Direct Analysis of Opioids and Metabolites in Oral Fluid by Mixed-mode μElution SPE Combined with UPLC-MS/MS for Forensic Toxicology

Importance of the topic

Forensic toxicology increasingly relies on rapid and reliable detection of natural and synthetic opioids in biological matrices. Oral fluid analysis offers a non-invasive alternative to urine and blood, reflecting recent drug intake and reducing privacy and adulteration concerns. Direct measurement of both parent opioids and their glucuronide metabolites without hydrolysis eliminates potential false negatives and streamlines laboratory workflows.

Objectives and Study Overview

This study aimed to develop and validate a sensitive UPLC-MS/MS method for simultaneous quantification of 26 opioid drugs and metabolites in oral fluid. Key goals included:

• Avoiding enzymatic or chemical hydrolysis by direct metabolite measurement.
• Achieving rapid analysis with full resolution of isomeric pairs.
• Using minimal sample volume to preserve specimen for additional testing.
• Demonstrating linearity, precision, accuracy, and adequate limits of quantitation.

Methodology and Instrumentation

Oral fluid samples were collected using a stabilization buffer device and equilibrated to simulate transit conditions. A mixed-mode cation exchange μElution SPE plate was employed for sample cleanup using acidification, washing, and elution steps. The dried eluate was reconstituted in aqueous solution and injected onto an ACQUITY UPLC BEH C18 column. Mass spectrometric detection used MRM transitions optimized for each analyte.

Used Instrumentation

• Oasis MCX μElution 96-well plate
• ACQUITY UPLC I-Class system with BEH C18, 1.7 µm, 2.1 × 100 mm column
• XEVO TQD triple quadrupole mass spectrometer with positive ESI
• Waters MassLynx software v4.1

Main Results and Discussion

The method separated all 26 compounds in under 5.5 minutes with baseline resolution of critical isomeric pairs. Recovery averaged 79 % using a 60:40 ACN:IPA elution solvent. Calibration was linear over 5–500 ng/mL (1.25–125 ng/mL for fentanyl), with R² values above 0.989. Quality control samples demonstrated accuracy within ±15 % and precision (%CV) under 15 % at four concentration levels. The μElution format required only 100 µL of oral fluid, preserving the remainder for subsequent analyses.

Benefits and Practical Applications

• Elimination of hydrolysis reduces sample preparation time and risk of incomplete metabolite release.
• Rapid chromatographic cycle enhances laboratory throughput.
• Low sample volume requirement suits limited-volume matrices like oral fluid.
• Comprehensive panel covers natural opiates, semi-synthetic and synthetic analgesics.

Future Trends and Opportunities

Advances may include automated micro-extraction devices and high-throughput UPLC-MS/MS platforms. Incorporation of high-resolution mass spectrometry could further improve specificity. Expanding panels to emerging synthetic opioids and monitoring additional biomarkers in oral fluid will support roadside testing, workplace screening, and clinical toxicology.

Conclusion

The described mixed-mode μElution SPE with UPLC-MS/MS method provides a fast, accurate, and sensitive approach for direct analysis of opioids and glucuronide metabolites in oral fluid. It combines minimal sample volume, robust recovery, and excellent analytical performance, making it well suited for forensic and clinical toxicology laboratories.

References

1. Goldberger B.A., Cone E.J. Confirmatory tests for drugs in the workplace by gas chromatography-mass spectrometry. J Chromatogr A. 1994;674(1-2):73-86.
2. Gustavsson E. et al. Validation of direct injection electrospray LC-MS/MS for confirmation of opiates in urine drug testing. J Mass Spectrom. 2007;42(7):881-889.
3. Murphy C.M., Huestis M.A. LC-ESI-MS/MS analysis for quantification of morphine, codeine and their glucuronides in human urine. J Mass Spectrom. 2005;40(11):1412-1416.
4. Edinboro L.E., Backer R.C., Poklis A. Direct analysis of opiates in urine by liquid chromatography-tandem mass spectrometry. J Anal Toxicol. 2005;29(7):704-710.
5. French D., Wu A., Lynch K. Hydrophilic interaction LC-MS/MS analysis of opioids in urine: significance of glucuronide metabolites. Bioanalysis. 2011;3(23):2603-2612.
6. Wang P. et al. Incomplete recovery of prescription opioids in urine using enzymatic hydrolysis of glucuronide metabolites. J Anal Toxicol. 2006;30(8):570-575.