Laboratory of Clinical Pharmacy and Experimental Therapeutics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences.
Chem Pharm Bull (Tokyo). 2022;70(5):375-382. doi: 10.1248/cpb.c21-00982.
Evaluation of endogenous melatonin (MEL) secretion using its urinary metabolites is useful for the treatment of circadian rhythm sleep disorders. The primary melatonin metabolites excreted in the urine are 6-hydroxymelatonin (6-O-MEL) sulfate (S-O-MEL) and 6-O-MEL glucuronate, which result from sequential MEL metabolism by phases I and II drug metabolizing enzymes. To determine the accurate MEL secretion level, these urinary metabolites should be enzymatically deconjugated and converted into MEL. Furthermore, the use of LC-tandem mass spectrometry (LC-MS/MS) is preferable for the precision of this determination. Therefore, as part of our ongoing efforts to ultimately determine the level of MEL secretion, we herein aimed to develop an LC-MS/MS-based quantification method for 6-O-MEL and optimize deconjugation conditions. We determined the LC-MS/MS conditions of 6-O-MEL measurement and optimized the conditions of enzymatic reactions. The most efficient S-O-MEL deconjugation (102.1%) was achieved with Roche Glucuronidase/Arylsulfatase (from Helix pomatia) at 37 °C, pH-4.0 reaction buffer, and 60 min of reaction time. For human urine samples, the minimum amount of the enzyme required was 5944 units. Under these conditions, the accuracy and precision values of the 6-O-MEL determination (relative errors and standard deviation) were -3.60--0.47% and <6.80%, respectively. Finally, we analyzed the total amount of MEL metabolites excreted in 24-h urine samples; it was 6.70-11.28 µg in three subjects, which is comparable with the values reported till date. Thus, we have established a new method of measuring the total 6-O-MEL in human urine samples using an LC-MS/MS coupled with the prerequisite deconjugation reaction.
评估内源性褪黑素（MEL）的分泌情况，使用其尿液代谢物进行评估是治疗昼夜节律睡眠障碍的有效方法。尿液中主要排泄的 MEL 代谢物是 6-羟褪黑素（6-O-MEL）硫酸盐（S-O-MEL）和 6-O-MEL 葡糖苷酸，它们是由 I 相和 II 相药物代谢酶对 MEL 进行连续代谢产生的。为了确定准确的 MEL 分泌水平，这些尿代谢物应该进行酶解和转化为 MEL。此外，使用液相串联质谱（LC-MS/MS）可以提高该测定的精密度。因此，作为我们正在进行的努力的一部分，最终确定 MEL 分泌水平，我们旨在开发一种基于 LC-MS/MS 的 6-O-MEL 定量方法，并优化解偶联条件。我们确定了 6-O-MEL 测量的 LC-MS/MS 条件，并优化了酶反应条件。在 37°C、pH-4.0 反应缓冲液和 60 分钟的反应时间下，Roche Glucuronidase/Arylsulfatase（来自 Helix pomatia）实现了最有效的 S-O-MEL 解偶联（102.1%）。对于人尿样，所需的最小酶量为 5944 单位。在这些条件下，6-O-MEL 测定的准确度和精密度值（相对误差和标准偏差）分别为-3.60%至-0.47%和<6.80%。最后，我们分析了 24 小时尿样中排泄的 MEL 代谢物总量；在 3 个受试者中，其排泄量为 6.70-11.28μg，与迄今为止报道的值相当。因此，我们建立了一种新的方法，使用 LC-MS/MS 结合必需的解偶联反应来测量人尿样中的总 6-O-MEL。
