Evaluation of micro volume sample preparation technology newly designed for forensic toxicology with High Resolution Accurate Mass Spectrometry

Importance of the topic

Rapid and reliable identification of emerging synthetic drugs in biological matrices is critical for forensic toxicology. Traditional sample preparation methods often require large volumes, lengthy procedures, and may fail to detect novel analogs at low concentrations. Micro-volume pretreatment combined with high-resolution accurate mass spectrometry (HRAM) addresses these challenges by reducing sample size requirements, improving throughput, and enhancing mass accuracy for confident compound identification.

Study objectives and overview

This work evaluates a newly designed micro-volume QuEChERS extraction protocol applied to whole blood samples for forensic toxicology. The combined workflow with an LC-Q-TOF-MS system aims to demonstrate:

• Efficient extraction of low-volume (100 µL) blood samples spiked with drugs and metabolites.
• High mass accuracy (< 0.5 mDa) in MS and MS/MS for reliable formula prediction.
• Linearity, recovery, and quantitative performance across a range of analytes.

Methodology and instrumentation

Sample pretreatment follows a seven-step micro-volume QuEChERS protocol:

1. Add 100 µL whole blood to 200 µL distilled water in a pre-packed QuEChERS tube containing 80 mg MgSO4 and 20 mg CH3COONa.
2. Introduce 300 µL acetonitrile and shake to mix.
3. Centrifuge to separate layers.
4. Collect the supernatant and evaporate under N2.
5. Reconstitute residue in 40 µL methanol with 0.1% TFA.
6. Inject into LC–MS system for analysis.

The analytical platform includes:

• UHPLC: Nexera X2 with Phenomenex Kinetex XB-C18 column (2.1×100 mm, 2.6 µm).
• Mass spectrometry: Shimadzu LCMS-9030 Q-TOF in positive ESI mode, MS scan m/z 100–1000, MS/MS acquisition.
• Software: LabSolutions LCMS for data processing and MS Workbook Suite for automated fragment formula assignment.

Main results and discussion

Spike-and-recovery tests at concentrations from 1 to 100 ppb yielded calibration curves with R² ≥ 0.999 for all target compounds (etizolam, triazolam, acetyl fentanyl and metabolites). Quantitative accuracy ranged 95–108%, and mass errors for precursor and fragment ions remained below 0.5 mDa. Extracted-ion chromatograms confirmed selective detection of isomeric pairs by high resolving power. MS/MS spectra revealed characteristic fragment ions at m/z 130.065, 136.075, and 219.149 for fentanyl analogs; unique ions at m/z 150.091 for hydroxylated metabolites enabled structural differentiation. Automated formula prediction matched theoretical fragments, streamlining identification of unknowns.

Benefits and practical applications

This protocol offers significant advantages for forensic and clinical toxicology:

• Minimal sample volume reduces invasiveness and preserves precious specimens.
• Simple workflow suitable for routine laboratory implementation.
• High mass accuracy and resolving power improve confidence in identifying novel psychoactive substances.
• Automated data processing accelerates reporting and reduces manual interpretation.

Future trends and potential applications

Advances may include further miniaturization and automation of extraction, expansion to broader drug panels, and integration of retention time prediction models. Coupling HRAM with machine learning-based spectral libraries could accelerate discovery of new analogs. Portable HRAM instruments may enable field-deployable forensic screening.

Conclusion

The combination of micro-volume QuEChERS extraction and a robust HRAM platform demonstrates high efficiency, sensitivity, and accuracy for forensic toxicology applications. This workflow enables rapid detection and confident structural elucidation of emerging synthetic drugs from minimal biological samples.

References

No references were provided in the source document.