Application of Vacuum Jacketed UHPLC Columns Coupled with Mass Spectrometry in High Throughput Pharmaceutical Analysis

Significance of the Topic

The implementation of sub-2 µm particle UHPLC columns has driven advances in sensitivity, resolution and throughput in pharmaceutical analysis. However, high backpressures (up to 18,000 psi) generate frictional heating, leading to band broadening and loss of chromatographic performance. Vacuum-jacketed column (VJC) technology mitigates these effects by isolating the column in a low-pressure environment, reducing post-column dispersion and enabling ultra-fast separations without compromising data quality.

Objectives and Study Overview

This work evaluates the performance of vacuum-jacketed UHPLC columns coupled to a Xevo G2-XS Q-ToF mass spectrometer (VJC/MS) for high-throughput analysis of verapamil and its microsomal metabolites. Key aims include:

• Comparing separation speed, peak shape and sensitivity against a conventional UHPLC/MS setup
• Demonstrating sub‐minute analysis times suitable for pharmaceutical screening workflows

Methods and Instrumentation

Liquid Chromatography System

• Waters ACQUITY I-Class UHPLC
• Flow rate: 0.6 mL/min
• Columns: 2.1 × 50 mm VJC and conventional housings packed with 1.7 µm BEH C18
• Mobile phase A: 0.1 % formic acid in water
• Mobile phase B: 0.1 % formic acid in acetonitrile
• Gradient: linear, optimized for sub-minute elution
• Injection volume: 2 µL

Mass Spectrometry System

• Waters Xevo G2-XS Q-ToF
• Acquisition mode: MSE, 50–1000 Da
• Capillary voltage: 2 kV; cone voltage: 50 V
• Source temperature: 140 °C; desolvation temperature: 600 °C
• Desolvation gas: 1000 L/h; cone gas: 50 L/h

Sample Preparation

• Microsomal incubations with 1 mg/mL protein and 50 µM substrate in phosphate buffer at 37 °C
• Reaction initiated by UDPGA and NADPH (20 mM)
• Quenched with cold acetonitrile, centrifuged and diluted with water

Results and Discussion

VJC/MS achieved complete separation of verapamil and its metabolites in under one minute, whereas conventional columns required longer runtimes. Total ion chromatograms revealed significantly narrower peak widths and increased peak heights with the VJC setup, indicating reduced thermal dispersion and enhanced sensitivity. Rapid gradient methods provided clear resolution of primary hydroxylated metabolites, confirming suitability for high-throughput screening.

Benefits and Practical Applications

• Sub-minute analysis time increases sample throughput and instrument availability
• Improved chromatographic resolution enhances quantitation accuracy for low-abundance metabolites
• Plug-and-play column housing simplifies maintenance and method transfer
• Seamless integration with MS detection benefits pharmaceutical R&D, drug metabolism studies and QA/QC workflows

Future Trends and Opportunities

The integration of VJC technology with high-resolution and high-mass-accuracy MS platforms could further broaden compound coverage and structural elucidation capabilities. Expansion into large-molecule and biologics analysis, combined with automated sample handling and real-time data processing, will drive next-generation high-throughput analytical workflows in pharmaceutical and clinical laboratories.

Conclusion

Vacuum-jacketed UHPLC columns coupled to Q-ToF MS effectively address frictional heating challenges at high pressures, delivering sub-minute separations with superior resolution and sensitivity. This approach offers a robust, high-throughput solution for pharmaceutical analysis, streamlining metabolite profiling and QA/QC operations.

References

• Gritti F. Vacuum-Jacketed Columns: Maximum Efficiency, Easy Deployment Without Oven, and Improved LC–MS Performance. LCGC. 2019;32(5):8–13.
• Plumb RS, et al. High-Throughput UHPLC/MS/MS-Based Metabolic Profiling Using a Vacuum Jacketed Column. Anal Chem. 2021;93(30):10644–10652.