative reactions, and products were analyzed online/offline by LC-MS/MS.
- EC Simulation (Phase II Conjugation):
- The effluent from the Phase I EC cell was mixed with a solution of the conjugation agent, Glutathione (GSH), in a reaction coil.
- The resulting mixture was analyzed by LC-MS/MS to identify GSH-conjugated metabolites.
- In Vitro Incubation (HLM):
- Drugs were incubated with Human Liver Microsomes (CYP450) and the NADPH cofactor (20 mM) in phosphate buffer at 37 °C.
- Reactions were quenched by immersion in an ice bath and addition of ice-cold Acetonitrile (ACN), followed by centrifugation (5000 rpm) for supernatant analysis.
- Biological Sample Preparation (MEPS):
- Patient plasma samples were isolated and enriched using Microextraction by Packed Sorbent (MEPS) with C18 sorbent.
- Optimal extraction involved two cycles, followed by elution using a ternary mixture of acetonitrile:methanol:water (5:3:2, v/v/v).
- LC-MS/MS Analysis: All samples (EC TPs, HLM incubates, and patient plasma) were analyzed using a validated LC-MS/MS method (ACE C18 column, 0.4 mL/min flow) operating in ESI(+) mode to quantify drugs and identify metabolites via MRM transitions.
| Industry/Sector | Application Area | Technical Relevance |
|---|
| Pharmaceutical Drug Discovery | High-Throughput Metabolic Screening | EC-MS provides rapid, non-enzymatic prediction of oxidative metabolites, accelerating the early-stage screening of new drug candidates for metabolic stability and potential toxicity. |
| Clinical Diagnostics & TDM | Development of Bioanalytical Assays | The EC-MS approach generates and characterizes key metabolites, providing necessary fragmentation data (MRM transitions) for developing sensitive and specific LC-MS/MS methods used in Therapeutic Drug Monitoring (TDM). |
| Toxicology and Safety | Identification of Reactive Intermediates | EC-MS is uniquely suited to simulate single-electron oxidation, enabling the detection and structural proposal of short-lived, reactive metabolites (e.g., GSH conjugates) that are often responsible for adverse drug reactions. |
| Bioanalytical Instrumentation | Advanced Electrode Technology | Validates the use of BDD electrodes in complex bioanalytical flow systems, demonstrating their stability and wide electrochemical window for simulating biological redox processes. |
| Regulatory Science | Alternative In Vitro Testing | EC-MS offers an ethical and instrumental alternative to traditional HLM or animal testing for predicting Phase I metabolism, potentially reducing the need for extensive in vivo studies. |
View Original Abstract
Introduction Psychotropic drugs strongly affect the human psyche through their ability to modulate the neurotransmitter activity and to treat mental disorders and diseases. Monitoring of psychotropic drugs in clinical studies is significant. Thus. establishing methodologies for analyzing these drugs and their pharmacologically active metabolites in biological matrices is essential for patientsâ safety. Therefore, therapeutic drug monitoring (TDM) of these drugs in patients receiving pharmacotherapy in psychiatric hospitals is necessary to avoid medical complications, psychiatric adverse effects, or poisoning. In addition to TDM, the main factor in pharmacokinetics that should be monitored along with the drug is its metabolic pathway. The literature on transformation products (TPs) resulting from the psychotropic drug degradation is limited. Hence, to investigate the potential TPs of target compounds, electrochemistry (EC) and liver microsome assays were used to generate TPs, which were further characterized using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results obtained by EC-(LC)-MS and liver microsome assays were compared with conventional in vivo studies by analyzing biological samples (human plasma) from patients. Methods The electrochemical mimicry of the oxidative phase I and II metabolism was achieved in a thin-layer cell equipped with a boron-doped diamond (BDD) working electrode under controlled potential conditions. Structures were proposed for the electrochemically generated products based on the MS/MS experiments. Moreover, in order to examine the proposed metabolic pathways of target compounds, the incubation with human liver microsomes was applied. Additionally, a sensitive, specific, and rapid LC-MS/MS method was developed and validated to quantify selected drugs and their metabolites in biological samples. The preparation of biological samples was accomplished through microextraction by a packed sorbent (MEPS). Finally, the results from LC-MS/MS analysis of biological samples, liver microsomes and electrochemical TPs were compared to evaluate the quality of electrochemical metabolism mimicry. Results and discussion Data from in vivo experiments agreed with the data from electrochemical oxidation, which predicted some of the potential metabolites found in the human liver microsomes. EC-(LC)-MS is well-suited for the simulation of the oxidative metabolism of selected psychotropic drugs and acts as the orthogonal source of information about drug metabolites compared to liver microsomes and biological matrices. EC-(LC)-MS enables the direct identification of reactive TPs, circumvents time-consuming sample preparation and is ethically advantageous because it reduces the need for animal experiments.
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