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

Importance of the Topic

The continuous emergence of novel psychoactive substances and the complexity of biological matrices demand robust screening methodologies in forensic toxicology. High-resolution mass spectrometry combined with data independent acquisition ensures comprehensive detection of both known and unknown analytes in urine at trace levels.

Objectives and Overview of the Study

This study evaluates the performance of a hybrid quadrupole multi-reflecting time-of-flight (MRT) mass spectrometer with MSE data independent acquisition for routine urinary drug screening. It aims to demonstrate enhanced specificity, part-per-billion mass accuracy, and improved informatics output to reliably identify drugs of abuse, prescription medications, metabolites, dietary and endogenous compounds.

Methodology and Used Instrumentation

Authentic human urine samples were diluted and analyzed alongside a 10-component QC test mix. Separation was achieved with reversed-phase UPLC using an ACQUITY UPLC HSS C18 column and a formate formic acid gradient at 50 °C.

• Instrument: SELECT SERIES MRT hybrid quadrupole multi-reflecting time-of-flight mass spectrometer
• Acquisition mode: MSE with alternating low and high collision energy (15–40 eV)
• Resolution: >200 000 FWHM (up to 130 000 for fragment ions)
• Mass range: m/z 50–2400; acquisition rate: 10 Hz
• Software: MassLynx 4.2, Waters Connect, TIBCO Spotfire 6.0

Main Results and Discussion

The system achieved routine part-per-billion mass accuracy (RMS error ~500 ppb) for both precursor and fragment ions under stringent processing tolerances (±2 ppm precursor, ±0.2 mDa fragment). QC mix analytes and all library entries were correctly identified. In sample 103, true positives such as methamphetamine (87 ppb error) and methadone (–83 ppb error) were confirmed, while false positives like oxycodone were excluded. Sample 51 demonstrated polydrug use with 14 drug compounds, 7 metabolites, and common urinary constituents identified.

Benefits and Practical Applications of the Method

The high resolving power improves ion selectivity and reduces matrix interferences, enhancing detection confidence in complex samples. Unbiased DIA datasets enable retrospective library searches and non-targeted workflows, supporting both routine forensic screening and research applications.

Future Trends and Applications

Integration of expanding high-resolution spectral libraries and advanced informatics will further streamline non-targeted screening of emerging psychoactive substances. Continuous software improvements and coupling with orthogonal techniques may enhance structural elucidation and quantitative capabilities.

Conclusion

The combination of high-resolution MRT mass spectrometry and MSE DIA offers reliable, high-confidence identification of a broad range of analytes in urine. Stringent mass accuracy tolerances and robust software tools enable efficient forensic toxicology workflows with minimal false detections.

Reference

• Rosano TG, 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.
• Twohig M, Aubin AJ, Hudalla CJ. Waters Corporation Application Note 720007720. 2022.
• Bonn B, Leandersson C, Fontaine F, Zamora I. Enhanced Metabolite Identification with MSE and Semi-Automated Software for Structural Elucidation. Rapid Commun Mass Spectrom. 2010;24(21):3127–3138.