Toxicological Drug Screening Using the LC Screener Tool with High-Resolution LC/Q-TOF

Significance of the Topic

Screening biological fluids for a wide array of toxic substances is fundamental in forensic toxicology, clinical diagnostics, and workplace safety. High-resolution LC/Q-TOF combined with data-independent acquisition (DIA) enables comprehensive detection, retrospective data mining, and confident identification of both known and emerging compounds through accurate-mass measurements and fragmentation patterns.

Objectives and Study Overview

This application note presents a complete workflow for toxicological drug screening in plasma and urine using the Agilent Revident LC/Q-TOF MS and the embedded LC Screener tool. The aim is to demonstrate sample preparation, untargeted suspect screening of 32 drugs and 16 isotopically labeled standards over a broad concentration range, and rapid data review using MassHunter and ChemVista software.

Methodology and Instrumentation

• LC System: Agilent 1290 Infinity II (high-speed pump, cooled multisampler, multicolumn thermostat)
• Mass Spectrometer: Agilent Revident LC/Q-TOF with Dual AJS ESI source
• Software: MassHunter Acquisition 12.0; MassHunter Quantitative Analysis 12.1 with LC Screener tool; ChemVista Library Manager 1.0

Sample Preparation:
Plasma samples underwent EMR–Lipid cartridge SPE cleanup followed by reconstitution in 60:40 methanol:water. Urine samples were diluted 10:1 with the same solvent. Both matrices were spiked with 32 scheduled drugs at eight levels (1–100 ng/mL) plus 16 heavy-labeled internal standards at 50 ng/mL.

Chromatographic Conditions:

• Column: Poroshell 120 EC-C18, 2.1 × 100 mm, 2.7 µm
• Mobile Phase A: H₂O + 0.1% formic acid + 5 mM ammonium formate + 0.05 mM ammonium fluoride
• Mobile Phase B: MeOH + 0.1% formic acid + 5 mM ammonium formate + 0.05 mM ammonium fluoride
• Flow Rate: 0.5 mL/min; Column Temperature: 55 °C; Injection Volume: 4 µL
• Gradient: 5% B at 0 min, ramp to 98% B by 8.5 min, hold to 9.5 min, re-equilibrate to 5% B at 10 min
• Acquisition: DIA All Ions at 0, 20, and 40 V collision energies; reference ions for mass accuracy locking

Main Results and Discussion

Across plasma and urine, all 32 analytes exhibited excellent linearity (R² ≥ 0.99), average mass accuracies within ±1 ppm, and chromatographic precision with %RSDs below 20% at concentrations from 1 to 100 ng/mL. The LC Screener tool automatically classified compounds as identified, questionable, or not detected based on retention time alignment, mass match score, mass accuracy (<5 ppm), signal-to-noise (>3), and verified ion count. Spectral matching of coeluting fragment ions enhanced identification confidence, and the ChemVista library provided curated high-resolution MS/MS spectra and structure-based tags for rapid searching.

Benefits and Practical Applications

• Unbiased, retrospective screening for emerging or unexpected substances
• High-confidence identifications through accurate mass and fragmentation data
• Streamlined data review with the LC Screener user interface
• Scalable workflows for forensic casework, clinical toxicology, and QA/QC laboratories
• Robust library management and expansion via ChemVista

Future Trends and Opportunities

Continued expansion of high-resolution spectral libraries, incorporation of novel psychoactive substances, and integration of machine learning for automated spectral interpretation will further enhance untargeted screening. Advances in ion mobility, hybrid acquisition modes, and cloud-based data sharing promise even greater coverage and collaboration among forensic laboratories.

Conclusion

The described workflow combines Agilent’s Revident LC/Q-TOF, MassHunter software suite, and ChemVista library manager to deliver rapid, reliable untargeted drug screening in complex biological matrices. High mass accuracy, reproducible chromatography, and the LC Screener tool facilitate confident identifications, making this approach highly suitable for forensic and toxicological applications.

References

1. Zhao L. Quantitative Determination of Drugs of Abuse in Human Whole Blood by LC/MS/MS Using Agilent Captiva EMR—Lipid Cleanup. Agilent Technologies Application Note. 2018;5991-9251EN.
2. Yannell KE, Gomez M. Drug Screening in Whole Blood Using the Agilent 6546 LC/Q-TOF and the LC Screener Tool with Automated Sample Preparation. Agilent Technologies Application Note. 2020;5994-1744EN.
3. Simmermaker C, Durham SD, Stone P, Bertram L. Toxicological Drug Screening Using the LC Screener Tool with High-Resolution LC/Q-TOF. Agilent Technologies Application Note. 2024;5994-7850EN.