Di(2-ethylhexyl) phthalate (DEHP), the most widely used plasticizers in the world, has been regarded as an endocrine disrupting chemical with serious adverse health outcomes. Accumulating evidence strongly suggests that the undesirable biological effects of DEHP are meditated by its metabolites rather than itself. However, the metabolic footprints of DEHP in vivo are still unclear. Here we developed a click chemistry-assisted mass spectrometry (CC-MS) strategy for in-depth profiling DEHP metabolites in rats. An alkyne-modified DEHP analogue (alkyne-DEHP) was synthesized as a tracer for in vivo tracing, and a pair of MS probes (4-azido-nphenylbenzamide, 4-ANPA, and its deuterated reagent d₅-4-ANPA) were prepared to specifically label the alkyne-DEHP metabolites, and prominently improve their detection sensitivity and selectivity. Using the CC-MS strategy, we successfully screened 247 alkyne-DEHP metabolites from rat urine, feces, and serum, including many unrevealed metabolites, such as oxidized phthalate diester metabolites and glucuronides of phthalate monoester metabolites. The discovery of new DEHP metabolites provides additional insights for understanding the metabolism of DEHP, which may be beneficial in exploring the mechanism underlying DEHP induced-toxicity in the future.

邻苯二甲酸二（2-乙基己基）酯 (DEHP) 是世界上使用最广泛的增塑剂，被认为是一种内分泌干扰化学品，会对健康造成严重不良后果。越来越多的证据强烈表明，DEHP 的不良生物学效应是由其代谢物而非其自身引起的。然而，DEHP在体内的代谢足迹仍不清楚。在这里，我们开发了一种点击化学辅助质谱 (CC-MS) 策略，用于深入分析大鼠中的 DEHP 代谢物。合成了一种炔烃修饰的 DEHP 类似物 (alkyne-DEHP) 作为体内示踪剂，并合成了一对 MS 探针（4-叠氮基-nphenylbenzamide、4-ANPA 及其氘代试剂d ₅-4-ANPA）制备用于特异性标记炔烃-DEHP代谢物，显着提高其检测灵敏度和选择性。使用 CC-MS 策略，我们成功地从大鼠尿液、粪便和血清中筛选出 247 种炔烃-DEHP 代谢物，包括许多未揭示的代谢物，例如氧化的邻苯二甲酸二酯代谢物和邻苯二甲酸单酯代谢物的葡萄糖醛酸苷。新的 DEHP 代谢物的发现为理解 DEHP 的代谢提供了额外的见解，这可能有助于未来探索 DEHP 诱导毒性的潜在机制。