Agilent ASMS 2020 Posters Book

Significance of the Topic

High-resolution mass spectrometry (HRMS) enables rapid, broad-spectrum drug screening in complex biological matrices. Recent advances in data-independent acquisition (DIA) and streamlined sample preparation can transform routine forensic testing by improving confidence in compound identification while maximizing laboratory throughput.

Objectives and Overview

This study aimed to develop a fast, robust workflow for multiresidue drug screening in whole blood using an Agilent 6546 LC/Q-TOF with DIA and a novel software screening tool. Key goals were:

• Achieve sensitive, broad-scope detection of drugs (153 analytes).
• Simplify sample preparation with automated liquid handling and solid-phase extraction.
• Demonstrate confident identifications using strict criteria (mass accuracy, retention time, fragment co-elution).
• Streamline data review with the LC Screener software integrated into MassHunter Quantitative Analysis.

Methodology and Instrumentation

Sample Preparation:

• Whole blood spiked with 153 target drugs and 10 unknowns.
• Automated protein precipitation and cleanup using Captiva EMR-Lipid 96-well plates on a Bravo liquid handler.
• No concentration step—samples diluted post-elution.

Chromatography and MS Acquisition:

• 10-minute reversed-phase LC on an Agilent 1290 Infinity II UHPLC.
• Agilent 6546 LC/Q-TOF in positive DIA mode (m/z 40–1000, 8 spectra/sec).
• Collision energies of 20 eV and 40 eV to capture fragment ions.
• Reference mass correction for <5 ppm mass accuracy.

Main Results and Discussion

Sample Cleanup and Matrix Effects:

• Captiva EMR-Lipid cleanup reduced matrix interferences; 77% of analytes exhibited <10% ion suppression.
• Overall recoveries exceeded 70% for 91% of analytes.

Chromatography and Sensitivity:

• 153 drugs separated in 10 minutes with baseline resolution of key isobaric pairs (e.g., morphine vs. codeine).
• DIA at 8 Hz produced ≥12 data points per peak, ensuring robust quantitation and fragmentation spectra.

Data Processing with LC Screener:

• Custom Personal Compound Database and Library for 153 analytes generated.
• Identification criteria: mass accuracy (±5 ppm), retention time (±10%), signal-to-noise ≥3, ≥2 co-eluting fragments.
• Software auto-flags positives (green), needs review (orange), negatives (red). Review panels display chromatograms and spectra for rapid decision making.
• Simultaneous quantitation achieved for calibration standards (e.g., diphenhydramine R² ≥ 0.995, LLOQ = 1 ng/mL).

Benefits and Practical Applications

• Rapid, high-throughput screening of hundreds of drugs without method redevelopments.
• Automated sample prep reduces manual errors and increases reproducibility.
• High-resolution DIA ensures confident identification of unexpected or novel compounds.
• Integrated software tool expedites data review and reduces training burden.

Future Trends and Possibilities

• Expansion of the Personal Compound Database to include emerging designer drugs.
• Integration of retrospective data mining for historical sample re-analysis.
• Coupling DIA workflows with advanced chemometric or machine learning for forensic intelligence.
• Adaptation to other complex matrices (urine, oral fluid, tissues) with minimal method changes.

Conclusion

This work demonstrates a streamlined, high-confidence method for drug screening in whole blood using a 6546 LC/Q-TOF with DIA and automated cleanup. Fast chromatography, low matrix effects, and advanced software screening enable forensic laboratories to detect and confirm a broad panel of drugs in a single analysis with minimal manual intervention.

References

1. VanAernum et al. Rapid Online Buffer Exchange for Native MS. ChemRxiv (2019).
2. Agilent Application Note 5991-0685: Automated Metabolite Extraction.
3. Agilent MassHunter Quantitative Analysis User’s Guide.