Streamlined Unknown Screening for Postmortem Analysis

Importance of the Topic

The rapid proliferation of novel psychoactive substances (NPS), designer drugs and prescription medication misuse poses a major challenge for forensic toxicology. Comprehensive and sensitive postmortem drug screening is essential for determining cause of death, informing legal investigations and understanding ante-mortem events. High-resolution mass spectrometry (HRMS) with data-independent acquisition provides a robust digital record that can be re-interrogated as new targets emerge.

Objectives and Overview of the Study

This work describes the development of a streamlined workflow for unknown compound screening in postmortem blood using the SCIEX X500R QTOF system with SWATH® acquisition. Key goals included:

• Establishing a targeted panel of 151 drugs and NPS with sub-ng/mL detection limits
• Simplifying sample preparation to accelerate throughput
• Demonstrating comprehensive coverage and retrospective data mining

Methodology and Used Instrumentation

Sample preparation consisted of protein precipitation with a methanol/acetonitrile mixture, followed by evaporation and reconstitution in aqueous methanol. Chromatographic separation exploited an ExionLC AC system and a Phenomenex Kinetex Phenyl-Hexyl column under an 8.5-minute gradient. Mass spectrometry utilized the SCIEX X500R QTOF with positive-mode electrospray and SWATH® acquisition, capturing full TOF-MS scans and overlapping MS/MS windows. Data processing employed SCIEX OS Software with four confidence criteria (mass error, retention time, isotope ratio, library match) combined into a composite score.

Key Results and Discussion

The optimized method achieved limits of detection in the sub-ng/mL range for all 151 panel compounds. Representative analytes such as buspirone, norfentanyl and scopolamine were identified with clear extracted ion chromatograms, accurate mass spectra and high-quality fragment ions at low concentration levels. Postmortem blood samples processed under this workflow yielded confident identification and quantification of common drugs (e.g., acetaminophen, codeine, diazepam) and low-level opioids like fentanyl. Retrospective interrogation of the SWATH® data enabled detection of unexpected targets without additional analysis steps.

Advantages and Practical Applications

By integrating SWATH® acquisition with HRMS, the workflow offers:

• Rapid, high-throughput screening with minimal sample handling
• Comprehensive coverage of known and emerging compounds
• Automated data processing with intuitive “traffic-light” scoring for case reporting
• Ability to re-process archived data for new analytes, reducing sample reinjection

This approach enhances forensic laboratory efficiency, sensitivity and confidence in determining toxicological causes of death.

Future Trends and Possibilities

Continued expansion of spectral libraries and targeted panels will further improve detection of rare NPS. Integration of machine learning for more sophisticated spectral deconvolution and anomaly detection may streamline unknown screening. Advances in ultrafast chromatography and ambient ionization could reduce run times and sample preparation steps, enabling near-real-time forensic analysis.

Conclusion

The described workflow using the SCIEX X500R QTOF system with SWATH® acquisition and streamlined sample preparation delivers a powerful platform for postmortem drug screening. It achieves high sensitivity, broad compound coverage and flexible retrospective analysis, addressing the evolving demands of forensic toxicology.

Reference

1. Elmiger M. P., Poetzsch M., Steuer A. E., Kraemer T. Anal Bioanal Chem 409, 6495–6508 (2017)
2. Roemmelt A. T., Steuer A. E., Poetzsch M., Kraemer T. Anal Chem 86, 11742–11749 (2014)