A strategy for assessing peak purity of pharmaceutical peptides in reversed-phase chromatography methods using two-dimensional liquid chromatography coupled to mass spectrometry. Part I: Selection of columns and mobile phases

• Published in: Journal of Chromatography A 2023-03, Vol.1693, p.463874, Article 463874
• Main Authors: Petersson, Patrik, Buckenmaier, Stephan, Euerby, Melvin R., Stoll, Dwight R.
• Format: Article
• Language: English
• Subjects: 2D-RPC-MS; Mobile phase; Peak purity; Peptides; Stationary phase
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2023.463874

•2D-RPC-MS strategy for main peak purity analysis of pharmaceutical peptides.•Selection of columns / mobile phases with complementary selectivity in 1D and 2D.•2D-LC allows the determination of isomers that cannot be differentiated by LC-MS.•Results support previously reported peptide RPC column characterisation protocol.•TFA found to reduce selectivity differences for investigated peptides / columns. The current study describes the development of a 2D-LC-MS-based strategy for assessing main peak purity in the analysis of pharmaceutical peptides. The focus is on 2D-LC using reversed-phase (RP) separations in both dimensions, and particularly peptide isomer selectivity, since compounds with the same mass to charge ratio are not readily differentiated by mass spectrometry and therefore must be separated chromatographically. Initially, 30 column / mobile phase combinations were evaluated for both general separation performance (i.e., selectivity and peak shape) and isomer selectivity using forcibly degraded peptide samples and mixtures of synthetic diastereomers. A ranking of more than 300 UV and MS chromatograms suggests that when developing a new method, screening a set of four columns and four volatile mobile phases with differing characteristics should be adequate to both cover the selectivity space, and yield good separation performance. When 2D-LC-MS is to be used to evaluate peak purity for a new method, our results show that a second-dimension separation comprising a C8/C18 column possessing no ionic functionality, and an acetic acid / ammonium acetate mobile phase buffered at pH 5, provides good selectivity at 25 °C for peptide isomers with a MW 10 kDa). In the current dataset which also contain smaller peptides it received the highest ranking for 40% of the column and mobile phase combinations due to better selectivity and/or peak shape. The r