Department of Laboratory Medicine, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
Department of Endocrinology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
Clin Chem Lab Med. 2020 Jul 16;59(1):187-195. doi: 10.1515/cclm-2020-0455.
Regulators of circadian rhythm, including melatonin, influence fundamental biological processes. Measuring the melatonin metabolite 6-sulfatoxymelatonin in urine can estimate melatonin production. 6-sulfatoxymelatonin is mainly analyzed by immunoassays, but these methods are hampered by cross-reactivity and poor reproducibility when used to analyze small molecules. Therefore, we validated a high-throughput liquid chromatography with tandem mass spectrometry (LC-MS/MS) method to quantify 6-sulfatoxymelatonin in urine. We evaluated age-dependent 24-h excretion of 6-sulfatoxymelatonin into urine and the biological variation of urinary excretion in healthy individuals.
The online solid phase extraction method combined with LC-MS/MS was validated according to international guidelines, and used to measure the excretion of 6-sulfatoxymelatonin into urine of 240 healthy individuals. Biological variation of 6-sulfatoxymelatonin excretion was examined in 10 healthy individuals.
Urinary 6-sulfatoxymelatonin results were well within the validation criteria (interassay coefficient of variation: <5.4%, quantification limit: 0.2 nmol/L). There was an age-related decrease in 6-sulfatoxymelatonin excretion into 24-h urine [F(5, 234)=13.9; p<0.001]. Within-subject variation of 6-sulfatoxymelatonin was 39.2% in day urine, 15.1% in night urine, and 12.2% in 24-h urine. Between-subject variation was 39.1% in day urine, 37.9% in night urine, and 36.8% in 24-h urine.
This MS-based method enables straightforward, reproducible, and sensitive quantification of 6-sulfatoxymelatonin in urine. Urinary 6-sulfatoxymelatonin levels decreased with age. Biological variation of 6-sulfatoxymelatonin excretion into urine was high between subjects and lower within subjects, indicating that repeated measurements of 6-sulfatoxymelatonin in 24-h urine are needed in future studies.
生物钟调节因子，包括褪黑素，会影响基本的生物过程。通过测量尿液中的褪黑素代谢产物 6-硫酸褪黑素，可以评估褪黑素的生成情况。6-硫酸褪黑素主要通过免疫分析法进行分析，但这些方法在分析小分子物质时，由于交叉反应和重现性差而受到阻碍。因此，我们验证了一种用于定量尿液中 6-硫酸褪黑素的高通量液相色谱-串联质谱（LC-MS/MS）方法。我们评估了年龄相关的 24 小时尿液中 6-硫酸褪黑素的排泄情况，以及健康个体尿液排泄的生物学变异性。
根据国际指南对在线固相萃取方法与 LC-MS/MS 联合方法进行验证，并用于测量 240 名健康个体尿液中 6-硫酸褪黑素的排泄情况。在 10 名健康个体中检查了 6-硫酸褪黑素排泄的生物学变异性。
尿液中 6-硫酸褪黑素的结果完全符合验证标准（批内变异系数：<5.4%，定量下限：0.2 nmol/L）。6-硫酸褪黑素在 24 小时尿液中的排泄量与年龄相关，呈下降趋势[F(5, 234)=13.9;p<0.001]。6-硫酸褪黑素在日间尿液中的个体内变异性为 39.2%，夜间尿液为 15.1%，24 小时尿液为 12.2%。个体间变异性在日间尿液中为 39.1%，夜间尿液中为 37.9%，24 小时尿液中为 36.8%。
该基于 MS 的方法能够直接、可重复且灵敏地定量尿液中的 6-硫酸褪黑素。尿液中的 6-硫酸褪黑素水平随年龄增长而下降。尿液中 6-硫酸褪黑素排泄的个体间生物学变异性高，个体内变异性低，表明在未来的研究中需要对 24 小时尿液中的 6-硫酸褪黑素进行重复测量。
