Automatic, simultaneous and rapid analysis of 46 drugs of abuse in saliva by on line SPE and UHPLC-MS/MS

Significance of the Topic

Saliva drug testing offers a non-invasive and easily collected sample matrix that reflects recent intake of parent compounds. Compared with blood or urine, saliva levels are lower and represent only the unmetabolized drugs, demanding highly sensitive analytical approaches. Rapid and automated methods are essential to process large numbers of samples within the critical 24-hour window.

Objectives and Study Overview

This work aims to develop a robust, fully automated method for simultaneous quantification of 46 drugs of abuse in human saliva. The method must be rapid enough to handle hundreds of samples per day while maintaining high sensitivity, accuracy, and reproducibility.

Methodology and Instrumentation

Sample Preparation and Online SPE

• Saliva collection on Floqswab support
• Ultrasonic extraction with 2 mL pH 6.4 buffer for 5 minutes, spiked with calibration standards
• Automated on-line solid-phase extraction coupled directly to UHPLC-MS/MS

Chromatographic and Mass Spectrometric Conditions

• UHPLC system: Shimadzu Nexera X2 with LCMS-8060 triple quadrupole
• Column: ACE C18 AR, 100 mm × 2.1 mm i.d., 2 µm particle size, 30 °C
• Mobile phases: A: water with 0.002% formic acid and 2 mM ammonium formate; B: 90/10 methanol/acetonitrile
• Flow rate: 0.6 mL/min; Injection volume: 80 µL
• Detection in MRM mode with fast polarity switching

Results and Discussion

All 46 target compounds—including amphetamines, opioids, cannabinoids, stimulants, and novel psychoactive substances—were separated within a 15-minute cycle time that covers sample extraction, chromatographic run, column rinsing, and injector cleaning. Calibration curves demonstrated linearity across pg/mL ranges for each analyte, with accuracy between 80% and 115%. Representative chromatograms confirmed clear resolution and reproducible retention times. The method showed low limits of quantification, allowing detection of trace levels of each drug in saliva.

Benefits and Practical Applications

The presented approach offers high throughput and minimal manual intervention, making it suitable for forensic toxicology, roadside testing, workplace compliance, rehabilitation monitoring, and quality control in clinical and research laboratories. The protocol can be adapted to blood analysis by replacing swab extraction with protein precipitation without altering chromatographic conditions.

Future Trends and Applications

Ongoing developments may include expansion to emerging new psychoactive substances, integration of artificial intelligence for data review, microfluidic sampling for point-of-care testing, and coupling with laboratory information management systems to enhance throughput and traceability.

Conclusion

A fully automated on-line SPE UHPLC-MS/MS method was developed and validated for simultaneous analysis of 46 drugs of abuse in saliva. The workflow combines speed, sensitivity, and robustness, supporting high-volume testing needs in diverse analytical settings.

References

• Patent WO 2016/098169 A1