Accurate quantification of EVT201 and its two metabolites in human urine using UHPLC-MS/MS method with solid phase extraction

A sensitive and accurate ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of EVT201 and its two metabolites, Ro46-1927 and Ro18-5528, in human urine. Different sample preparation methods were compared, and solid-phase extraction (SPE) was finally employed. Separation conditions and mass spectrometry parameters were optimized to achieve complete separation and enough sensitivity. Finally, the three analytes were separated on an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with a gradient mobile phase. The gradient elution consisted of acetonitrile (containing 0.1% formic acid) and 10 mM ammonium formate (containing 1% acetonitrile and 0.1% formic acid) at a flow rate of 0.50 mL/min. Detection was performed on an electrospray ionization (ESI) source in positive mode with multiple reaction monitoring (MRM). The method was fully validated in accordance with the bioanalysis guidance in Chinese Pharmacopoeia. It showed satisfying linearity, accuracy, and precision in the range of 0.2–200 ng/mL for all the three analytes. The mean extraction recoveries were 85.2%, 65.6%, 87.9%, and 86.4% for EVT201, Ro46-1927, Ro18-5528, and the IS, respectively. The method was successfully applied to the bioanalysis of 833 urine samples to determine the concentration of EVT201 and its two metabolites simultaneously.