(T1430-10-55) Manual 2D LC-MS for Peak Purity Assessment of Atropisomeric Compound

Purpose: To introduce a new manual 2D LC-MS process and provide a case study for the peak purity assessment of an atropisomeric compound.

Methods: PDA-facilitated peak purity assessment (PPA) is the most common approach to demonstrate adequate selectivity, which examines changes in the UV absorbance spectrum throughout the peak. However, the UV spectral PPA has a limitation¹ when: - impurities are present at much lower concentrations (absorbances) than the main compound, the spectra for the main compound and the impurity are identical or very similar, and the impurity is coeluted with the main compound with a retention profile that has the same shape and retention time as the main compound. The PPA using PDA for class 2 atropisomeric compound (as a mixture due to an axis of rotation) is even more challenging as on-column atropisomeric interconversion occurs. Two-dimensional (2D) liquid chromatography (LC) and MS can be used to address the above issue to evaluate peak purity². In addition, the 2D LC-MS offers an advantage in quantitation of the co-eluting impurities. The combination of two different separation mechanisms results in improved resolution and separation of components in complex samples, where the two sets of conditions are joined through a “fraction collecting” process. This process first separates components under the “normal” (or “nominal”) chromatographic conditions (1D, scaling to preparative LC method from analytical LC method), then collect the fractions of the peak(s) of interest, lyophilize the fractions, prepare fraction samples, and inject to the second separative system (2D) with UV and MS detectors. Conceptual view of the process is shown in the Figure 1.

Results: LEN (GS-6207) has two atropisomers and shows in the 1D LC method conditions as two peaks with a bridge -the raised baseline level between the atropisomer peaks indicating the on-column interconversion in 1D method conditions as shown in Figure 2. The two atropisomer peaks (LEN.1 and LEN. 2) have slight differences in the UV. The purity angle of the main component (LEN.1 and LEN.2) was less than the threshold, respectively. However, the purity curve is not below the threshold curve across especially in the bridging area. Thus, manual 2D LC-MS was applied to further confirm the peak purity. 1D LC method conditions was scaled up accordingly to collect the fractions from LEN.1 to LEN.2 including the bridging area. Each collected fraction is labeled in the preparative chromatogram. Fractions collected from fractions 1-6 are considered as LEN.1, Bridge 1 is fractions 7-8, Bridge 2 is fractions 9-10, and LEN.2 is fractions 11-19. The fractions were lyophilized to yield white solids. 2D LC-MS Method was developed using different column chemistry and mobile phases. A peak with m/z 950 was observed at about 7.6 min in the Bridge 1 Fraction (Figure 3).  Further 2D LC quantitation on the level of the m/z 950 peak in the sample is ND (0.014%). PDA-facilitated PPA has the limitation to detect the m/z 950 impurity from the main component because the impurity is present at much lower concentration than the main compound, though it has different UV.

Conclusion: The work demonstrates that the manual 2D LC-MS process is a powerful tool for peak purity assessment in the most complex sample - the class 2 atropisomeric compound in case Agilent 2D-LC system is not available. Using sequential chromatographic systems with orthogonal conditions and/or columns expands the separative ability of each system, offering superior overall selectivity that can separate coeluting peaks. Orthogonal detection systems, providing additional information about a sample in dedicated areas of the chromatogram–for an extra level of confidence.

References: 1. Dwight R. Stoll, Sarah C. Rutan C. J. Venkatramani; Peak Purity in Liquid Chromatography, Part I: Basic Concepts, Commercial Software, and Limitations; LCGC North America, Feb 2018, volume 36, Issue 2, pages: 100-110
2. Pascal Marillier, Neal Adams, Steven W. Baertschi, John M. Campbell, Chris Foti, Jucara Ribeiro Franca, Simon Hicks, Dorina Kotoni, Christian Laue, Stacey Marden, Liping Meng, Ana Claudia de Oliveira Santos, Mariah Ultramari, An Van Cleempoe, Chloe Wang, Todd Zelesky, Zongyun Huang; Liquid Chromatographic Peak Purity Assessments in Forced Degradation Studies: An Industry Perspective; LCGC International, Jan 2024, volume 1, Issue 1, pages: 22-31

Acknowledgements: Thank Simone Ellison for the help in the work and thank Jungjoo Yoon and Wendy for reviewing the poster.

Manual 2D LC-MS process

Two atropisomers of LEN and on-column conversion in 1D LC method conditions

2D LC-MS for checking the fractions and quantitation of impurity