Improving Results in Forensic Testing

Importance of the Topic

Forensic toxicology laboratories face growing challenges from emerging novel psychoactive substances and the evolving opioid crisis. Reliable detection of a wide range of compounds at ever-lower concentration levels is essential for accurate casework, public safety and legal outcomes. Advancements in sample preparation techniques and mass spectrometric analysis have become critical to address these demands, reducing turnaround times and improving data quality in forensic investigations.

Objectives and Study Overview

This collaborative research, funded by the U.S. National Institute of Justice and led by Boston University School of Medicine in partnership with Research Triangle Institute, aimed to evaluate and compare different sample preparation methods for forensic toxicology. Two main projects were defined:

• Stability study of synthetic cannabinoids in human matrices (blood, urine), examining degradation over time, temperature effects and the role of preservatives.
• Head-to-head comparison of liquid–liquid extraction (LLE), solid-phase extraction (SPE), supported liquid extraction (SLE+) and phospholipid depletion, with full method validation across key drug classes (synthetic cannabinoids, benzodiazepines, barbiturates, opioids, amphetamines, cocaine and more).

Methodology and Instrumentation

Sample preparation methods were optimized and validated according to forensic guidelines. Key instrumentation and consumables included:

• Biotage® ISOLUTE SLE+ 1 mL columns for supported liquid extraction.
• Conventional LLE and SPE cartridges covering a broad polarity range.
• Phospholipid depletion plates to enhance matrix cleanup.
• High-performance liquid chromatography coupled to triple quadrupole mass spectrometry (HPLC-MS/MS) for targeted quantification of up to 39 compounds in one method.

Human biological matrices comprised blood, urine and oral fluid. Method development addressed analyte recovery, matrix effects, limits of detection (LODs) and run-to-run reproducibility.

Main Results and Discussion

Supported liquid extraction (SLE+) demonstrated rapid processing times (up to 50 % reduction in preparation) while maintaining high recoveries across all tested drug classes. Phospholipid depletion improved chromatographic performance by reducing background interferences. Stability experiments revealed that certain synthetic cannabinoids degrade significantly at room temperature, underscoring the importance of controlled storage and use of preservatives.
Comparative data with RTI partners are under consolidation, but preliminary observations confirm that modern extraction approaches can match or outperform traditional LLE/SPE in sensitivity and throughput. Student involvement accelerated data collection and contributed to method refinement.

Benefits and Practical Applications

Adoption of streamlined sample preparation workflows offers forensic laboratories:

• Enhanced throughput: faster sample turnaround and higher daily capacity.
• Improved sensitivity and specificity: reliable detection of low-level analytes.
• Greater method robustness: consistent recoveries and reduced matrix effects.
• Resource efficiency: lower solvent usage and reduced labor time.

These advances support forensic casework, post-mortem toxicology, human performance testing and routine QA/QC analyses.

Future Trends and Potentials

Emerging opportunities include:

• Automation of sample preparation using 48- or 96-well plate formats to further increase throughput and reproducibility.
• Integration of high-resolution mass spectrometry (HRMS) for non-targeted screening of unknown substances.
• Development of miniaturized and on-site testing platforms for rapid field analysis.
• Application of machine learning for method optimization and data interpretation.

Conclusion

This study highlights the impact of advanced sample preparation technologies combined with sensitive LC-MS/MS analysis in modern forensic toxicology. By comparing SLE+, SPE, LLE and phospholipid depletion, laboratories can select workflows that best balance speed, recovery and sensitivity. Continued innovation and automation will be key to addressing the dynamic landscape of emerging drugs and ensuring accurate, efficient forensic testing.