An artifactual solution degradant of pregabalin due to adduct formation with acetonitrile catalyzed by alkaline impurities during HPLC sample preparation.

During the HPLC related substances testing of pregabalin API, an unknown peak was observed at a level exceeding the identification threshold. Preliminary investigation revealed that this impurity is not a process impurity but rather an artifactual solution degradant or "ghost peak" during the HPLC analysis. By using a strategy that combines LC-PDA/UV-MS with mechanism-based stress studies, the unknown peak was rapidly identified as a covalent adduct formed between pregabalin and acetonitrile (the latter is a component of the HPLC sample diluent), which is structurally an ethylamidine derivative of pregabalin. It appeared that the formation of this solution degradant was catalyzed by alkaline impurities during the sample preparation. This plausible mechanism was verified by a mechanism-based forced degradation study, in which a base was added into the sample diluent and consequently, the pregabalin-acetonitrile adduct was produced extremely efficiently at a level of ˜92%. Subsequently, the structure of the solution degradant was confirmed as an ethylamidine derivative of pregabalin through characterization by 1D and 2D NMR; the formation of the ethylamidine moiety is apparently via a nucleophilic attack on the cyano group of acetonitrile by the amino group of pregabalin. Due to the extensive presence of primary and secondary amine moieties in drug substances, this kind of artifactual solution degradation would likely occur during the sample preparations of these amine drugs in their HPLC analyses. In a GMP environment, such an event would trigger undesirable out-of-specification (OOS) investigations. The results of this study should help resolve such OOS investigations or prevent their happening from the very beginning. Furthermore, the somewhat surprising finding of the rather facile reaction that produces the ethylamidine moiety using simple alkylnitrile reagents, such as acetonitrile, may be of practical value in the synthesis of alkylamidines.