Isolation and chemical structure elucidation of a new praziquantel degradant and BHT crosslinked impurity in a liquid finished drug product

During the stability test of a liquid finished drug product (topical liquid transdermal formulation), an unexpected unknown peak was observed in the High Performance Liquid Chromatography (HPLC) chromatogram. This finished drug product is used to control mixed infestations of ectoparasites, nematodes, and cestodes in cats. This topical liquid drug product contains three active pharmaceutical ingredients (APIs), esafoxolaner, eprinomectin, and praziquantel. The identity of any new unknown peak (above certain threshold) in any given finished product batch must be established to ensure that the unknown peak is correctly estimated against the parent compound (API). As there are three APIs in this product, an incorrect assignment of the origin and identity of any given impurity will cause either overestimation or underestimation of the impurity, including misleading information on safety. Therefore, the chemical structure and the correct origin/source(s) of this unknown peak need to be determined. Although High Resolution Mass Spectrometry (HRMS) provides valuable information for determining the structure of the impurity associated with this peak, its structure cannot be fully established by HRMS data alone. Hence, this impurity peak was isolated and purified from aged finished drug product stability samples by semi-prep HPLC. Its chemical structure was fully elucidated through Nuclear Magnetic Resonance (NMR) (¹H, ¹³C, and 2D). The results revealed that the impurity was formed through a cross-reaction between a praziquantel degradant and butylated hydroxytoluene (BHT), which is used as an antioxidant in the finished drug product. The experimental details and full structure elucidation data and related information of this unexpected new degradation product peak is reported in this paper. The mechanism of formation of this impurity is proposed based on its positively identified chemical structure. Based on an extensive literature search, it can be concluded that this is the first known report on the definitive identification of the chemical structure and formation mechanisms of this impurity.