Using UPLC-MS/MS for the Quantitation of Illicit or Prescription Drugs in Preserved Oral Fluid

Importance of the Topic

The reliable measurement of illicit and prescription drugs in oral fluid is critical for forensic toxicology, workplace safety, and roadside impairment testing. Oral fluid offers a non-invasive alternative to blood or urine, reducing privacy concerns and enabling supervised collection. Preserved oral fluid stabilizes target analytes, making it a valuable matrix for rapid, accurate drug screening.

Study Objectives and Overview

This work aimed to establish a simple, sensitive, and quantitative UPLC-MS/MS method for 14 common drugs and metabolites in preserved oral fluid. The focus was on achieving detection levels below recommended cut-offs, minimizing matrix effects, and facilitating high sample throughput for confirmation assays.

Methodology and Instrumentation

Sample Collection and Preparation

• Oral fluid was collected using the Quantisal device, yielding 1 mL of sample diluted fourfold with stabilizing buffer.
• Each 350 µL aliquot was spiked with deuterated internal standards and acidified with 4% phosphoric acid.
• Analytes were extracted via mixed-mode µElution SPE on Oasis MCX 96-well plates, with conditioning (methanol, water), washing (formic acid, methanol), and elution (acetonitrile/propan-2-ol with ammonium hydroxide).
• Eluates were dried under nitrogen, reconstituted in water/acetonitrile, and vortexed prior to analysis.

Instrumentation

• UPLC: Waters ACQUITY I-Class with BEH C18 column (2.1×100 mm, 1.7 µm) at 40 °C, gradient elution (0.1% formic acid and acetonitrile), 0.4 mL/min, 7 min cycle.
• MS/MS: Xevo TQD triple quadrupole, ESI positive, capillary 1 kV, desolvation 500 °C, MRM transitions optimized for 14 analytes with compound-specific deuterated ISTDs.

Main Results and Discussion

Linearity and Calibration

• Calibration curves over 0–500 ng/mL (neat) showed R2 > 0.995 with 1/x quadratic fitting.

Limits of Detection and Quantitation

• LOD for all analytes was below 0.1 ng/mL in neat fluid.
• LLOQ ranged from 0.16 to 4 ng/mL, with the lowest QC levels between 1.5 and 7.5 ng/mL.

Precision, Accuracy, and Matrix Effects

• Interday precision (%RSD) was below 10%, and accuracy within ±15% at low, medium, and high QC levels.
• Recoveries matched internal standards and matrix effects were below 25% variability, controlled by use of deuterated ISTDs.

Chromatographic Performance

• A representative chromatogram demonstrated clear separation of 14 analytes within a 7 min run, meeting retention time and ion-ratio criteria for confident identification.

Benefits and Practical Applications

This method combines rapid analysis, minimal sample preparation, and robust quantitation, making it suitable for high-volume laboratories performing workplace and roadside drug confirmations. The low detection limits exceed current cut-off requirements, while preserved oral fluid enables straightforward, non-invasive sampling.

Future Trends and Potential Applications

Future developments may include expansion to additional drug classes, full automation of SPE workflows, portable UPLC-MS platforms for on-site testing, and integration with advanced data processing or machine learning for faster decision support. Multi-analyte panels could be adapted to monitor emerging substances of abuse.

Conclusion

The presented UPLC-MS/MS approach offers a fast, sensitive, and reliable solution for quantifying a broad range of drugs in preserved oral fluid. Its robustness and throughput align with the demands of forensic and workplace testing, providing a solid foundation for further validation and implementation.

References

1. European Workplace Drug Testing Society Guidelines. ewdts.org (accessed May 29, 2015)
2. Danaceau R et al. Direct analysis of opioids and metabolites in oral fluid by mixed-mode µElution SPE combined with UPLC-MS/MS for forensic toxicology. Waters Application Note 720004838EN, 2013