Comparison of Sample Preparation Techniques for the Analysis of Drugs of Abuse in Oral Fluids

Significance of the Topic

Oral fluid testing is gaining traction in forensic toxicology and workplace drug screening due to its non­invasive sampling, fast collection and direct link to recent drug intake. However, challenges such as buffer components and surfactants can compromise analytical performance and column longevity. Establishing reliable sample preparation workflows is essential to deliver robust quantitation of drugs of abuse in this matrix.

Objectives and Study Overview

This study compares three sample preparation techniques for the analysis of 68 drugs of abuse and novel psychoactive substances in oral fluid by LC­MS/MS. The aim is to evaluate salt­assisted liquid­liquid extraction (SALLE) and supported liquid extraction (SLE) against a simple dilute­and­shoot approach, assessing recovery, matrix effects and overall suitability for routine high­throughput testing.

Methodology and Used Instrumentation

Sample Preparation Workflows:

• SALLE: Addition of saturated NaCl and acetonitrile to oral fluid/buffer, vortex, centrifugation and organic layer collection.
• SLE: Load fluid onto supported liquid extraction cartridge, allow binding, two elutions with dichloromethane/isopropanol, evaporation and reconstitution.
• Dilute­and­shoot: Direct addition of buffer, internal standard, dilution and injection.

Chromatographic and Detection Conditions:

• LC Column: Biphenyl stationary phase (50 × 2.1 mm, 2.7 µm) with guard cartridge.
• Mobile Phases: Water and methanol each containing 0.1 % formic acid with a gradient from 15 % to 100 % organic over 9 minutes.
• Flow Rate: 0.5 mL/min; Column Temperature: 40 °C; Injection Volume: 5 µL.
• Detection: Triple quadrupole MS in positive MRM mode covering 136 transitions and achieving baseline resolution of eight isobaric pairs within a 10-minute cycle.

Main Results and Discussion

Sample clean­up had a pronounced impact on analyte recovery and signal intensity.

• Both SALLE and SLE outperformed dilute­and­shoot for all 68 compounds, improving peak areas and reducing matrix suppression.
• Mitragynine (kratom) exhibited roughly twofold higher response with SALLE, while norfentanyl and other opiates showed better recovery using SLE.
• Linearity was excellent for all analytes, with r2 values ≥ 0.99 using 1/x weighted regression.
• Interday precision and accuracy met QC criteria of ± 15 % across low, mid and high concentration levels.

Benefits and Practical Applications

This comparison demonstrates that tailored sample preparation enables:

• High throughput screening of a broad panel of drugs and NPS in a single ten-minute LC-MS/MS run.
• Improved method robustness and column lifetime by reducing surfactant and matrix load.
• Reliable quantitation suitable for forensic, clinical and workplace testing environments.

Future Trends and Potential Applications

Advancements likely to further enhance oral fluid analysis include:

• Automation of SALLE and SLE workflows to increase throughput and reproducibility.
• Development of novel sorbent materials and microextraction approaches to miniaturize sample preparation.
• Integration with high-resolution mass spectrometry for broader non­targeted screening of emerging psychoactive substances.

Conclusion

Supported liquid extraction and salt­assisted liquid­liquid extraction significantly improve recovery and data quality compared to dilute­and­shoot for a wide range of drugs in oral fluid. Their implementation supports accurate quantitation in under 10 minutes per sample, offering a robust platform for modern toxicology workflows.

References

• Valente IM, Poole CF, et al. Another glimpse over the salting-out assisted liquid-liquid extraction in acetonitrile/water mixtures. Journal of Chromatography A. 2013;1308:58–62.
• Majors RE. Salting-Out Liquid-Liquid Extraction (SALLE). LCGC International. 2009;27(7):526–533.