A Workflow for Comprehensive Analysis of Forensic Samples Using Electron and Chemical Ionization High Resolution Time-of-Flight Mass Spectrometry

Importance of the Topic

This workflow addresses the growing need for reliable, comprehensive profiling of complex forensic samples such as seized botanicals, synthetic drugs and hallucinogenic mushrooms. High-resolution GC-TOFMS with both electron ionization (EI) and chemical ionization (CI) modes enables robust identification of volatile and nonvolatile constituents, supporting forensic investigations, quality control and intelligence gathering.

Study Objectives and Overview

The primary goal was to develop a unified analytical sequence combining headspace solid-phase microextraction (HS-SPME) and solvent extraction/derivatization, coupled to a high-resolution time-of-flight mass spectrometer. The workflow was demonstrated on a wide variety of seized materials, including marijuana, hash, khat, pokeweed, magic mushrooms and synthetic cannabinoids (e.g., XLR-11).

Methodology and Instrumentation

Sample Preparation

• HS-SPME: 0.05 g crushed sample with NaCl and water or salt solution in a vial, incubated and extracted with a DVB/CAR/PDMS fiber.
• Solvent Extraction/Derivatization: 0.025 g sample in methanol, sonication, filtration, drying under N₂, derivatization with methoxyamine (MEOX) and MSTFA.

Instrumentation

• GC: Agilent 7890 with Gerstel MPS, Restek Rxi-5 MS column (30 m × 0.25 mm × 0.25 µm).
• MS: LECO Pegasus GC-HRT TOFMS with EI (70 eV, 250 °C) and CI (140 eV, 200 °C) sources, resolution up to 50 000, acquisition at 6 spectra/s, mass range EI 35–510, CI 60–510 m/z.
• Calibration: Internal PFTBA; reagent gas 5 % ammonia in methane.
• Data Processing: High Resolution Deconvolution™ (HRD) for accurate mass and spectral similarity searches against NIST/Wiley.

Main Results and Discussion

HS-SPME of volatile profiles revealed key terpenes (α-pinene, β-myrcene, d-limonene, ocimene, fenchyl alcohol, isoborneol, caryophyllene, α-selinene, α-guaiene, cadalene, cembrene) with excellent mass accuracy (<1 ppm) and deconvoluted spectra. Solvent extracts after derivatization captured amino acids (alanine, valine, leucine, etc.), organic acids, sugars, fatty acids, sterols and trace synthetic stimulants (cathinone, cathine). CI mode provided complementary adduct information for confident formula assignment. The workflow successfully differentiated isomers and identified novel analogs in complex matrices.

Benefits and Practical Applications

• Comprehensive fingerprinting of both volatiles and polar, high-boiling analytes in a single workflow.
• High confidence identification through combined spectral library matching and accurate mass measurement.
• Efficient sample throughput suitable for routine forensic and QA/QC laboratories.
• Capability to detect emerging synthetic compounds and natural toxins in seized materials.

Future Trends and Possibilities

The next frontier includes coupling GC-HRT with multidimensional separations (GC×GC), integration with ambient ionization sources for rapid field screening, expansion of high-resolution libraries for novel psychoactive substances, and application of machine learning for automated deconvolution and compound annotation.

Conclusion

The presented EI/CI high-resolution TOFMS workflow delivers a powerful, versatile platform for forensic analysis of botanicals, synthetic stimulants and hallucinogens. By leveraging complementary ionization modes, rigorous sample preparation and advanced data processing, it ensures accurate, reproducible results essential for forensic investigations.