URINARY FORENSIC TOXICOLOGY DATA INDEPENDENT ANALYSIS SCREENING: USING HIGH RESOLVING POWER MULTI-REFLECTING TIME-OF-FLIGHT MASS SPECTROMETRY

Importance of the Topic

High resolution mass spectrometry (HRMS) is a critical tool in forensic toxicology, enabling comprehensive screening of complex biological matrices for illicit and prescribed substances. The continuous emergence of new psychoactive compounds demands robust analytical workflows that can both detect known targets and discover unknowns without bias. This study demonstrates how Multi-Reflecting Time-of-Flight (MRT) mass spectrometry coupled with data-independent acquisition (DIA) addresses these challenges by offering exceptional mass accuracy and resolving power.

Study Objectives and Overview

The primary objective was to evaluate a screening workflow based on SELECT SERIES™ MRT technology for untargeted detection of drugs and metabolites in authentic urine samples. The workflow integrates high resolving power (>200 000 FWHM) and stringent mass accuracy criteria to minimize false identifications and maximize confidence in compound assignments. Anonymized human urine specimens and system suitability test (SST) mixes were analyzed to benchmark performance against a forensic toxicology library of nearly 2000 entries.

Methodology and Instrumentation

The analytical approach combined chromatographic separation on an ACQUITY UPLC I-Class Premier system with Multi-Reflecting TOF mass spectrometry operated in DIA (MSE) mode. Key parameters included:

• Sample preparation: 1:10 dilution of authentic urine and serial dilutions of SST mix (2.5–500 pg/μL).
• Chromatography: HSS C18 column, reversed-phase gradient (87–5% aqueous mobile phase A over 15 min), 0.4 mL/min flow, 50 °C column temperature.
• Mass spectrometry: ESI+ ionization, capillary voltage 0.8 kV, desolvation 500 °C, source 120 °C, cone voltage 20 V, collision energy ramp 15–40 eV, mass range 50–2400 m/z, MSE acquisition at 10 Hz.
• Data processing: 2 ppm precursor and 0.2 mDa fragment ion tolerances, retention time window ±0.35 min, library matching for 1975 analytes.

Key Results and Discussion

The workflow achieved routine mass measurement accuracy in the ppb range, with an RMS error around 500 ppb across calibration standards and authentic samples. In real urine specimens, 14 drug molecules, 7 metabolites, and common endogenous/dietary markers were confidently identified. Optimization of source temperature reduced in-source fragmentation for labile compounds such as amphetamine, enhancing precursor ion detection. False positive identifications (e.g., oxycodone in one sample) were successfully excluded through precise mass filtering. The diversity of detected drug classes underscores the need for unbiased DIA screening to capture the evolving forensic landscape.

Benefits and Practical Applications

• Enhanced selectivity and confidence in compound identification due to high mass resolution and ppb-level accuracy.
• Reduced false detection rates through stringent processing parameters and retrospective data interrogation.
• Efficient non-targeted screening capable of handling complex matrices without extensive method redevelopments.
• Applicability to clinical, forensic, and regulatory laboratories requiring comprehensive toxicological surveillance.

Future Trends and Opportunities

Advancements in informatics and library curation will further streamline data interpretation and compound annotation. Integration with artificial intelligence and machine learning could enable automated pattern recognition for novel psychoactive substances. Continued improvements in resolving power and acquisition speed will allow deeper coverage of trace-level analytes and expanded applicability to other biological matrices.

Conclusion

The application of high resolving power MRT mass spectrometry with unbiased DIA enables robust, high-confidence screening of urine for a wide range of toxicologically relevant compounds. This approach addresses the challenges posed by emerging drug threats, offering a scalable workflow for forensic and clinical laboratories seeking comprehensive and reliable toxicological analyses.

References

1. Rosano G, Wood M, Ihenetu K, Swift TA. Drug Screening in Medical Examiner Casework by High-Resolution Mass Spectrometry (UPLC–MSE-TOF). J Anal Toxicol. 2013;37(8):580–593.
2. Twohig M, Aubin AJ, Hudalla CJ. Waters Corporation Application Note, Library No. 720007720. October 2022.
3. Bonn B, Leandersson C, Fontaine F, Zamora I. Enhanced Metabolite Identification with MSE and a Semi-Automated Software for Structural Elucidation. Rapid Commun Mass Spectrom. 2010;24(21):3127–3138.