Ultrafast Analysis of Methadone and Metabolite EDDP in Urine by the Agilent RapidFire High-Throughput Mass Spectrometry System

Importance of the Topic

Methadone is widely used in pain management and addiction therapy, making its accurate detection and that of its metabolite EDDP in urine essential for forensic toxicology and clinical monitoring. Rapid, high-capacity analysis is needed to address the growing demands on laboratory throughput.

Objectives and Study Overview

This application note describes the development of an ultrafast method for simultaneous quantitative analysis of methadone and EDDP in urine using the Agilent RapidFire High-Throughput Mass Spectrometry system. The study aimed to achieve a broad linear range (10–5000 ng/mL), robust precision and accuracy, and minimal sample cycle time.

Methodology

• Samples of urine, calibrators (10–5000 ng/mL) and QC standards were spiked with internal standards (methadone-d3 and EDDP-d3) and diluted 1:100 in 50:50 methanol:water.
• Automated dilute-and-shoot workflow employed RapidFire reversed-phase C18 SPE cartridges for cleanup.
• Multiple reaction monitoring (MRM) on a triple quadrupole MS, with an 11-second injection-to-injection cycle.

Instrumentation

• Agilent RapidFire 360 High-Throughput SPE system
• Agilent 6460 Triple Quadrupole Mass Spectrometer
• Agilent MassHunter software for data acquisition and quantitative analysis

Results and Discussion

• Excellent linearity for both analytes (R² > 0.995) over 10–5000 ng/mL.
• Intra- and inter-day accuracies within ±10% and CVs below 5% across QC levels.
• Throughput exceeding 300 samples per hour, over 10× faster than conventional LC/MS/MS.
• No significant carryover or matrix effects (both <10%), ensuring reliable performance in urine.

Benefits and Practical Applications

• Drastically reduced analysis times enhance workload capacity in forensic and clinical labs.
• Simplified dilute-and-shoot protocol minimizes preparation steps.
• High throughput supports routine screening, confirmation and large-scale studies.

Future Trends and Applications

• Expansion of ultrafast SPE/MS/MS workflows to other small-molecule panels.
• Integration with automated liquid handling for fully streamlined operations.
• Potential deployment in rapid clinical decision-making and point-of-care testing.
• Development of multiplexed assays combining therapeutic drug monitoring with toxicology screening.

Conclusion

The Agilent RapidFire/MS/MS method delivers a robust, precise, and ultra-fast workflow for methadone and EDDP quantitation in urine, matching LC/MS/MS performance while offering >10× speed enhancements, thereby addressing high-throughput forensic and clinical testing needs.

References

1. Feng J. et al. Simultaneous Determination of Multiple Drugs of Abuse and Relevant Metabolites in Urine by LC-MS/MS. Journal of Analytical Toxicology 2007;31:369-368.
2. Vlase L. et al. Bioanalysis of Methadone in Human Plasma and Urine by LC/MS/MS. Revue Roumaine de Chimie 2008;53(12):1157-1164.
3. Galloway ER; Bellet NF. Methadone Conversion to EDDP during GC-MS Analysis of Urine Samples. Journal of Analytical Toxicology 1999;23:615-619.