Metabolism study of the novel cathinone derivative 3-MMC combining three in vitro approaches, LC-HRMS acquisition and molecular networking tools

Importance of the Topic

New psychoactive substances (NPS) such as 3-methylmethcathinone (3-MMC) pose substantial challenges for clinical and forensic toxicology. Rapid emergence of novel compounds often outpaces availability of human in vivo metabolism data, complicating urine screening and risk assessment. Reliable in vitro models are essential to generate metabolic profiles that support detection of parent drugs and their transformation products with high confidence.

Objectives and Overview

This study aimed to characterize the metabolic fate of 3-MMC using three complementary in vitro systems.

• Human liver microsomes (HLM) – a cost-effective, enzyme-rich preparation to rapidly produce phase I metabolites.
• HepaRG cell line – a human hepatoma culture model expressing multiple drug-metabolizing enzymes over 48 h.
• HepaRG cells-on-chip – a 3D microfluidic organ-on-a-chip platform designed to better mimic liver physiology and sustain enzyme activity.

The combination of high-resolution LC-MS/MS, data-dependent acquisition (DDA-MS/MS) and molecular networking workflows provided an integrated strategy for metabolite identification and structural annotation.

Methodology and Instrumentation

Incubations were performed with 3-MMC at 5 µM and 50 µM concentrations in each model. Key analytical conditions included:

• Liquid Chromatography: Shim-pack Velox Biphenyl column (2.1×100 mm, 2.7 µm), binary gradient of water/methanol with 2 mM ammonium formate and 0.002 % formic acid, flow rate 0.3 mL/min.
• Mass Spectrometry: QTOF LCMS-9030, full-scan MS (m/z 70–1000) and DDA-MS/MS (m/z 40–1000, collision energy spread 5–55 V, cycle time <0.55 s).
• Data Processing: LabSolutions Insight Analyze for component detection with a targeted biotransformation list (±5 ppm mass tolerance); LabSolutions Insight Assign for MS/MS fragment verification; GNPS and MS-DIAL for molecular networking and discovery of unknown metabolites.

Main Results and Discussion

Across all models, the parent 3-MMC (m/z 178.12264 [M+H]+) and two primary metabolites (hydroxy-3-MMC at m/z 194.11750, demethylated 3-MMC at m/z 164.10700) were consistently detected. The HLM system generated all expected metabolites within 60 minutes, demonstrating rapid phase I turnover. HepaRG cells produced the same profile over 48 hours, confirming adequate enzyme expression. In contrast, the 3D HepaRG-on-chip model yielded parent and demethylated products but failed to generate the hydroxy metabolite, possibly due to medium dilution effects or altered oxygenation in the microfluidic environment. Molecular networking via GNPS corroborated the structural assignments and helped identify minor unknown features related to 3-MMC biotransformation.

Benefits and Practical Applications

The integrated workflow combining three in vitro platforms with HRMS and molecular networking offers:

• Comprehensive metabolite coverage to support forensic urine screening methods.
• Rapid generation of phase I profiles (microsomes) and more physiologically relevant data (cell culture).
• Enhanced confidence in metabolite identification through DDA-MS/MS and networked spectral annotation.

This approach aids laboratories in developing targeted screening assays for NPS and anticipating novel metabolic pathways.

Future Trends and Applications

Advancements in microphysiological systems and automation are expected to improve predictive power of in vitro models. Integration of phase II enzyme expression (UGTs, sulfotransferases) and incorporation of multi-organ chips could yield fuller human-relevant metabolic maps. Coupling ion mobility spectrometry and machine-learning-based spectral interpretation may accelerate discovery of low-abundance metabolites.

Conclusion

The study demonstrates that a tiered in vitro strategy—leveraging human liver microsomes, HepaRG cells, and organ-on-a-chip—paired with high-resolution LC-MS/MS and molecular networking, effectively elucidates the metabolic fate of 3-MMC. While simple microsomal incubations deliver rapid initial insights, cell-based and microfluidic models add physiological depth, guiding development of reliable toxicological screening methods for emerging NPS.

Reference

No specific literature list was provided.