Validation of a UPLC–MS/MS method for the simultaneous determination of E6005, a phosphodiesterase 4 inhibitor, and its metabolite in human plasma

• A bioanalytical method of E6005 and its metabolite in human plasma is reported.
• The method was developed using UPLC–MS/MS with the LLOQ of 1 ng/mL.
• The validation study demonstrated that the method is simple and reproducible.
• Stability including conversion was thoroughly assessed.
• The validated method was successfully applied to support clinical trials.

E6005, a novel phosphodiesterase 4 inhibitor, is currently under clinical development for the treatment of atopic dermatitis. As ER-392710 (M11), a hydrolyzed metabolite, is a main metabolite, a simultaneous assay method for quantification of E6005 and M11 in human plasma has been developed and validated using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS). E6005, M11, and each deuterium-labeled compound used as internal standard were extracted from 100 μL human plasma by solid phase extraction then chromatographed on an Acquity UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm) under gradient elution. The analytes were detected by selected reaction monitoring in the positive ion mode with the mass transition of m/z 473.1/163.0 and m/z 459.1/149.0 for E6005 and M11, respectively. E6005 and M11 were quantifiable ranging from 1 to 200 ng/mL with no carryover. Accuracy and precision in intra- and inter-batch reproducibility assays were within the acceptance criteria recommended by the regulatory bioanalytical guidelines. Various stability assessments including possible conversion of E6005 to M11 were thoroughly performed to demonstrate the stability of E6005 and M11 in human blood and plasma. The method was successfully applied to support clinical trials.