Department of Pharmaceutical Candidate Optimization, Bristol Myers Squibb, Princeton, NJ, USA.
AB Sciex, Redwood City, CA, USA.
Rapid Commun Mass Spectrom. 2024 Oct 30;38(20):e9890. doi: 10.1002/rcm.9890.
This study focuses on the advantage of using the novel electron-activated dissociation (EAD) technology on the QTOF system for structural elucidation of conjugation metabolites. In drug metabolite identification, conceptual "boxes" are generally used to represent potential sites of modifications, which are proposed based on MS/MS data. Electron-activated dissociation (EAD) provides unique fragmentation patterns, potentially allowing for more precise localization of the metabolic modification sites compared to CID, particularly for conjugations.
Known compounds were incubated with rat liver microsomes in the presence of nicotinamide adenine dinucleotide phosphate (NADPH), uridine dihosphate-glucuronic acid (UDPGA), and glutathione. Conjugation metabolites were analyzed using the QTOF system. High-resolution MS/MS spectra were collected using EAD and CID fragmentations along with TOF MS full scan for tested drugs and metabolites. Fragmentation patterns were compared to evaluate their efficiency in structural elucidation.
Metabolite profiling identified conjugation metabolites (glucuronides and GSH adducts), using characteristic mass shifts. A comparison of EAD and CID fragmentation revealed EAD-specific fragments for most conjugates. EAD was able to break the relatively stable bonds on parent drug motifs while keeping relatively weak conjugation bonds intact, despite the generally low intensity of EAD. EAD effectively narrowed the conceptual "box" representing modification sites, providing more definitive information on conjugation sites and facilitating the structural elucidation of conjugated metabolites.
EAD is a powerful tool for metabolite profiling in drug development, particularly for identifying conjugation sites. EAD-enabled MS/MS spectra offer a greater variety of signature fragments compared to CID, resulting in more comprehensive and unique structural information for metabolic modification analysis. Overall, EAD, complementary to CID, has the potential to narrow down potential modification sites, significantly enhancing the precision of conjugation metabolite structure elucidation.
本研究专注于在 QTOF 系统上使用新型电子激活解离（EAD）技术进行缀合代谢物结构阐明的优势。在药物代谢物鉴定中，通常使用概念“框”来表示潜在的修饰部位，这些部位是基于 MS/MS 数据提出的。电子激活解离（EAD）提供了独特的裂解模式，与 CID 相比，特别是对于缀合反应，可能更精确地定位代谢修饰部位。
将已知化合物与大鼠肝微粒体在烟酰胺腺嘌呤二核苷酸磷酸（NADPH）、尿苷二磷酸葡萄糖醛酸（UDPGA）和谷胱甘肽存在下孵育。使用 QTOF 系统分析缀合代谢物。使用 EAD 和 CID 碎裂以及 TOF MS 全扫描收集测试药物和代谢物的高分辨率 MS/MS 谱。比较裂解模式以评估它们在结构阐明方面的效率。
代谢物分析确定了缀合代谢物（葡萄糖醛酸苷和 GSH 加合物），使用特征质量位移。EAD 和 CID 碎裂的比较表明，大多数缀合物都有 EAD 特异性片段。EAD 能够打破母体药物结构中相对稳定的键，同时保持相对较弱的缀合键完整，尽管 EAD 的强度通常较低。EAD 有效地缩小了表示修饰部位的概念“框”，提供了更明确的缀合部位信息，促进了共轭代谢物的结构阐明。
EAD 是药物开发中代谢物分析的有力工具，特别是用于鉴定缀合部位。与 CID 相比，EAD 启用的 MS/MS 谱提供了更多种类的特征片段，为代谢修饰分析提供了更全面和独特的结构信息。总体而言，EAD 与 CID 互补，具有缩小潜在修饰部位的潜力，显著提高了缀合代谢物结构阐明的精度。
