Application of 2D-LC-MS for analysis of pharmaceutical peptides (Patrik Petersson, MDCW 2025)

🎤 Presenter: Patrik Petersson (Ferring Pharmaceuticals A/S, 2770 Kastrup, Denmark)

💾 PDF presentation

💡 Book in your calendar: 17th Multidimensional Chromatography Workshop (MDCW) 13 - 15. January 2026

Abstract

This contribution focus on peptide purity methods, i.e. RPC methods for the determination of peptide related impurities present in pharmaceutical peptides. There is a common misperception that peptides should be analyzed with TFA based eluents only. Salt based eluents, however, often provide both better peak shape and better selectivity something that we will exemplify. 2D-LC-MS allow almost real time MS data for salt-based eluents. Consequently, 2D-LC-MS is an important tool for the identification of peptide related impurities.

Irrespective if volatile or salt-based eluents are used 2D-LC-MS probably is the best technique for assessing if impurities are co-eluting with the main peak, so-called peak purity analysis. A strategy for 2D-LC-MS-based peak purity analysis of pharmaceutical peptides will be presented. Our focus is on 2D-LC separations using reversed-phase separations in both dimensions, and particularly isomer selectivity, since isobaric compounds are not readily distinguished by MS and therefore must be separated chromatographically.

One problem with peptide analysis is that very shallow gradients typically are required and that peptides respond strongly to small fluctuations in organic modifier and temperature resulting. This may result in fluctuating 1D retention times and difficulties to capture peaks of interest for 2D analysis – a moving target. A solution to this problem will be presented. We will also show how 2D-LC-MS analysis can result in false identification of oxidation products and how this can be addressed.