Analysis of Tricyclic Antidepressants in Human Urine Using UltraPerformance Convergence Chromatography (UPC2)

Significance of the Topic

Tricyclic antidepressants (TCAs) remain widely used due to their cost-effectiveness and clinical efficacy in cases where newer agents are inadequate. Accurate, high-throughput quantification of TCAs in biological fluids is essential for therapeutic drug monitoring, pharmacokinetic studies, and compliance testing. UltraPerformance Convergence Chromatography (UPC²) introduces a greener, faster alternative to traditional reversed-phase LC, leveraging supercritical CO₂ to reduce solvent use and improve selectivity.

Objectives and Study Overview

This work presents a proof-of-concept Application Note demonstrating a rapid UPC²–MS/MS method for simultaneous analysis of five TCAs in human urine. The goals were to minimize organic solvent consumption, simplify sample preparation through direct injection of SPE eluates, and achieve sub-nanogram per milliliter sensitivity in a 3-minute chromatographic cycle.

Methodology and Instrumentation

Sample preparation employed Oasis WCX 96-well µElution plates for mixed-mode weak cation exchange extraction of acidified urine, followed by direct injection of combined elution aliquots without evaporation.

• Chromatography: Waters ACQUITY UPC² system with a BEH 2.1 × 50 mm, 1.7 µm column.
• Mobile phases: CO₂ (A) and methanol + 0.2% NH₄OH (B); gradient from 2% to 40% B in 2 min, total run time 3 min.
• Mass spectrometry: Xevo TQ-S triple quadrupole, positive ESI, optimized cone voltages and collision energies for each analyte.

Key Results and Discussion

The optimized method provided rapid separation with peak widths <2 s at base and low system pressures (<4200 psi). SPE recoveries ranged from 92% to 104% (RSD 3%–6%). Systematic screening identified the BEH column and NH₄OH modifier as optimal for sensitivity and peak shape. Calibration curves over 0.1–10 ng/mL were linear (R² > 0.998) with average accuracy within ±8%. LLOQs of 0.1 ng/mL met FDA bioanalytical criteria, with signal-to-noise ratios from 66:1 to 590:1.

• High throughput: 3 min cycle time without carryover.
• Excellent sensitivity: sub-ng/mL quantification in urine.
• Robust performance: reproducible retention times and peak areas.

Benefits and Practical Applications

• Green chemistry: supercritical CO₂ reduces organic solvent consumption and waste.
• Workflow efficiency: direct injection of SPE eluates eliminates evaporation and reconstitution steps.
• Wide applicability: suitable for therapeutic drug monitoring, pharmacokinetics, and toxicology screening.

Future Trends and Opportunities

UPC² technology is expanding its role in bioanalysis, offering orthogonal selectivity to reversed-phase LC and compatibility with diverse sample matrices. Potential advancements include:

• Implementation of buffered modifiers (e.g., ammonium formate) to improve peak shapes for polar compounds.
• Integration with fully automated sample preparation for large-scale studies.
• Extension to other drug classes and metabolite panels for comprehensive screening.

Conclusion

The UPC²–MS/MS method delivers a fast, accurate, and environmentally sustainable approach for quantifying tricyclic antidepressants in human urine. The combination of efficient mixed-mode SPE, optimized UPC² separation, and sensitive MS detection fulfills stringent bioanalytical requirements while enhancing throughput and reducing solvent usage.

References

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