Rapid and sensitive liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of yonkenafil and its major metabolites in rat plasma

Yonkenafil is a promising drug for treatment of male erectile dysfunction. Previous studies showed that the piperazine‐N,N’‐deethylation metabolite, piperazine‐N‐deethylation metabolite, and piperazine‐N‐deethylation‐N,N’‐deethylation metabolite were the major metabolites of yonkenafil after extensive metabolism. We developed a sensitive and selective method for the simultaneous quantification of yonkenafil and its major metabolites using high‐throughput liquid chromatography with tandem mass spectrometry. Analytes and internal standard were extracted from a small quantity of plasma (50 μL) using liquid–liquid extraction with diethyl ether/dichloromethane (60:40, v/v), and the baseline separation was achieved on Zorbax SB‐C₁₈ column using ammonia/water/methanol (0.2:20:80, v/v/v) as the mobile phase. The assay was performed with an electrospray positive ionization mass spectrometry through the multiple‐reaction monitoring mode within 2 min. Calibration curve of the method was linear within the range of 1.00–1000 ng/mL for all the analytes with the intra‐ and interday precisions of 4.0–5.2 and 4.0–5.3% for yonkenafil, 3.1–4.9 and 3.1–5.2% for the piperazine‐N,N’‐deethylation metabolite, 4.8–6.8 and 4.8–7.3% for the piperazine‐N‐deethylation metabolite, and 2.9–6.1 and 5.4–6.3% for the piperazine‐N‐deethylation‐N,N’‐deethylation metabolite, respectively. The recoveries were above 90% with low matrix effects. The validated assay was successfully applied to support a preclinical pharmacokinetic study in six rats using a single oral dose of yonkenafil (8 mg/kg).