Fully Automated Online Sample Preparation and LC-MS/MS Analysis of Drugs of Abuse in Oral Fluids

Significance of the Topic

Automated sample preparation is critical to meet growing demand for fast and reliable drug screening in forensic and clinical laboratories. Oral fluid testing offers a noninvasive collection method but presents challenges in consistent sample handling and high throughput. Integrating sample preparation directly with LC-MS/MS reduces manual steps, minimizes analytical variability, and accelerates workflow efficiency.

Study Objectives and Overview

This study describes the development and validation of a fully automated platform for online sample preparation and LC-MS/MS analysis of seventy-seven drugs of abuse in oral fluids. The CLAM-2000 module was seamlessly coupled to a Shimadzu Nexera UHPLC system and a Shimadzu 8050 triple quadrupole mass spectrometer to deliver rapid, reproducible, and high-throughput quantitation.

Methodology and Instrumentation

• Chemicals and reagents: Methanol, formic acid, distilled water.
• Mobile phases: A = 0.1% formic acid in water; B = 100% methanol; gradient from 10% to 60% B over 4.5 minutes.
• Matrix: Oral fluid diluent (Intercept i2HE) for samples, calibrators, and quality controls.
• Instrumentation:
  • CLAM-2000 automated sample preparation module with functionalities for pipetting, mixing, filtration, and heating; capable of parallel processing of four samples.
  • Shimadzu Nexera UHPLC system.
  • Shimadzu 8050 triple quadrupole mass spectrometer.
• Workflow: Addition of methanol, sample, and internal standard; 30-second shake; 90-second vacuum filtration; automated transfer of processed extract to LC-MS vial.

Main Results and Discussion

• Total cycle time of 11 minutes per sample, comprising 4 minutes of automated preparation and 7 minutes of chromatographic separation and detection.
• Quantitative analysis of 77 target compounds with calibration curves exhibiting R² ≥ 0.99 using 1/C² weighting.
• Limits of detection as low as 0.75 ng/mL for fentanyl and 10 ng/mL for morphine.
• High precision with relative standard deviations ≤ 5% across multiple runs.
• Representative chromatograms demonstrated clear separation of diazepam, morphine, fentanyl, and their deuterated internal standards, confirming method selectivity and sensitivity.

Benefits and Practical Applications

• Elimination of manual handling reduces human error and labor costs.
• Parallel processing of four samples maximizes instrument utilization and throughput.
• Robust reproducibility and sensitivity are ideal for forensic toxicology, clinical drug monitoring, and workplace testing.
• Seamless integration streamlines laboratory workflows and improves data quality.

Future Trends and Potential Applications

The automated sample preparation strategy can be extended to other biological matrices and broadened to include additional small molecules and peptide analytes. Future developments may integrate advanced extraction chemistries and high-resolution mass spectrometry to expand applications in clinical diagnostics, environmental analysis, and pharmaceutical research.

Conclusion

The CLAM-2000 LC-MS/MS platform offers a fully automated, rapid, and reliable solution for comprehensive drug-of-abuse screening in oral fluids. By combining online sample preparation with high-throughput chromatographic separation and sensitive mass spectrometric detection, this system meets the demands of modern analytical laboratories for speed, precision, and robustness.

Reference

• Emory J.F., DeFreitas N., Roberts M., Tyagi M., Boutaghou M.N., Feild B.J.; "Fully Automated Online Sample Preparation and LC-MS/MS Analysis of Drugs of Abuse in Oral Fluids"; ASMS 2017 TP-442; Shimadzu Scientific Instruments; First Edition: June 2017.