Drug Screen Suite: Streamlined LC-HRMS Toxicological Screening for Routine and Post-Mortem Casework

Significance of the topic

The rapid and reliable identification of drugs, therapeutic agents and new psychoactive substances in biological matrices is critical for clinical research, forensic casework and post-mortem investigations. High-resolution LC-MS screening workflows that combine accurate mass, retention time and HRMS/MS spectral matching address the need for high sensitivity and specificity in complex matrices while enabling laboratories to respond rapidly to emerging substances and high sample throughput demands.

Objectives and overview of the study

This report presents the Drug Screen Suite, a fully integrated LC-HRMS workflow developed to streamline routine and post-mortem toxicological screening from sample preparation through automated data processing and reporting. The evaluation aimed to assess sensitivity, limits of identification (LOI), robustness across matrices (urine, blood, serum, vitreous humor), performance on proficiency-test material, and applicability to real casework including post-mortem samples.

Methodology

The workflow couples UHPLC separation with data-dependent high-resolution tandem MS (AutoMS/MS) and an extensible spectral/fragmentation library and automated reporting. Key experimental elements include:

• Sample preparation: matrix-adapted approaches—liquid–liquid extraction for serum and blood (alkaline chlorobutane LLE for some serum workflows), cold acetonitrile (ACN) protein precipitation for urine and serum, and solid-phase extraction (SPE) for vitreous humor.
• Screening design: automated AutoMS/MS acquisition to collect MS/MS spectra for library matching; open library approach enables timely updates to include new substances/NPS.
• Analytical evaluation: determination of limits of identification using 120 commonly encountered analytes spiked into blank urine at 50, 10, 5 and 1 ng/mL; analysis of proficiency testing samples (2024 GTFCh panels) and reanalysis of complex post-mortem extracts to assess performance under high-matrix conditions.

Used Instrumentation

The evaluation used Bruker instrumentation and components as implemented in the Drug Screen Suite:

• LC: Bruker Elute+ UHPLC with a 20 min gradient (mobile phases reported as water with 0.2% buffer mix and methanol with 0.2% buffer mix).
• Column: Intensity Solo 1.8 C18-2, 100 × 2.1 mm.
• MS: Bruker OTOF high-resolution time-of-flight instrument.
• Ion source: VIP HESI (heated electrospray) source, primarily positive ion mode.
• Acquisition: AutoMS/MS at ~12 Hz, m/z range 30–1,000; library-search-based identification with mass tolerance set to ±5 mDa.
• Software: automated data analysis and reporting integrated into the Drug Screen Suite.

Main results and discussion

Performance highlights from spiked samples, proficiency tests and casework:

• Limits of identification: Approximately 80% of tested substances were identifiable at 10 ng/mL or lower in urine. LOI experiments used 120 common analytes spiked into 100 µL urine at multiple concentrations (50, 10, 5, 1 ng/mL).
• Proficiency testing: The suite successfully identified compounds across a range of GTFCh-style proficiency tests (urine screening, abstinence monitoring, general unknowns, benzodiazepine panels, neuroleptics, narcotics), demonstrating applicability to routine laboratory assessments.
• Casework and post-mortem matrices: In high-matrix post-mortem femoral blood, vitreous humor and urine extracts, the Drug Screen Suite identified essentially all substances detected by routine analyses (immunoassay and Toxtyper®) and targeted LC-MS/MS, with a few exceptions attributable to ionization or library limitations (e.g., ethyl glucuronide and ethyl sulfate required negative-mode acquisition).
• Sensitivity and spectral matching: HRMS and MS/MS library matching provided robust identifications; example reported purity/match scores (fentanyl shown with a high library match score). However, at very low concentrations, background low-m/z interferences can impede reliable library matching.
• Matrix/preparation limitations: Chlorobutane LLE simplifies serum extraction but is incompatible with some analytes (e.g., THC-COOH), which were better recovered with ACN precipitation. Compounds absent from the library or those requiring negative electrospray mode were not identified in the positive-mode-only workflow.

Benefits and practical applications

The Drug Screen Suite offers several operational advantages for forensic and clinical laboratories:

• Turnaround: Automated processing and reporting reduce evaluation bottlenecks in high-throughput environments.
• Sensitivity and specificity: HRMS lowers false positives and supports confident identifications through exact mass, isotope pattern and MS/MS matching.
• Flexibility: Open, updateable libraries allow timely inclusion of new psychoactive substances and emergent analytes.
• Comprehensive capability: Supports screening across common matrices (urine, blood, serum, vitreous humor) and workflows from broad screening to follow-up targeted quantitation (via LC-MS/MS).

Limitations and considerations

• Polarity coverage: The evaluation focused on positive-ion HRMS; analytes requiring negative-ion detection (e.g., EtG, EtS) were not identified unless alternative acquisition modes are implemented.
• Sample preparation trade-offs: Simplified extraction methods increase throughput but can reduce recovery for certain analytes or classes; matrix-specific methods may be required for optimal detection.
• Library dependence: Identification is limited by the contents and quality of the spectral library; novel substances not yet included will be missed until added.
• Low-level interference: At sub-ng/mL levels, background ions and coeluting interferences can reduce MS/MS match quality and identification confidence.

Future trends and applications

Potential directions to enhance and extend such HRMS screening suites include:

• Dual-polarity and switching acquisition to capture negative-mode analytes concurrently with positive-mode targets.
• Integration of ion mobility separation to improve isomer/isobar discrimination and reduce spectral interference.
• Continual expansion and curation of spectral libraries with community-sourced and vendor-curated spectra to cover NPS and metabolites.
• Incorporation of machine-learning approaches for deconvolution of mixed spectra, automated LOI estimation and prioritization of tentative identifications.
• Workflow automation: improved sample-prep robotics, LIMS integration and standardized reporting templates to accelerate throughput and ensure chain-of-custody in forensic labs.
• Validation pathways to extend the suite from research use toward accredited diagnostic and forensic casework, including formal stability and matrix-effect studies.

Conclusion

The Drug Screen Suite demonstrates a practical, sensitive and adaptable LC-HRMS screening solution for routine toxicology and post-mortem casework. By combining optimized sample workflows, high-resolution AutoMS/MS acquisition and automated library-based identification, the suite reduces evaluation bottlenecks and improves detection coverage for a broad array of drugs and metabolites. Remaining gaps (negative-mode analytes, library completeness and certain matrix/prep incompatibilities) can be addressed through targeted method extensions and ongoing library curation to maintain relevance in a rapidly evolving forensic landscape.

References

The source document is an abstract/presentation from ASMS 2026 (THP 206) authored by Birgit Schneider, Juergen Kempf, Laura M. Huppertz, Carsten Baessmann, Eva Niehaus and Sam Putnam with instrumentation and data provided by Bruker Daltonics. No additional literature references were provided in the supplied material.