Center for Drug Metabolism and Pharmacokinetics Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Anal Chim Acta. 2013 Jul 25;788:89-98. doi: 10.1016/j.aca.2013.06.022. Epub 2013 Jun 20.
Idiosyncratic reactions are one of the major causes of drug treatment limitations or market withdrawals, and likely involve the formation of reactive metabolites. Because of their unstable nature, reactive species are usually discovered as stable conjugates by glutathione (GSH) trapping rather than by direct detection. The GSH conjugates are then detected by neutral loss scanning of 129 Da or precursor ion scanning at m/z 272, but the conjugation sites can only be identified by comparison with reference standards. In the present study, the fragmentation behaviors of 52 GSH conjugates belonging to five structural classes (aliphatic, aromatic, benzylic, disulfide, and thioester) were investigated in both positive and negative electrospray ionization modes by high-resolution mass spectrometry with suitable collision energies such that the relative abundance of the parent ion was approximately 50% of the most abundant product ion. Several structural-diagnostic fragmentations were identified: aliphatic conjugates gave i/j-type ions upon cleavage of the C-S bond between the drug and GSH, and d/k-type ions formed by the cleavage of the cysteinyl C-S bond, with approximately equal intensity, in both positive and negative modes, whereas aromatic conjugates only possessed d/k-type ions, and benzylic conjugates primarily yielded i/j-type ions. Disulfide conjugates typically produced dehydrogenated GS(-) fragment ions ([i-2H]-type) in negative mode, and thioester conjugates displayed sequential losses of pyroglutamic acid and water ([e-H2O]-type) in positive mode. A fragmentation-based method was thus established to facilitate the discrimination of these five classes of GSH conjugates, thereby providing insight into the bioactivation mechanisms and supporting lead optimization.
个体反应是药物治疗受限或市场撤出的主要原因之一，可能涉及到反应性代谢物的形成。由于它们的不稳定性质，反应性物质通常通过谷胱甘肽 (GSH) 捕获被发现为稳定的轭合物，而不是通过直接检测。然后通过中性丢失扫描 129 Da 或在 m/z 272 处的前体离子扫描来检测 GSH 轭合物，但只能通过与参比标准比较来鉴定结合部位。在本研究中，通过高分辨率质谱法在正、负离子喷雾模式下，用适当的碰撞能量研究了 52 种属于五个结构类别（脂肪族、芳香族、苄基、二硫键和硫酯）的 GSH 轭合物的碎裂行为，使得母体离子的相对丰度约为最丰富产物离子的 50%。鉴定了几种结构诊断性碎裂：脂肪族轭合物在 GSH 与药物之间的 C-S 键断裂时产生 i/j-型离子，在正、负离子模式下均以相等的强度形成由半胱氨酸 C-S 键断裂产生的 d/k-型离子，而芳香族轭合物仅具有 d/k-型离子，苄基轭合物主要产生 i/j-型离子。二硫键轭合物通常在负离子模式下产生脱氢 GS(-)片段离子 ([i-2H]-型)，硫酯轭合物在正离子模式下连续失去焦谷氨酸和水 ([e-H2O]-型)。因此，建立了一种基于碎裂的方法来区分这五类 GSH 轭合物，从而深入了解生物活化机制，并支持先导化合物优化。
