Rapid postmortem analysis of novel psychoactive substances using a high-resolution Orbitrap mass spectrometry method

Significance of the topic

Illicit drug toxicity and deaths associated with novel psychoactive substances (NPS) continue to pose an urgent public‑health and forensic challenge. Rapid, sensitive and high‑throughput toxicological screening of postmortem blood is essential to inform coroners, public‑health authorities and harm‑reduction efforts about emerging compounds and population exposure. High‑resolution accurate‑mass LC‑MS workflows that also support retrospective interrogation of acquired data increase the likelihood of detecting newly introduced analogs that are not part of routine targeted panels.

Goals and overview of the study

The British Columbia Provincial Toxicology Centre (PTC), in collaboration with instrument vendors, developed and validated an LC‑HRAM‑MS workflow for routine quantitative postmortem blood screening and for retrospective detection of NPS. The immediate aims were to (1) shorten turnaround time for routine case reporting, (2) provide robust quantitation for forensic interpretation, and (3) enable re‑interrogation of archived high‑resolution datasets to detect NPS introduced after initial analysis. A demonstration application was the retrospective identification and quantification of the designer benzodiazepine bromazolam in postmortem blood samples.

Methodology

Key aspects of sample preparation and analysis:

• Sample set: Postmortem blood from suspected illicit‑drug fatalities (cases between mid‑2020 and 2022; retrospective search for >83 compounds including bromazolam).
• Extraction: Salt‑assisted liquid–liquid extraction with ice‑cold methyl tert‑butyl ether/acetonitrile (1:9, v:v); evaporation and reconstitution in methanol:water (1:1) prior to injection.
• Chromatography: Fast UHPLC separation (total run time 12.5 min) on a Phenyl‑Hexyl stationary phase (2.1 × 100 mm) with a gradient mobile phase containing ammonium formate/formic acid and acetonitrile:methanol organic modifier; column and autosampler temperatures controlled.
• Mass spectrometry: Positive ESI full‑scan with targeted data‑dependent MS2 on a high‑field Orbitrap platform for accurate‑mass precursor and fragment detection.
• Quantitation strategy: Standard‑addition calibration using the individual case matrix to compensate for postmortem matrix effects; validation assessed linearity, LOD/LOQ, recovery, matrix effects, carryover and interferences.

Instrumentation used

The workflow employed a high‑resolution quadrupole‑Orbitrap mass spectrometer coupled to a UHPLC system capable of robust, high‑throughput separations. The platform provided fast scan speeds and high resolving power required for both reliable quantitation and archival data quality suitable for later retrospective screening.

Main results and discussion

Retrospective interrogation identified bromazolam in 41 postmortem cases. Of these, 27 cases had concentrations above the validated lower limit of quantitation (0.5 ng/mL); seven additional cases contained bromazolam between the LOD (0.1 ng/mL) and the LOQ. Observed concentrations spanned 0.5–319.3 ng/mL with a median ≈1.6 ng/mL and mean ≈11.4 ng/mL (large SD reflecting a few high‑level cases). Co‑detections were common: fentanyl was present in ~88% of bromazolam‑positive cases, and stimulants (methamphetamine/amphetamine) were also frequent. Across a three‑year surveillance window PTC reported 2,865 NPS detections, with designer benzodiazepines and fentanyl analogs among the most prevalent categories. Temporal trends showed bromazolam emerging in early 2021 and becoming the dominant benzodiazepine detected in postmortem blood by late 2022.

Method performance metrics included linear calibration fits (r2 > 0.99 by back‑calculation), acceptable recovery and matrix effect assessments using case‑matched standard addition, no significant carryover in blank injections following calibrators, and fragment/retention time criteria with strict mass accuracy filters (<10 ppm) for presumptive identifications. The fast 12.5‑minute run allowed same‑day reporting (results to coroners within ~24 hours) while producing full‑scan HRAM datasets suitable for later re‑interrogation to discover NPS not included in the initial target list.

Benefits and practical applications

Practical advantages highlighted by the work:

• High throughput: Short LC cycle time and fast Orbitrap scans increased daily sample capacity and accelerated case reporting to coroners and public‑health stakeholders.
• Sensitivity and specificity: HRAM data coupled with data‑dependent MS2 improved confidence in presumptive identifications of low‑abundance NPS in complex postmortem matrices.
• Retrospective surveillance: Archival full‑scan data enabled detection of emerging NPS after their introduction to the population, supporting public‑health alerts and forensic investigations.
• Forensic quantitation: Use of standard addition mitigated matrix effects typical of postmortem blood, producing defensible concentration estimates for interpretive purposes.

Future trends and opportunities

Key directions to strengthen surveillance and forensic toxicology capability:

• Routine inclusion of high‑resolution full‑scan acquisition to create searchable population‑scale archives that can be re‑queried as new NPS are characterized.
• Automated retrospective screening pipelines and curated NPS databases to accelerate discovery and minimize analyst time per re‑query.
• Integration of quantitative approaches (e.g., case‑specific standard addition) into high‑throughput workflows to retain robust concentration data for forensic interpretation.
• Continued method updates to monitor rapidly evolving fentanyl analogs, nitazenes and designer benzodiazepines; regular review of seizure data to align screening panels with the illicit supply.

Conclusion

The validated LC‑HRAM‑MS workflow demonstrates that rapid, high‑sensitivity postmortem screening combined with retrospective data analysis is a powerful approach for detecting and quantifying emerging NPS such as bromazolam. The ability to report timely results to coroners and to re‑interrogate archival datasets enhances public‑health surveillance and enables more rapid recognition of shifts in the illicit drug supply. Continued adoption of full‑scan HRAM acquisition and retrospective data mining will be essential to track the evolving landscape of designer drugs and support harm‑reduction and forensic objectives.

References

1. United Nations Office on Drugs and Crime. Early Warning Advisory on New Psychoactive Substances. 2023.
2. Mérette, S.A.M.; Thériault, S.; Piramide, L.E.C.; Davis, M.D.; Shapiro, A.M. Bromazolam Blood Concentrations in Postmortem Cases — A British Columbia Perspective. Journal of Analytical Toxicology. 2023;47(4):385–392. doi:10.1093/jat/bkad005.
3. Skinnider, M.A.; Mérette, S.A.M.; Pasin, D.; Rogalski, J.; Foster, L.J.; Scheuermeyer, F.; Shapiro, A.M. Identification of Emerging Novel Psychoactive Substances by Retrospective Analysis of Population‑Scale Mass Spectrometry Data Sets. Analytical Chemistry. 2023;95(47):17300–17310.
4. University media report: New process for screening old urine samples reveals previously undetected designer drugs. 2023.