On-line H/D exchange LC–MS strategy for structural elucidation of pharmaceutical impurities

Structural elucidation of pharmaceutical impurities in drug substances and drug products is an important task in pharmaceutical analysis in various phases of drug development. Liquid chromatography–mass spectrometry (LC–MS) technologies play a key role in this task owing to their general attributes of superior selectivity, sensitivity and speed. Full scan and product ion scan analysis, providing molecular weight information and fragmentation data, respectively, offer rich structural information and allow proposal of candidate structures rather quickly. However, these proposed structures often lack certainty especially when dealing with structural isomers. On-line hydrogen/deuterium (H/D) exchange by LC–MS using D2O as the mobile phase component is a powerful tool for identifying active hydrogen atoms, thus constituting a simple strategy for distinguishing between isomeric structures which are sometimes difficult by product ion spectral data or accurate mass data. This review describes the typical experimental setup we use routinely in the laboratories for performing H/D exchange LC–MS experiments in conjunction with representative applications of the strategy in structural elucidation of pharmaceutical impurities.