Development of radioactive tracing coupled with LC/MS-IT-TOF methodology for the discovery and identification of diaveridine metabolites in pigs.

We developed a sensitive and reliable method by coupling radiotracing with LC/MS-IT-TOF to identify diaveridine metabolites. Tritium-labeled diaveridine was orally administered to pigs and their organs, blood, bile, and excreta were collected. Under optimized conditions, radioactive recovery was >90% and the highest numbers of metabolites were detected. MCX-based solid-phase extraction was conducted for urine, plasma, and bile purification. Methanol-chloroform 1:1 (v/v), methanol-chloroform 6:1 (v/v), methanol, methanol-chloroform 1:1 (v/v), and methanol were used as solvents to extract feces, liver, kidney, fat and muscle, respectively. The method validation confirmed satisfactory 3H-H exchange efficiency (<5%), chromatographic column efficiency (≥97.5%), LOQ (10.73 µg/kg), and analytical accuracy (97.6-107.8%) and precision (RSD < 5%). Moreover, novel in vivo metabolites were detected in the pigs, including D2 (3'-desmethyl-diaveridine monoglucuronide), D3 (diaveridine monoglucuronide). Hence, the analytical method developed herein lays an empirical foundation for further systematic studies of the diaveridine metabolism.