A Triple Quadrupole GC/MS MRM Database for Forensic and Toxicological Workflows

Significance of the Topic

Systematic toxicological analysis in forensic investigations requires highly sensitive and selective methods to detect an expanding array of toxicants at trace levels. The development of a curated multiple reaction monitoring (MRM) database for gas chromatography–triple quadrupole mass spectrometry (GC/TQ) addresses critical challenges, including low analyte concentrations, growing analyte lists, and limited availability of reference standards. By streamlining method creation and improving detection confidence, this resource enhances routine forensic and toxicological workflows.

Goals and Overview of the Study

This work aimed to build a comprehensive MRM transition database encompassing 175 entries (154 unique compounds and their derivatives) relevant to forensic and toxicological applications. The database supports rapid screening and quantitation method development on Agilent GC/TQ platforms and was validated using real post-mortem blood samples. It is scheduled for public release in July 2024.

Methodology and Instrumentation Used

The database was compiled using Agilent MassHunter Optimizer for GC/TQ with a Start from Scan workflow and MassHunter Unknowns Analysis for compound identification against a curated spectral library. Key instrument parameters included:

• Agilent 7000 Series GC/TQ mass spectrometer with dMRM mode
• Agilent J&W DB-5ms column (30 m × 0.25 mm × 0.25 µm)
• Multimode inlet, splitless injection of 2 µL with 25 psi pulse
• Oven program: 80 °C hold 1 min, ramp 20 °C/min to 290 °C, 8 min hold
• Helium carrier gas at 1 mL/min, transfer line 300 °C, source 230 °C
• Electron energy 70 eV, quadrupoles at 150 °C

Database curation steps:

• Full-scan GC acquisition optimization
• Precursor and product ion selection
• Collision energy optimization across 1,794 transitions
• Export of MRM list as CSV for import into MassHunter Optimizer

Main Results and Discussion

The final database contains:

• 175 entries: 123 underivatized, 32 trimethylsilylated, 20 acetylated
• 154 unique compounds covering benzodiazepines, antidepressants, opioids, and drugs of abuse
• 1,794 optimized MRM transitions (3–12 per compound)
• Retention times locked to cocaine at 12.26 min for system alignment

Proof-of-concept analysis of 25 archived post-mortem blood samples compared full-scan versus MRM acquisition. The MRM approach detected all target compounds, including fentanyl quantified at 1.7 ng/mL, whereas the full-scan method missed low-level toxicants.

Benefits and Practical Applications of the Method

• Rapid creation of sensitive and selective screening and quantitation methods
• High confidence in compound identification via multiple optimized transitions
• Time savings by avoiding manual method development for each analyte
• Applicability to routine forensic, clinical toxicology, QA/QC, and industrial analyses

Future Trends and Opportunities

• Expansion of the database to include emerging synthetic opioids and novel psychoactive substances
• Integration with high-resolution MS libraries for hybrid workflows
• Automation of method updates and real-time retention time correction
• Application to environmental and clinical biomonitoring studies

Conclusion

The curated GC/TQ MRM database provides a valuable tool for forensic and toxicology laboratories, enabling enhanced sensitivity, selectivity, and efficiency in detecting a broad range of analytes. Its application to authentic samples demonstrated superior performance over conventional full-scan methods, underlining its potential to become a standard resource in forensic workflows.

Reference

• Lokits K, Ciotti R, Diaz H. QuickProbe Dual Configurations for Forensic Workflows: Providing flexibility and robustness on a single GC/MS system. Agilent Technologies Application Note 5994-6889EN, 2023.
• Lokits K, Willey A. Evaluation of Hydrogen Carrier Gas and the Agilent HydroInert Source for Forensic Street Drug Analysis. Agilent Technologies Application Note 5994-6982EN, 2023.
• Maurer HH, Pfleger K, Weber AA. Mass Spectral Library of Drugs, Poisons, Pesticides, Pollutants, and Their Metabolites, 3rd Edition, 2007.