Development and Validation of an Ultra‐Fast UPLC–MS/MS Method for Simultaneous Quantification of Lodenafil and Lodenafil Carbonate in Human Liver Microsomes: Depletion Rate and Metabolic Stability Assessment

ABSTRACT A rapid UPLC–MS/MS method was developed and validated for the simultaneous quantification of lodenafil (LDN) and its prodrug lodenafil carbonate (Helleva; LDC), a phosphodiesterase type 5 (PDE5) inhibitor developed by Cristália and approved by ANVISA for the treatment of erectile dysfunction in Brazil, in HLMs. Chromatographic separation was conducted utilizing Eclipse plus RP‐C18 (1.8 μm, 50 × 2.1 mm), under isocratic elution with 0.1% HCOOH in water and 0.1% HCOOH in ACN (30:70, v/v) at a 0.4 mL/min for 1.0 min. The method was linear from 1 to 4000 ng/mL ( R 2 = 0.9991; y = 0.2466x − 0.3277, 1/x weighting) for LDN and 1 to 4000 ng/mL ( R 2 = 0.9931; y = 5.5928x + 0.0311, 1/x weighting) for LDC. Substrate depletion of LDN in HLMs +NADPH yielded an in vitro t½ of 23.09 min and a Cl int of 35.11 mL/min/kg. LDC depletion in HLMs −NADPH yielded an in vitro t½ of 17.24 min and a Cl int of 47.02 mL/min/kg. LDC depletion proceeded at a comparable rate with and without NADPH. This finding is consistent with NADPH‐independent, esterase‐catalyzed hydrolysis of the carbonate linkage. Metabolic stability of the active moiety (LDN) and the depletion kinetics of the prodrug (LDC) were investigated.