Determination of Urinary Opioids by Solid-phase Extraction LC-MS/MS for Clinical Research: Comparison of Automated and Manual Sample Preparation

Significance of the Topic

Monitoring opioid and metabolite levels in human urine is crucial for clinical research, therapeutic drug monitoring and ensuring patient compliance. Solid-phase extraction (SPE) combined with LC-MS/MS offers high sensitivity, selectivity and throughput. Automating sample preparation can further enhance laboratory efficiency, consistency and safety while reducing analyst-to-analyst variability and transcription errors.

Objectives and Study Overview

The study compared manual and automated SPE workflows for quantifying 21 opioids and related metabolites in human urine. Over three days, both approaches were evaluated for calibration linearity, inter-assay precision and accuracy, carryover and total processing time. Automated sample handling used a Tecan Freedom EVO 100 liquid handler integrated with a MassLynx File Converter for sample list generation.

Methodology and Instrumentation

Samples (150 µL urine) were spiked with deuterated internal standards and acidified with phosphoric acid. SPE was performed on an Oasis MCX µElution 96-well plate with conditioning, equilibration, sample loading, two wash steps and dual elution in basic methanol–acetonitrile. Following evaporation and reconstitution, extracts were analyzed by UPLC–MS/MS.

• UPLC system: Waters ACQUITY UPLC with BEH C18 column (2.1 × 100 mm, 1.7 µm) at 40 °C
• Mobile phases: 0.1% formic acid in water (A) and acetonitrile (B); gradient from 2% to 45% B over 4 min
• MS/MS: Waters Xevo TQD with ESI in positive MRM mode (see Table of transitions)
• Automation: Tecan Freedom EVO 100 with 4-tip liquid handling arm, plate shaker, wash station, vacuum manifold and non-disposable tip washing; PosID barcode reader; MassLynx File Converter for automated sample list creation

Main Results and Discussion

Both manual and automated workflows met typical bioanalytical acceptance criteria:

• Linearity: R² ≥ 0.99 for all analytes
• Precision: inter-assay CV ≤ 10% across low, mid and high QC levels
• Accuracy: mean deviation within ±10% of nominal concentrations
• Carryover: < 4% for all compounds

Total processing time for 96 samples was comparable: manual pipetting ~45 min (plus 5 min extraction, 11 min drying/reconstitution) versus automated SPE ~55 min (plus 21 min sample transfers, 11 min drying/reconstitution). Use of the MassLynx File Converter eliminated 5–20 min of manual sample list generation and minimized transcription errors.

Benefits and Practical Applications

Automated SPE LC-MS/MS offers:

• Reduced hands-on time and ergonomic stress
• Consistent, error-free sample processing and tracking
• Improved laboratory safety by limiting solvent exposure and repetitive motions
• Streamlined data handling via automated sample list import into MassLynx

Future Trends and Potential Applications

Advances may include integration of online SPE with UPLC for full automation, expansion to larger analyte panels, miniaturized lab-on-chip platforms, AI-driven method optimization and remote monitoring of automated workflows. These developments will further increase throughput, data quality and flexibility in clinical and forensic laboratories.

Conclusion

The automated SPE workflow using a Tecan liquid handler and MassLynx File Converter matched manual preparation in accuracy, precision and linearity while enhancing traceability and reducing preparation time and human error. This approach supports high-throughput, reliable opioid monitoring in clinical research settings.

References

None provided.