Evaluation of vardenafil's metabolic stability in human liver microsomes through ultra-fast UPLC-MS/MS for quantitative analysis, including assessments of greenness, ADME properties, DEREK alerts, and metabolic lability

Abstract

Vardenafil is a highly potent and selective phosphodiesterase 5 (PDE5) inhibitor that was well-tolerated in men with erectile dysfunction. A UPLC-MS/MS method was developed to quantify VRF in human liver microsomes (HLMs) and evaluate its in vitro metabolic stability. The method demonstrated good greenness, as indicated by the AGSA (65.28) score. P450 and DEREK software were used to assess VRF's metabolic lability and chemical structure alerts. The linear range was 1–4000 ng mL⁻¹, with a runtime of 1 min. VRF and tadalafil (internal standard) were chromatographically resolved on an Eclipse Plus 1.8 µm C8 column (50 mm × 2.1 mm) using a mobile phase of 0.1% HCOOH in water (60%, pH 3.2) and acetonitrile (40%). The intra- and inter-day precision and accuracy ranged from −3.17% to 8.0% and −4.99% to 7.25%, respectively. The in vitro half-life of VRF was 21.91 min, with a moderate intrinsic clearance of 37.0 mL min⁻¹ kg⁻¹. In silico studies indicate that minor changes to the 4-ethyl piperazine ring (98%) could enhance the metabolic stability and safety of upcoming VRF derivatives.