Fast LC/MS/MS Analytical Method with Alternate Column Regeneration for the Analysis of 125 Various Drugs and Their Metabolites in Urine in Clinical Research

Importance of the Topic

Rapid multi-analyte detection in clinical research is essential for high-throughput screening, safety monitoring, and pharmacokinetic studies. Combining targeted quantitation of diverse drug classes with fast turnaround supports decision-making in laboratories requiring sensitivity and specificity.

Study Goals and Overview

This study aimed to develop and validate a single rapid LC–MS/MS method for simultaneous quantitation of 125 drugs and metabolites in urine. By integrating an Alternate Column Regeneration (ACR) system, the method sought to enhance throughput while maintaining analytical performance across a broad dynamic range.

Methodology and Instrumentation

The workflow involved preparing calibration standards in drug-free human urine (1–1000 ng/mL) with internal standards at 125 ng/mL. A 1 µL aliquot was injected into an Agilent 1290 Infinity II UHPLC coupled to an Agilent 6470 Triple Quadrupole MS. Separation employed two Poroshell 120 EC-C18 columns (2.1×100 mm, 2.7 µm) under a gradient from 12 % to 98 % organic within 2.9 min at 0.35 mL/min. The ACR valve allowed one column to regenerate while the other performed analysis, reducing cycle time from 7.35 to 5.1 min.

Results and Discussion

Calibration exhibited linear response (R² > 0.99) for 112 analytes across 1–1000 ng/mL; remaining compounds were fitted with quadratic/quartic models. Signal-to-noise ratios at the 1 ng/mL level exceeded 10 for 104 analytes. Retention time reproducibility between columns showed negligible variation. Representative TIC overlays confirmed consistent peak shapes and retention across 10 sequential injections. Within-run precision was below 15 % CV for all analytes.

Benefits and Practical Applications

• High throughput: 30 % reduction in analysis time via ACR.
• Comprehensive panel: quantitation of 125 compounds in a single run.
• Low detection limits: reliable quantitation down to 1 ng/mL.
• Robustness: high linearity and reproducibility support clinical and forensic workflows.

Used Instrumentation

• UHPLC: Agilent 1290 Infinity II with dual binary pumps, multisampler (5 °C), column compartment (55 °C).
• MS: Agilent 6470 Triple Quadrupole with AJS ESI source.
• Columns: 2×Poroshell 120 EC-C18, 2.1×100 mm, 2.7 µm.
• Mobile phases: A: H₂O + 5 mM ammonium formate + 0.01 % formic acid; B: MeOH + 0.01 % formic acid.
• Flow rate: 0.35 mL/min; injection volume: 1 µL; needle wash: IPA:MeOH:ACN:H₂O (50:20:20:10).

Future Trends and Potential Applications

Further work may explore matrix interferences across diverse urine sources, expand panels to emerging drugs, and integrate high-resolution MS for untargeted screening. Automation of sample preparation and implementation in diagnostic laboratories could broaden applicability.

Conclusion

A fast, sensitive, and specific LC–MS/MS method with alternate column regeneration enables reliable quantitation of 125 drugs and metabolites in urine within a 5.1 min cycle. The approach offers substantial time savings without sacrificing analytical performance, making it suitable for high-throughput clinical and forensic applications.

References

• 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.