Forensic Toxicology Data-Independent Analysis Screening Using Xevo MRT Mass Spectrometer Delivering Routine Parts-per- Billion (ppb) Mass Accuracy

Significance of the topic

Forensic toxicology laboratories require rapid, specific and cost-effective screening of complex biological matrices to support public health and legal decisions. High-resolution, data-independent acquisition (DIA) workflows with parts-per-billion (ppb) mass accuracy can substantially reduce ambiguous identifications, lower confirmatory testing burden, and accelerate case turnaround. The work summarized here demonstrates how the Xevo MRT multi-reflecting QTof mass spectrometer improves routine mass accuracy and specificity within a DIA (MSE) forensic screening workflow, enabling more defensible and efficient toxicology screening.

Objectives and study overview

• Demonstrate the capability of the Xevo MRT Mass Spectrometer to deliver routine ppb-level mass accuracy in DIA (MSE) acquisitions for forensic toxicology screening.
• Showcase how high mass accuracy combined with guided informatics (waters_connect and UNIFI) improves selectivity, reduces false positives and streamlines laboratory workflows.
• Provide operational metrics (mass error distribution, detection consistency) using real-world-like samples to illustrate practical benefits.

Methodology

• Sample preparation: a system suitability test (SST) mix at 25 ng/mL in 5 mM ammonium formate (pH 3) was used. Ten anonymized urine samples were prepared by 1:10 dilution with the mobile phase.
• Chromatography: ACQUITY UPLC I-Class PLUS system with a 15-minute gradient was used for separations.
• Mass spectrometry acquisition: DIA implemented as MSE mode (alternating low- and elevated-collision-energy scans) with ESI positive ionization to collect comprehensive precursor and fragment information without precursor isolation.
• Data processing: waters_connect Software and the UNIFI Application performed guided workflows including automated peak detection, deconvolution, library matching (2,000+ compounds), retention time and mass-accuracy checks, and confidence scoring.
• Identification criteria emphasized retention time agreement, precursor and product ion presence, and stringent mass-accuracy tolerances (reported precursor mass ≤2 ppm; fragments down to ≤0.2 mDa in examples).

Used instrumentation

• Waters ACQUITY UPLC I-Class PLUS System (15-min gradient)
• Xevo MRT Mass Spectrometer (multi-reflecting QTof architecture)
• Electrospray ionization in positive mode (ESI+)
• Data acquisition: MSE (DIA) mode
• Informatics: waters_connect Software and UNIFI Application for guided processing and library searching

Main results and discussion

• Mass accuracy performance: across 160 precursor detections spanning m/z 122–478 the root mean square (RMS) mass error was 571 ppb, with 94% of precursors detected within ±1 ppm. For product ions (92 fragments from 23 substances, m/z 80–268), 96% of fragment detections fell within ±1 ppm.
• These routinely observed ppb-level mass errors provided robust precursor and fragment matching across the mass range, supporting high-confidence compound identification in complex urine matrices.
• Impact on false positives: tightening fragment-ion mass-accuracy tolerances from broader thresholds (≤2 mDa) to more stringent values (≤0.2 mDa) resolved an ambiguous match between anabasine and nicotine in an example case—eliminating a potential false positive by removing an outlier fragment and revealing the correct assignment.
• Operational benefits: ppb mass accuracy combined with MSE and guided informatics reduced manual review workload, shrank reliance on secondary confirmatory testing, shortened reporting times, and decreased outsourcing and consumable costs. Environmental and sustainability gains were noted due to fewer re-analyses and reduced sample transport.
• Limitations: as an application brief, detailed experimental parameters and broader validation statistics (e.g., limits of detection/quantification across many analytes, inter-day reproducibility across labs) are not provided; laboratories should perform local validation for forensic casework acceptance.

Contributions and practical applications

• Demonstrates how routine ppb mass accuracy in a DIA workflow raises selectivity for forensic toxicology screening, reducing ambiguous hits and lowering confirmatory testing need.
• Shows that guided informatics can standardize processing, enabling less experienced analysts to produce reproducible results and improving throughput.
• Supports adoption of a single-injection broad-screening approach (DIA/MSE) for high-throughput toxicology laboratories seeking to consolidate assays and shorten turnaround.

Future trends and potential applications

• Expansion of compound libraries (including new psychoactive substances and metabolites) and retention time prediction will further increase screening coverage and confidence.
• Deeper integration of machine learning and AI to prioritize candidate identifications, flag atypical spectra, and reduce operator intervention.
• Automation of end-to-end workflows, from sample prep to reporting, will enable higher throughput and reproducibility in forensic labs.
• Inter-laboratory validation and standardization efforts will be necessary to establish regulatory acceptance for ppb-level HRMS screening as frontline forensic evidence.
• Potential coupling with targeted confirmatory workflows (e.g., orthogonal LC methods or tandem MS/MS quantitation) will provide a hybrid strategy balancing speed and legal defensibility.

Conclusion

The Xevo MRT Mass Spectrometer, when used in DIA (MSE) mode and combined with guided informatics (waters_connect and UNIFI), achieves routine ppb-level mass accuracy that materially improves forensic toxicology screening specificity and operational efficiency. Demonstrated improvements in precursor and fragment mass accuracy reduce false positives, lower confirmatory testing needs, and shorten case turnaround, supporting more sustainable and cost-effective laboratory operations. Local validation and broader performance characterization remain important for implementation in regulated forensic contexts.

Reference

1. N. S. Mistry, L. J. Calton, J. Cooper. The Utility of MSE for Toxicology Screening with waters_connect and the Xevo G3 QTof Mass Spectrometer. Waters Application Note. 720008045. Sept 2023.
2. N. S. Mistry, L. J. Calton, J. Cooper. The Ultimate Forensic Toxicology Screening Companion— Xevo G3 QTof. Waters Application Note. 720008033. Sept 2023.