Analytical Artifact Due to Residual HCN in Acetonitrile: Identification and Control Strategies

Mismatch in the potency from quantitative ¹H NMR (∼96%) and the calculated potency (∼94%) of an aldehyde intermediate led to the investigation of an unknown impurity peak observed in the chromatography. The HR-MS/MS analysis of the unknown impurity suggested it to be the cyanohydrin derivative of the corresponding aldehyde intermediate with the addition of ∼27 amu. Further investigation was performed using analogous 3-methyl iso-nicotinaldehyde as a model compound. A postcolumn hydrogen to deuterium exchange (H/D exchange) experiment further supported the proposed impurity structure as cyanohydrin. The source of HCN for the possible generation of this impurity was traced to certain brands of acetonitrile used duirng the analysis, where the presence of HCN as a contaminant was confirmed and quantified using ion chromatography. The aforementioned model compound was used to investigate the effect of other parameters like diluent composition, sample temperature and storage time, pH of the diluent, and duration of sonication, which impact the formation of such artifact impurity. Based on the results of all the experiments, mitigation strategies were proposed to avoid/control the formation of these impurities during the analytical processing such as use of methanol or HCN-free acetonitrile as a sample diluent, reduced composition of acetonitrile in the diluent, and use of freshly prepared solutions for injections to avoid longer storage time specially when certain sensitive substrates like aldehydes and ketones are analyzed. To evaluate if the formation of this impurity is limited to the compound of interest or if it is a common artifact peak for other similar compounds, various substrates involving aldehyde and ketone functional groups were analyzed under similar analytical conditions. The results indicated that aldehydes were more reactive than ketones, specifically the aldehydes containing a heterocyclic ring such as pyridine were prone to generate the cyanohydrin impurity.