Quantitation of drugs of abuse and their metabolites in urine using PaperSpray tandem mass spectrometry for clinical research and forensic toxicology

Importance of the Topic

Paper spray tandem mass spectrometry offers a rapid and simplified workflow for quantifying drugs of abuse and their metabolites in urine. Urinalysis is a cornerstone in forensic toxicology and clinical research, yet conventional chromatographic methods often involve lengthy sample preparation and separation steps. Adopting direct ionization techniques can greatly increase throughput while maintaining analytical rigor.

Objectives and Study Overview

This study aimed to develop and validate a single quantitative assay for 41 illicit drugs and metabolites in human urine using a PaperSpray ion source coupled to a triple-quadrupole mass spectrometer. Key goals included achieving established cutoff values, demonstrating accuracy and precision in line with forensic toxicology guidelines, and reducing per-sample cost and preparation time.

Methodology and Applied Instrumentation

Sample Preparation and Ionization:

• Eight microlitres of pooled human urine spiked with target analytes and isotopically labeled internal standards were spotted onto paper cartridges and air-dried for 30 minutes.
• No additional extraction, cleanup, or chromatographic separation was required.

Mass Spectrometry Setup:

• Thermo Scientific TSQ Fortis triple-quadrupole mass spectrometer operated in positive ion mode.
• Thermo Scientific VeriSpray PaperSpray ion source with automated plate loader accommodating up to 240 samples.
• Spray solvent: 90% acetonitrile, 10% water with 0.1% acetic acid; spray voltage ramped to 3.8 kV for 1.1 seconds.
• Selected reaction monitoring transitions optimized for each analyte, with Q1 and Q3 resolutions set at 0.7 Da FWHM.
• Data acquisition and quantitation performed using Thermo Scientific TraceFinder software with 1/x weighted linear regression.

Main Results and Discussion

• Calibration models over nine concentration levels yielded correlation coefficients (R2) exceeding 0.98 for all compounds.
• Lower limits of quantitation were at or below regulatory cutoffs, with signal-to-noise ratios above four.
• Within- and between-run precision (%CV) and accuracy (%Bias) values met the acceptance criteria (≤20%) defined by SWGTOX guidelines.
• Total analysis time per dried urine spot, including extraction and MS acquisition, was approximately 2.3 minutes.

Benefits and Practical Applications

The PaperSpray MS/MS approach eliminates extensive sample preparation and chromatographic separation, reducing labor, solvent use, and instrument runtime. High throughput and robust performance support its use in busy forensic and clinical laboratories for routine drug screening, compliance monitoring, and pharmacokinetic studies.

Future Trends and Potential Applications

Advancements may include expansion to additional biological matrices such as blood or saliva, integration with automated sample handling robots, and real-time on-site testing capabilities. Coupling this approach with high-resolution mass spectrometry and machine learning algorithms for peak annotation could further enhance specificity and data processing efficiency.

Conclusion

The validated PaperSpray tandem MS method on the TSQ Fortis platform provides a rapid, accurate, and cost-effective solution for quantifying a broad panel of drugs of abuse in urine. Its minimal sample preparation, high sensitivity, and compliance with forensic toxicology standards make it a practical alternative to traditional chromatographic assays.

References

1. Liu J, Wang H, Manicke NE, Lin J-M, Cooks RG, Ouyang Z. Development, characterization, and application of paper spray ionization. Analytical Chemistry. 2010;82(6):2463–2471.
2. SWGTOX. Scientific Working Group for Forensic Toxicology standard practices for method validation in forensic toxicology. Journal of Analytical Toxicology. 2013;37:452–474.