2D-LC as an Automated Desalting Tool for MSD Analysis Direct Mass Selective Detection of a Pharmaceutical Peptide from an MS-Incompatible USP Method

Significance of the Topic

Peptide and protein therapeutics require precise impurity profiling to ensure safety and efficacy. Many chromatographic methods for these biomolecules use nonvolatile buffers incompatible with mass spectrometry, limiting direct MS detection. Automated online desalting via two-dimensional liquid chromatography (2D-LC) overcomes this obstacle and streamlines analytical workflows.

Objectives and Study Overview

This application demonstrates the use of multiple heart-cutting 2D-LC as an inline desalting tool to enable direct MS detection of glucagon following the USP 39 method. The goal is to couple an MS-incompatible first-dimension separation with a second-dimension desalting step and MS analysis without manual intervention.

Instrumentation

The system comprised Agilent InfinityLab 1290 Infinity II modules (two High Speed Pumps, Multisampler, Multicolumn Thermostat, Variable Wavelength Detector), multiple valve drives with heart-cutting and diverter valves, an Agilent 6150 Single Quadrupole LC/MS with Jet Stream ESI source, a ZORBAX Eclipse Plus C18 analytical column, an AdvanceBio Desalting-RP trap column, and Agilent OpenLAB CDS ChemStation software.

Methodology

The first dimension used a phosphate buffer/acetonitrile gradient on C18 at 0.5 mL/min and 45 °C with UV detection at 214 nm. Glucagon peaks were captured via time- or peak-based heart-cutting into 40 µL loops and matrix salts were diverted to waste using a diverter valve. The second dimension employed an RP trap cartridge with 0.1% formic acid water/acetonitrile gradient at 0.4 mL/min for 2 min before mass selective detection. MS parameters targeted positive ions in the 600–1350 m/z range with full data acquisition.

Main Results and Discussion

The method met USP 39 suitability criteria: retention time RSD 0.27%, area RSD 0.11%, tailing factor 1.3, and resolution ≥ 2.3 between glucagon and the first desamido impurity. Glucagon was detected as [M+5H]5+, [M+4H]4+, and [M+3H]3+ ions with no salt adducts, confirming effective desalting. Four desamido-glucagon impurities (+1 Da) were resolved and their identities confirmed by mass spectra, demonstrating comprehensive inline impurity profiling.

Benefits and Practical Applications

Inline desalting reduces manual sample preparation, minimizes analyte loss and contamination, and increases throughput in QC and R&D settings. It enables direct MS detection of peptides from existing USP methods without altering established first-dimension protocols.

Future Trends and Potential Applications

Expanding 2D-LC desalting to a wider range of biologics including monoclonal antibodies
Integration with high-resolution mass spectrometry for enhanced impurity characterization
Development of automated multi-heart-cut routines for comprehensive profiling in regulated environments

Conclusion

Automated multiple heart-cutting 2D-LC offers a robust online desalting solution, seamlessly coupling MS-incompatible peptide methods to mass spectrometry. This approach satisfies regulatory requirements, enhances data quality, and streamlines peptide impurity analysis.

References

• Luo H et al. Two-dimensional LC as an on-line desalting tool allowing peptide identification directly from MS-unfriendly HPLC methods. Journal of Pharmaceutical and Biomedical Analysis 2017, 137, 139–145.
• Petersson P, Haselmann K, Buckenmaier S. Multiple heart-cutting 2D-LC-MS: towards real-time determination of related impurities of biopharmaceuticals in salt-based separation methods. Journal of Chromatography A 2016, 1468, 95–101.
• Suresh BCV, Ravindra G. Agilent AdvanceBio Desalting-RP Cartridges for Online Desalting in 2D-LC/MS mAb Analysis. Agilent Technologies Application Note 5991-7066EN, 2016.
• Joshi AB, Rus E, Kirsch LE. The degradation pathways of glucagon in acidic solutions. International Journal of Pharmaceutics 2000, 203, 115–125.
• United States Pharmacopeia USP 39. Glucagon Monograph, 2016.