Unraveling the bridging region of Lenacapavir atropisomers by two-dimensional liquid chromatography

Atropisomers are stereoisomers that arise from restricted bond rotation and undergo dynamic conformational change depending on time and temperature. The interconversion of atropisomers could result in a non-baseline "plateau" or "bridging area" between the isomer peaks in liquid chromatography (LC), which diminishes the chromatographic resolution and reduces the assurance of the analyte's purity. Conventional one-dimensional LC (1D-LC) separation relies on one or two major interactions among the analyte, mobile and stationary phase in an analytical column, which may not be sufficient for deciphering the bridging area. In contrast, two-dimensional LC (2D-LC) leverages orthogonal separation mechanisms in two dimensions, significantly enhancing peak capacity to resolve co-eluting impurities and ensure peak purity. Herein, we present a comprehensive 2D-LC study to investigate the on-column interconversion of Lenacapavir (LEN), which exists as a mixture of two atropisomers. Firstly, different columns, temperature, mobile phase pH, and cation additives were investigated using 1D-LC to assess their potential effects toward on-column interconversion of LEN atropisomers. The bridging area of LEN atropisomers was then unraveled using the heart-cutting mode in 2D-LC, presenting varying ratios for the two atropisomers across the retention time window of LEN. Furthermore, orthogonal conditions for the second dimension were developed to disentangle possible impurities in the bridging area using a regioisomer as surrogate. Lastly, and most importantly, by implementing the developed second-dimension methods, a co-eluting regioisomer of LEN can be separated using 2D-LC. With the aid of high-resolution mass spectrometry (HRMS), we were able to distinguish the co-eluting regioisomer down to 0.05 % (w/w).