Exploring Extra Sensitivity Using ionKey/MS with the Xevo G2-XS QTof HRMS for Small Molecule Pharmaceutical Analysis in Human Plasma

Importance of the Topic

The integration of microfluidic separation with high-resolution mass spectrometry addresses the ongoing demand for enhanced sensitivity and selectivity in small molecule quantitation in bioanalysis. High-resolution MS (HRMS) platforms capable of sub-femtogram detection support critical decision points in drug discovery, development, and therapeutic monitoring while reducing sample and solvent consumption.

Objectives and Study Overview

This study evaluates the sensitivity gains and operational flexibility of the ionKey/MS System coupled to a Xevo G2-XS QTof HRMS for quantifying pharmaceutical compounds in human plasma. Three experimental configurations were compared:

• Direct injection with ToF-MRM acquisition
• Single-pump trap-and-elute workflow
• Dual-pump trap-and-elute workflow with guard-column trapping and wash-out capabilities

Methodology and Instrumentation

Sample preparation involved protein precipitation of human plasma with acetonitrile (3:1), centrifugation, and 1:4 dilution with water (final ~19% ACN). Test analytes (propranolol, verapamil, buspirone, clopidogrel) were spiked and serially diluted from 100 ng/mL to sub-pg/mL levels.

• LC platform: ACQUITY UPLC M-Class with ionKey/MS System
  
• Separation device: iKey HSS T3 (150 µm×50 mm, 1.8 µm)
  
• Trap column: ACQUITY UPLC M-Class HSS T3 trap (300 µm×50 mm, 5 µm)
• MS: Xevo G2-XS QTof, ESI+ in sensitivity mode, 50–1 200 m/z scan range
• Acquisition: ToF-MRM for direct injections; full scan for trap-and-elute evaluations

Main Results and Discussion

Direct injection (1 µL, ToF-MRM) achieved sub-femtogram on-column quantitation limits: buspirone 1.5 fg, clopidogrel 0.8 fg, verapamil 3.1 fg, with linear ranges spanning >3 logarithmic decades and R² > 0.99. Signal-to-noise ratios at LOQ ranged from 9 to 78.

Single-pump trap-and-elute mitigated peak distortion observed for early-eluting compounds at larger injection volumes (5 µL). Early compounds showed fronting in direct injection but returned to Gaussian shapes after trapping, confirming effective sample focusing.

Dual-pump trap-and-elute allowed up to 20 µL plasma injections without loss of resolution or shape, equating to 23× column volume. Linear area responses (R² > 0.995) were maintained for all analytes. Trap-mobile-phase composition influenced recovery for lipophilic compounds: increasing ACN from 0.5% to 5% restored full elution of clopidogrel.

Benefits and Practical Applications

• Sub-fg sensitivity in routine bioanalysis
• Solvent use reduced by >90% vs. analytical LC
• High sample load capability for low-concentration targets
• Integrated microfluidics improve ionization and reduce matrix effects
• Trap column acts as guard to protect the separation device and MS

Future Trends and Applications

Emerging directions include automation of trap-and-elute sequences, broader adoption for metabolomics and clinical assays, expansion to multiplexed assays, and integration with data-independent acquisition workflows to leverage ultra-high resolution for complex matrices.

Conclusion

The ionKey/MS System with the Xevo G2-XS QTof HRMS offers unparalleled sensitivity and flexibility for small molecule pharmaceutical analysis in plasma. Direct injection and trap-and-elute modes deliver sub-femtogram quantitation, extended dynamic range, and robust peak performance while minimizing solvent usage and instrument contamination.

Reference

1. Alelyunas YW, Wrona MD, Mortishire-Smith RJ, Tomczyk N, Rainville PD. Quantitation by High-Resolution Mass Spectrometry: Using Target Enhancement and ToF-MRM to Achieve Femtogram-Level On-Column Sensitivity for Drugs in Human Plasma. Waters Application Note, 2014, 720005182EN.
2. Snyder LR, Dolan JW. High-Performance Gradient Elution: The Practical Application of the Linear-Solvent-Strength Model. Wiley, 2006, pp. 190–193. ISBN 978-0-471-70646-5.