Novel, rapid and precise RP-HPLC–DAD method was developed, validated and successfully applied for determination of metabolic changes of ethyl 5-(4-bromophenyl)-1-(3-(2-(2-hydroxybenzylidene)hydrazinyl)-3-oxopropyl)-2-methyl-1H-pyrrole-3-carboxylate (12b) in isolated rat hepatocytes. The analytes were detected by a simple DAD detector at 279 nm wavelength. A single-step extraction method was implemented to enable fast purification and extraction from cellular culture, resulting in a complete recovery. Thereafter, the method was adequately transferred to a LC–MS system for identification of unknown products. Additionally, network metabolism evaluation was performed to predict the structures of major metabolites with their isotope mass through BioTransformer 3.0. The data from the LC–MS analysis and the online server were compared for comprehensive identification. The results indicated formation of four metabolic products, obtained through processes of hydrolysis (12 and b), hydroxylation in the structure 12b (M1) and O-dealkylation (M2).

开发、验证并成功应用新型、快速、精确的 RP-HPLC-DAD 方法测定乙基 5-(4-溴苯基)-1-(3-(2-(2-羟基亚苯亚甲基)肼基)-3 的代谢变化离体大鼠肝细胞中的-氧代丙基)-2-甲基-1 H-吡咯-3-羧酸酯 ( 12b )。通过简单的 DAD 检测器在 279 nm 波长下检测分析物。采用单步提取方法可以从细胞培养物中快速纯化和提取，从而实现完全恢复。此后，该方法被充分转移到 LC-MS 系统中以鉴定未知产物。此外，还进行了网络代谢评估，通过 BioTransformer 3.0 预测主要代谢物的结构及其同位素质量。将 LC-MS 分析数据与在线服务器数据进行比较，进行全面鉴定。结果表明形成了四种代谢产物，通过水解（12和b）、结构12b中的羟基化（M1）和O-脱烷基化（M2）过程获得。