Structure elucidation and quantification of the active pharmaceutical ingredient in a non-approved drug and in cat serum using QTRAP-MS/MS and ZenoTOF-MS/MS

Feline infectious peritonitis (FIP) is a fatal systemic disease in cats caused by a feline coronavirus (FCoV). Recent studies have shown that Xraphconn, an unapproved veterinary drug, has significantly increased survival rates in cats with FIP. However, the lack of transparent information regarding the chemical structure and quantity of the active pharmaceutical ingredient (API) necessitates a thorough investigation of this drug. In this study, the API from Xraphconn's plant-based matrix was successfully isolated and purified using weak cation-exchange solid-phase extraction (WCX-SPE). Preliminary structure elucidation was conducted using mass spectra simulations, multiple reaction monitoring-information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) scans from a 6500+ QTRAP-MS, and both collision-induced dissociation (CID) and electron-activated dissociation (EAD) fragmentation techniques with the ZenoTOF 7600. Conclusive structural identification of the Xraphconn API was achieved by ¹³C and ¹H nuclear magnetic resonance spectroscopy. A 4-minute RP HPLC-MS/MS method was optimized to achieve satisfactory separation of the API peak from an isobaric contaminant. The limit of detection (LOD) and limit of quantification (LOQ) were established at 2.11 nM and 6.39 nM in water, and 7.21 nM and 21.84 nM in plasma, respectively. The intra- and inter-day accuracy was found to range between 96 % and 105 %, with a coefficient of variation (CV%) of ≤ 5.51 %. The API content was determined to be 21.56 ± 0.98 mg per tablet employing ¹³C₅-GS-441524 as an internal standard to account for potential matrix effects. The API concentration was measured in plasma and serum samples collected at four different time points from six treated cats.