Melatonin (N-acetyl-5-methoxytryptamine) is implicated in physiologic changes related to light-dark cycles and has been recently found to display antioxidant properties. It is known that the reaction of melatonin with certain reactive oxygen and nitrogen species, such as hydrogen peroxide and singlet oxygen, produces N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK). We report herein on the development of a new liquid chromatography/tandem mass spectrometry (LC/ESI/MS-MS) assay to quantitatively determine melatonin and AFMK. The stable isotopic internal standard of melatonin-D3 was synthesized by the reaction of 5-methoxytryptamine with deuterated acetyl chloride (CD3COCl). Labeled AFMK (AFMK-D3) was obtained after photooxidation of melatonin-D3. The predominant ion [M + H]+ in the full scan mass spectra of melatonin, melatonin-D3, AFMK and AFMK-D3 were located, respectively, at m/z = 233, 236, 265 and 268. The collision-induced dissociation of the molecules revealed a predominant fragment at m/z = 174 for melatonin and melatonin-D3 (loss of the N-acetyl group), and at m/z = 178 for AFMK and AFMK-D3 (loss of both the N-acetyl and the N-formyl groups). The m/z transitions from 233 to 174 (melatonin), from 236 to 174 (melatonin-D3), from 265 to 178 (AFMK), and from 268 to 178 (AFMK-D3) were therefore chosen for the multiple reaction monitoring detection experiments, ensuring a high specificity and an accurate quantification of melatonin and AFMK in human plasma.

中文翻译：

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使用在线液相色谱-电喷雾串联质谱系统合成褪黑激素及其代谢产物N1-乙酰基-N2-甲酰基-5-甲氧基尿嘧啶的内部标记标准品，以对其进行定量。

褪黑激素（N-乙酰基-5-甲氧基色胺）涉及与明暗循环有关的生理变化，最近发现它具有抗氧化特性。众所周知，褪黑激素与某些活性氧和氮物种（例如过氧化氢和单线态氧）的反应会生成N1-乙酰基-N2-甲酰基-5-甲氧基尿嘧啶（AFMK）。我们在这里报告新的液相色谱/串联质谱分析（LC / ESI / MS-MS）测定法的发展，以定量测定褪黑激素和AFMK。通过5-甲氧基色胺与氘代乙酰氯（CD3COCl）的反应合成了褪黑激素-D3稳定的同位素内标。褪黑激素-D3光氧化后获得标记的AFMK（AFMK-D3）。褪黑素，褪黑素-D3的全扫描质谱图中的主要离子[M + H] + AFMK和AFMK-D3分别位于m / z = 233、236、265和268。碰撞诱导的分子解离显示褪黑激素和褪黑素-D3的主要片段位于m / z = 174（ （N-乙酰基）和AFMK和AFMK-D3的m / z = 178（N-乙酰基和N-甲酰基均丢失）。因此，选择m / z从233转变为174（褪黑激素），从236转变为174（褪黑激素-D3），从265转变为178（AFMK），从268转变为178（AFMK-D3）进行多反应监测检测实验，以确保人类血浆中褪黑激素和AFMK的高特异性和准确定量。而AFMK和AFMK-D3的m / z = 178（N-乙酰基和N-甲酰基均丢失）。因此，选择m / z从233转变为174（褪黑激素），从236转变为174（褪黑激素-D3），从265转变为178（AFMK），从268转变为178（AFMK-D3）进行多反应监测检测实验，以确保人类血浆中褪黑激素和AFMK的高特异性和准确定量。而AFMK和AFMK-D3的m / z = 178（N-乙酰基和N-甲酰基均丢失）。因此，选择m / z从233转变为174（褪黑激素），从236转变为174（褪黑激素-D3），从265转变为178（AFMK），从268转变为178（AFMK-D3）进行多反应监测检测实验，以确保人类血浆中褪黑激素和AFMK的高特异性和准确定量。