Tandem UHPLC operation for high-throughput LC-MS peptide mapping analyses

Significance of the Topic

Given the high demand for rapid and reliable peptide mapping in biopharmaceutical quality control and research, reducing downtime from column reconditioning is critical. Tandem UHPLC approaches that alternate analytical and reconditioning gradients on parallel columns can significantly boost throughput without modifying existing methods.

Study Objectives and Overview

This study demonstrates the Thermo Scientific Vanquish Duo UHPLC system configured for tandem operation, enabling parallel gradient runs and simultaneous column reconditioning for peptide mapping of a monoclonal antibody. The primary goal is to assess improvements in productivity, chromatographic precision, and carryover suppression while maintaining robust performance.

Methodology and Instrumentation

• Sample Preparation: Infliximab monoclonal antibody digested using the SMART Digest Kit (trypsin digestion at 70 °C for 45 min, followed by centrifugation and dilution to 100 ng/µL).
• UHPLC Setup: Vanquish Horizon Duo with two Binary Pump H modules, Split Sampler HT, Column Compartment H, Variable Wavelength Detector, and biocompatible 2-position/6-port valves for automated column switching.
• Columns: Two Acclaim Vanquish C18, 2.1 × 250 mm, 2.2 µm particles; active pre-heaters and capillaries as per the Vanquish Duo Tandem LC Kit.
• Mobile Phases: A) water + 0.1% formic acid; B) water/acetonitrile (10:90) + 0.1% formic acid.
• Gradient Programs: Analytical pump 1 used a 40 min gradient from 1% to 45% B at 0.4 mL/min; reconditioning pump applied multi-step washes up to 90% B to eliminate carryover and re-equilibrate the column.
• Mass Spectrometry: Q Exactive HF Orbitrap with HESI-II source, full MS range 140–2000 m/z, resolution 15 000 (FWHM at m/z 200), spray voltage 3.5 kV, capillary at 350 °C.

Main Results and Discussion

The tandem LC configuration reduced idle time by overlapping analytical gradients and column reconditioning, achieving up to 40% higher throughput. Overlay of five replicate total ion chromatograms for each column showed retention time RSDs below 0.11% and peak area RSDs around 2.5%. Retention time shifts between columns averaged only 0.023 min (0.18%), confirming consistent peptide identification. UV monitoring during reconditioning demonstrated effective removal of residual peptides.

Benefits and Practical Applications

• Enhanced sample throughput without altering validated gradient methods.
• Efficient use of laboratory space via a single system handling two columns.
• Automated column regeneration reduces manual labor and instrument downtime.
• High reproducibility in retention times and quantitative data supports QC and research workflows.

Future Trends and Opportunities

Emerging valve technologies and ultra-high-pressure pump modules will further decrease cycle times and expand tandem LC applicability to diverse biotherapeutic and proteomic analyses. Integration with intelligent data systems may enable dynamic method adaptation and predictive maintenance, promoting broader adoption in high-throughput laboratories.

Conclusion

The Vanquish Duo UHPLC tandem LC approach provides a robust, high-throughput peptide mapping solution by minimizing reconditioning idle time while preserving chromatographic quality and reproducibility. Its compatibility with existing methods and flexible configuration makes it ideal for both research and QC environments.

References

1. Fletcher MR, Foley JP. Kinetic Model of HPLC Column Re-Equilibration after Gradient Elution. Eastern Analytical Symposium 2015.
2. Van Middlesworth BJ, Dorsey JG. Re-equilibration Time of Superficially Porous Silica Columns in Gradient Elution. J Chromatogr A. 2011;1218:7158–7165.
3. Freiser HH, Nowlan MP, Schmuck MN. Use of Stabilized Silica Support with Short Alkyl Chains for Preparative Protein Chromatography. Micra Scientific Bulletin 10, 1988.