An Application Kit for the Screening of Samples for Analytes of Forensic Toxicological Interest using TOF or Q-TOF LC/MS with a Personal Forensic Toxicology Database

Significance of the Topic

The screening of forensic toxicological samples for a broad range of analytes is critical in legal, clinical and public health contexts. Accurate mass LC–MS techniques using TOF or Q-TOF instruments enable non-targeted, high-resolution detection of hundreds to thousands of potential toxins in a single run. A comprehensive database with accurate mass and retention time information enhances confidence in compound identification, reduces false positives and accelerates laboratory workflows.

Objectives and Study Overview

This application note describes the development and implementation of the Agilent MassHunter Personal Forensic Toxicology Database Kit. Its primary goals are:

• To provide an accurate-mass database of ~6 700 forensic analytes.
• To demonstrate sample preparation and LC–MS screening of a test mix containing 25 representative compounds.
• To illustrate database searching workflows—manual single-peak search, batch screening via Molecular Feature Extractor (MFE) and Find by Formula (FBF).
• To show how users can customize and automate workflows, including retention time updates and report generation.

Methodology

Sample preparation involved diluting a 1 µg/mL standard mix to 100 ng/mL in LC–MS grade methanol, yielding a 100 pg on-column injection. Chromatography employed an Agilent ZORBAX Eclipse Plus C18 column with a gradient from 90:10 aqueous (5 mM ammonium formate/0.01% formic acid) to 5:95 aqueous:acetonitrile over 6 minutes at 0.5 mL/min. MS acquisition used Agilent 6230 TOF (or Q-TOF) with electrospray ionization in positive mode, mass range 50–1 050 m/z, scan rate 3 Hz, and continuous reference lock masses.

Instrumentation Used

• Agilent 1200 Series HPLC: binary pump, degasser, autosampler, column compartment.
• Agilent 6230 TOF or compatible Q-TOF mass spectrometer with Jet Stream ESI source.
• Agilent MassHunter Workstation software with Personal Forensic Toxicology Database Kit (G6855AA).

Main Results and Discussion

Using the 25-component forensic test mix, all analytes were detected and identified via:

• Manual single-mass search: Phencyclidine (PCP) identified at 244.2058 m/z with 0.85 ppm error and composite score >99.
• MFE batch screening: Over 3 000 features extracted, 25 true positives matched by accurate mass (≤5 ppm), retention time and isotopic pattern. Isobaric distinctions (e.g. codeine vs. hydrocodone) were made by RT differences.
• FBF batch screening: Empirical formulas for the test mix yielded accurate mass, abundance and spacing matches, confirming all 25 compounds including detection of multiple adducts.

Benefits and Practical Applications of the Method

The combined high‐resolution LC–MS and PCDL workflow offers:

• Comprehensive non-targeted and targeted screening for a large analyte panel.
• Customizable databases with user-defined retention times for specific methods.
• Automatable data-analysis and reporting via MassHunter Workstation for high throughput.
• Enhanced confidence through multi-metric scoring: mass error, isotope pattern and retention time.

Future Trends and Potential Applications

Advances may include expanding the database with new synthetic drugs, incorporating MS/MS spectral libraries for deeper confirmation, and integrating AI-driven data mining for complex matrices. Cloud-based database sharing and real-time data analytics could further streamline forensic workflows.

Conclusion

The MassHunter Personal Forensic Toxicology Database Kit, combined with TOF/Q-TOF LC–MS and MassHunter software, enables rapid, accurate, and flexible screening of forensic samples. Its customizability, robust scoring metrics and automation capabilities support reliable identification of a wide array of toxicologically relevant compounds in varied forensic applications.

References

1. Agilent Technologies Application Note 5990-4253EN. Multi-Residue Pesticide Analysis with Dynamic Multiple Reaction Monitoring and Triple Quadrupole LC–MS/MS.
2. Agilent Technologies Technical Note 5990-3976EN. Pesticide Personal Compound Database for Screening and Identification.
3. Agilent Technologies Quick Start Guide G6855-90003. Agilent Personal Forensic Toxicology Database Kit.
4. Agilent Technologies Quick Start Guide 5990-4264EN. MassHunter Personal Forensic Toxicology Database Kit.
5. Agilent Technologies Familiarization Guide G3335-90060. MassHunter Workstation Software Qualitative Analysis.