Forensic Toxicology Application Notebook - APPLICATION NOTEBOOK

Importance of the Topic

Oral fluid provides an easily collected, noninvasive matrix that closely reflects recent drug use. The direct analysis of opioids and their glucuronide metabolites in oral fluid supports forensic investigations such as impaired-driving cases and overdose events.

Objectives and Study Overview

This study developed a fast, direct UPLC-MS/MS method for simultaneous determination of 22 opioids and phase II glucuronide metabolites in oral fluid, eliminating the need for enzymatic hydrolysis and enabling robust forensic toxicology screening.

Methodology and Instrumentation

• Sample preparation: Oral fluid was mixed with phosphate buffer and deuterated internal standards, precipitated with acetonitrile, and processed through Oasis MCX µElution plates (300 µL load). Plates were washed with water and water/methanol, then eluted with acetonitrile/isopropanol containing ammonium hydroxide. Eluates were evaporated and reconstituted in aqueous formic acid.
• Chromatography: ACQUITY UPLC I-Class with BEH C18 column (2.1 × 100 mm, 1.7 µm). Gradient: 2%–52.8% acetonitrile over 6 min, total cycle 8 min.
• Detection: Xevo TQD operated in MRM mode with optimized cone voltages and collision energies for each analyte.

Main Results and Discussion

• All 22 analytes (e.g., morphine-3-glucuronide, codeine-6-glucuronide, norbuprenorphine-glucuronide) were baseline separated within a 5.5 min window.
• Recoveries averaged >74% (lowest glucuronides excluded) with mean intra-day %CVs <10% and matrix effects ranging from –49% to +32%.
• Calibration curves were linear (R² > 0.97) over 5–500 ng/mL (1.25–125 ng/mL fentanyl), with mean biases <2% and %CVs <10% for 23/26 analytes.
• Limits of quantitation of 5 ng/mL yielded signals >20× blank, demonstrating high sensitivity.

Benefits and Practical Applications

• Direct determination of glucuronide metabolites removes the variability of enzymatic hydrolysis.
• µElution SPE in a 96-well format simplifies workflow, reduces sample handling, and concentrates analytes.
• Fast UPLC-MS/MS assay with sub-6 min chromatography is ideal for high-throughput forensic and clinical settings.
• Comprehensive coverage of parent drugs and metabolites supports roadside, workplace, and postmortem toxicology applications.

Future Trends and Potential Applications

As new synthetic opioids emerge, the adaptable µElution SPE–UPLC-MS/MS workflow can be rapidly updated to include novel analytes, sustaining its utility for comprehensive oral fluid screening in forensic toxicology.

Conclusion

A robust, high-throughput protocol for the direct analysis of opioids and glucuronide metabolites in oral fluid was established. Combining mixed-mode µElution SPE with UPLC-MS/MS delivers sensitive, reproducible results without hydrolysis, making it well suited for forensic investigations.

References

• Goldberger BA, Cone EJ. Confirmatory tests for drugs in the workplace by GC-MS. J Chromatogr A. 1994;674(1-2):73-86.
• Murphy CM, Huestis MA. LC-ESI-MS/MS quantification of morphine and glucuronides in urine. J Mass Spectrom. 2005;40(11):1412-1416.
• French D, Wu A, Lynch K. HILIC LC-MS/MS of opioids in urine: significance of glucuronide metabolites. Bioanalysis. 2011;3(23):2603-2612.