Characterization and quantitative determination of henagliflozin metabolites in humans

Henagliflozin is a highly specific inhibitor of sodium-glucose co-transporter-2 (SGLT2) proposed as a more efficient medication for type 2 diabetes mellitus (T2DM). In this work, henagliflozin metabolic profile was investigated in human plasma and urine samples using a newly developed high-performance liquid chromatography coupled with time-of-flight mass spectrometry (HPLC/Q-TOF MS) method. A total of 8 metabolites were observed, while the structures of four major metabolites, including M1 (O-deethylation metabolite), M5-1 (2-O-β-glucuronide conjugate), M5-2 (6-O-β-glucuronide conjugate), and M5-3 (3-O-β-glucuronide conjugate) were confirmed in our study after comparison with the reference standards. The principal henagliflozin metabolic pathways were identified as glucuronidation and O-deethylation in humans. The principal form of henagliflozin in human plasma was parent drug, followed by M5-1; while it was M5-3 and M5-1 in urine. Subsequently, an accurate and simple LC-MS/MS method was developed for simultaneously determine M5-1, M5-2, and M5-3 in human plasma. After optimization of this method, three M5 isomers were successfully separated and quantified using chromatography. Acetonitrile-induced protein precipitation method was adapted for extracting the analytes from human plasma. Separation was conducted using Gemini C₁₈ column under gradient elution with 5 mM aqueous ammonium acetate (A) and acetonitrile (B) mobile phases. Negative electrospray ionization was conducted using a selective reaction monitoring with the same transition of m/z 629→321 for detection of three M5 isomers. The method showed good linearities for M5-1, M5-2, and M5-3 within the range of 1.00-150 ng/mL, 0.500-75.0 ng/mL, and 1.00-150 ng/mL, respectively. Conclusively, the method has been applied successfully to assess phase I henagliflozin pharmacokinetics and pharmacodynamics and providing effective safety evaluations.