Investigation of nirmatrelvir epimerization occurred in analytical sample solution by using high resolution LC-MS, NMR, and hydrogen/deuterium exchange study.

During the related substances testing of nirmatrelvir, an unknown peak was observed and the level of the peak increased over time during the storage of the sample solution. By using a strategy including LC-PDA/UV-MS analysis, the degradant was rapidly identified as an epimer of nirmatrelvir, a solution degradation product that is caused by the trace amount of alkaline impurities leaching from the glass HPLC vials. In addition, by using hydrogen/deuterium exchange NMR spectroscopy analysis, the epimerization position was determined to be the carbon α to the adjacent cyano group. Further investigation indicated that the occurrence of the solution degradation can be suppressed when the glass HPLC vials were replaced by plastic HPLC or mass spectrometric grade vials.