Automated and High-throughput Urine Drug Screening using Paper Spray Mass Spectrometry

Importance of the Topic

Rapid, reliable urine drug screening is essential in forensic toxicology, clinical toxicology, and workplace testing. Traditional immunoassays can suffer from limited specificity, while chromatographic methods often require extensive sample preparation and longer analysis times. Automated paper spray mass spectrometry (PS-MS/MS) addresses these limitations by combining minimal sample handling with high throughput and broad compound coverage.

Study Objectives and Overview

The primary goal of this work was to develop and validate an automated, high-throughput PS-MS/MS workflow for the detection of 40 illicit drugs and metabolites in urine. Key aims included achieving rapid turnaround (under two minutes per sample), simplified sample preparation via on-plate enzymatic hydrolysis, and method validation in line with SWGTOX guidelines.

Methodology

Sample Preparation and Hydrolysis

• Urine aliquots (50 µL) were combined with a premixed solution of buffer, isotopically labeled internal standards, and IMCSzyme®RT recombinant β-glucuronidase.
• After 15 minutes of room-temperature incubation to cleave glucuronide conjugates, 12 µL of the mixture was spotted onto a paper spray sample cassette.

Instrumental Configuration

• VeriSpray™ paper spray ion source (Thermo Fisher Scientific) coupled to a TSQ Altis™ triple quadrupole mass spectrometer.
• Spray voltage: +4000 V; ion transfer tube temperature: 300 °C; CID gas at 1.5 mTorr.
• Data acquisition in SRM mode with two transitions per analyte and nine isotopically labeled internal standards for quantitation.
• Automated sample cassette processed up to 240 samples per run at ~2 min/sample.

Main Results and Discussion

Validation and Analytical Performance

• Limits of quantification (LOQs) ranged from 0.7 to 41 ng/mL, well below typical forensic and regulatory cutoffs.
• Calibration linearity (R2 ≥ 0.98) was achieved across 10–1000 ng/mL or broader ranges for various analytes.
• Spike-and-recovery experiments in clinical urine demonstrated recoveries above 70% for oxazepam, morphine, and codeine glucuronide.
• Enzyme inhibition by urine constituents was rare; no recoveries fell below 40%.

Screening Throughput

• Up to 240 samples processed in a single automated run.
• Average analysis time of ~2 minutes per sample, comparable to high-volume immunoassay workflows.
• Simultaneous screening for 40 targets without chromatographic separation.

Practical Benefits

• Minimal sample preparation reduces labor and consumables.
• Rapid turnaround supports time-sensitive forensic and clinical decisions.
• Broad analyte panel enhances detection coverage beyond standard immunoassays.
• Automated workflow improves reproducibility and throughput in high-volume laboratories.

Future Trends and Potential Applications

• Expansion of analyte panels to include emerging synthetic drugs and novel metabolites.
• Integration with high-resolution mass spectrometry for untargeted screening and retrospective data mining.
• Coupling with laboratory information management systems (LIMS) for streamlined data handling and reporting.
• Application to other biological matrices (blood, saliva) for broader toxicology testing.

Conclusion

The automated PS-MS/MS workflow offers a rapid, cost-effective, and high-throughput alternative to conventional urine drug screening. Validation against SWGTOX guidelines demonstrates excellent sensitivity, linearity, and recovery for 40 common drug targets. This approach can significantly enhance lab efficiency while maintaining rigorous analytical performance standards.

Reference

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