Extraction of a Drugs of Abuse Panel from Oral Fluid by ISOLUTE® SLE+ After Collection with the Quantisal™ Collection Device Prior to UPLC-MS/MS Analysis

Importance of the Topic

Oral fluid testing offers a non-invasive, rapid sampling approach with a short detection window, making it a valuable matrix for forensic, clinical, and roadside drug screening.
Supported liquid extraction (SLE+) reduces emulsion formation and enhances analyte recovery compared to traditional liquid–liquid extraction.

Objectives and Overview of the Study

This study aimed to develop an efficient SLE+ protocol using ISOLUTE SLE+ columns to extract 47 drugs of abuse from oral fluid collected on Quantisal devices, followed by UPLC–MS/MS analysis. The method was optimized for both 200 µL and 500 µL sample volumes to evaluate extraction performance and assay sensitivity.

Methodology

• Sample Collection and Pre-treatment: Oral fluid was collected with Quantisal devices, buffered, and adjusted with concentrated ammonium hydroxide.
• Supported Liquid Extraction: Two column formats (400 µL and 1 mL) were loaded with 200 µL or 500 µL sample, respectively, allowed to absorb, then eluted with dichloromethane under gravity and vacuum.
• Post-elution Processing: Extracts were stabilized by adding 50 mM HCl in methanol before evaporation, dried under nitrogen or air at 40 °C, and reconstituted in mobile phase.
• Chromatography: UPLC separation was performed on a C18 column (1.7 µm, 100 × 2.1 mm) using a gradient of 5 mM ammonium acetate in water (A) and methanol (B) at 0.3 mL/min.
• Mass Spectrometry: Detection was by ESI–MS/MS in positive-ion MRM mode on a triple quadrupole instrument with optimized cone voltages and collision energies for each analyte.

Instrumentation

• UPLC: Waters ACQUITY UPLC system with ACE EXCEL 1.7 µm C18 column.
• MS: Premier XE triple quadrupole mass spectrometer with electrospray interface.

Main Results and Discussion

Extraction recoveries for all classes ranged from approximately 60 % to 110 % with RSDs of 1.3 %–9.3 % across both column formats. Calibration curves over 1–500 ng/mL showed excellent linearity (r² > 0.99) for deuterated and native analytes. Estimated limits of quantitation were typically below 0.1 ng/mL for most compounds on the 1 mL columns. Blowdown stability for amphetamines and ketamine was improved by in-situ salt formation with HCl in methanol.

Benefits and Practical Applications

• High analyte recovery and precision without emulsion issues.
• Low limits of quantitation suitable for trace-level detection.
• Streamlined workflow for high-throughput forensic and clinical toxicology.
• Scalable sample volumes for flexible assay design.

Future Trends and Applications

Integration of SLE+ extraction with automated platforms, extension to emerging psychoactive substances, miniaturized devices for point-of-care testing, and coupling with high-resolution mass spectrometry are anticipated advancements.

Conclusion

This optimized SLE+ protocol delivers a robust, reproducible, and sensitive method for multi-class drug screening in oral fluid. Its high recovery, low LOQ, and streamlined sample preparation make it well suited to forensic, clinical, and roadside applications.

Reference

Biotage. Application Note AN832: Extraction of a Drugs of Abuse Panel from Oral Fluid by ISOLUTE SLE+ after Collection with the Quantisal Device Prior to UPLC–MS/MS Analysis. 2014.