High Sensitivity Bioanalysis for Small Drug-Like Compounds in Human Plasma using Microflow LC and High Resolution Mass Spectrometry

Importance of the Topic

The development of highly sensitive bioanalytical methods for small drug-like molecules in human plasma is critical for pharmacokinetic-pharmacodynamic studies, early drug discovery and clinical trials. Microflow liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) addresses the need for low limits of quantification while reducing sample and solvent consumption.

Objectives and Study Overview

This work evaluates an integrated microfluidics LC/MS platform (ionKey/MS) combined with an advanced quadrupole time-of-flight instrument (Xevo G2-XS HRMS). Key goals include:

• Characterizing quantification performance for potent compounds in human plasma
• Determining lower limits of quantification, linear dynamic range and signal-to-noise ratios
• Assessing a trap-and-elute configuration to increase injection volume without compromising peak quality

Instrumentation Used

• ionKey/MS microflow LC/MS source
• Waters AQUITY M-Class UPLC system
• Xevo G2-XS QTof high-resolution mass spectrometer
• ionKey HSS T3 separation device (150 µm × 50 mm, 1.8 µm) at 45 °C
• HSS T3 trap column (300 µm × 50 mm, 1.8 µm)

Methodology

Test compounds (buspirone, propranolol, verapamil, clopidogrel) were spiked into human plasma and diluted. A generic gradient from 5 % to 60 % acetonitrile (both phases containing 0.1 % formic acid) was run over 3.5 minutes at 3 µL/min. Direct injections (1 µL) were acquired in MRM-like mode on the Xevo G2-XS QTof. Separately, a trap valve manager enabled trap-and-elute workflows with injection volumes of 1, 5, 10 and 20 µL to mimic analytical-scale loads.

Main Results and Discussion

Direct injection experiments demonstrated excellent linearity (log range 3.6 to 4.2) for verapamil and clopidogrel, with lower limits of quantification between 0.8 fg and 3 fg on column and signal-to-noise ratios of 9 to 79. Trap-and-elute tests showed:

• Complete sample recovery up to 20 µL injections (equivalent to 4 mL on a standard column) with R2 = 0.9987
• No peak distortion for polar analytes such as propranolol and buspirone, even in 20 % organic solvent
• Maintained peak shape and resolution comparable to direct injection

Benefits and Practical Applications

This microflow LC-HRMS system offers:

• Attomole-level sensitivity for small molecules in plasma
• Substantial reduction (up to 90 %) in solvent consumption
• Flexibility to switch between direct injection and trap-and-elute modes
• Robust performance for routine bioanalysis, PK/PD studies and QA/QC workflows

Future Trends and Opportunities

Anticipated developments include integrating automated sample preparation, expanding compound coverage for multi-analyte panels, further miniaturization of microfluidic devices and leveraging AI-driven data analysis for rapid quantitation. Ultra-fast gradients and high-throughput capabilities will meet rising demands in drug discovery and clinical testing.

Conclusion

The combination of ionKey/MS microflow LC with Xevo G2-XS HRMS delivers outstanding sensitivity, linearity and robustness for bioanalysis of small drug-like molecules. The trap-and-elute option further enhances injection capacity without compromising chromatographic quality, positioning this platform as a powerful tool for modern pharmaceutical and clinical laboratories.