T-ReXing Synthetic Cannabinoids─Unveiling Metabolite Features Suitable for Urine Screening by Trapped Ion Mobility...

Area of Science:

• Forensic Toxicology
• Metabolomics
• Mass Spectrometry

Background:

• Urine analysis for synthetic cannabinoids (SCs) is crucial for toxicological screening.
• Detecting novel SCs requires rapid incorporation of their metabolite biomarkers.
• Scarcity of reference standards and difficulty in detecting parent SCs challenge forensic labs.

Purpose of the Study:

• To develop and validate a method for identifying phase I metabolites of prevalent SCs.
• To improve the detection and annotation of SC metabolites in urine using untargeted high-resolution mass spectrometry (HRMS).
• To integrate identified metabolites into targeted methods for casework analysis.

Main Methods:

• In vitro phase I metabolism using pooled human liver microsomes (pHLM).
• Analysis by trapped ion mobility spectrometry-time-of-flight mass spectrometry (timsTOF-MS) with parallel accumulation serial fragmentation (PASEF).
• Utilized MetaboScape software with T-ReX 4D workflow for in silico prediction, fragmentation, and collision cross section (CCS) forecasting.

Main Results:

• Successfully annotated predicted metabolites for ADB-BUTINACA, MDMB-BUTINACA, and MDMB-4en-PINACA, including monohydroxylations and ester hydrolysis.
• Qualitative findings from pHLM assays and urine samples showed good agreement.
• Identified key metabolites were incorporated into a targeted UHPLC-QTOF-MS method for analyzing casework samples.

Conclusions:

• The employed HRMS approach, combined with in silico tools, significantly accelerates the identification of SC metabolites.
• This method is less time-consuming and labor-intensive than manual evaluation.
• Despite challenges with complex biotransformations, the approach enhances forensic toxicology capabilities for emerging NPS.