Investigation of nirmatrelvir epimerization occurred in analytical sample solution by using high resolution LC-MSn, 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 n 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.