背景:长期睡眠状态对人的身心健康有重要影响。具有昼夜节律的褪黑素(MEL)和皮质醇被认为是潜在的睡眠生物标志物。考虑到MEL和皮质醇的快速代谢,它们的主要代谢产物可以作为替代指标,显示出更高的稳定性和可靠性。然而,目前还缺乏研究开发同时定量MEL的方法,皮质醇和它们在头发中的代谢产物。
目的:本研究旨在开发一种同时定量F,MEL及其主要代谢物(可的松;N-乙酰血清素,NAS;6-羟基褪黑素,6-O-MEL和6-磺基褪黑素,基于高效液相色谱-串联质谱的人发中S-O-MEL),然后探索生物标志物含量与睡眠状态之间的关系。
方法:在约27°C下,在1mL甲醇中从20毫克头发中提取分析物,然后在95%甲醇和5%5mM乙酸铵的流动相中分析,并用正离子模式的电喷雾电离源识别。从65名本科生中收集了最接近头皮的头发样本。睡眠状态是根据匹兹堡睡眠质量指数的参与者得分来测量的,Epworth嗜睡量表和晨曦/均匀性问卷。
结果:该方法在MEL的0.1-1000pg/mg范围内显示出良好的线性,相关系数的平方>0.99,NAS为0.4-1000pg/mg,对于6-O-MEL,1.0-1000pg/mg,S-O-MEL为1.0-1000pg/mg,皮质醇为0.5-1000pg/mg,可的松为1.0-1000pg/mg。它显示了六种分析物的检测限范围为0.05至0.3pg/mg,定量限范围为0.1至1.0pg/mg。日间和日间变异系数<20%。除S-O-MEL外,可在天然头发样品中检测到化合物。MEL的平均浓度为0.18pg/mg,对于NAS,3.5pg/mg,对于6-O-MEL,为3.8pg/mg,可的松为20.0pg/mg,皮质醇为2.8pg/mg。人群分析显示,头发可的松与睡眠质量之间存在正相关。
结论:本研究开发了一种同时定量MEL的LC-MS/MS方法,NAS,6-O-MEL,人类头发中的可的松和皮质醇。头发可的松可能是长期睡眠状态的有希望的生物标志物。
BACKGROUND: The long-term sleep state has an important influence on one\'s physical and mental health. Melatonin (MEL) and cortisol with circadian rhythm are deemed to be potential sleep biomarkers. Considering the rapid metabolism of MEL and cortisol, their main metabolites could be alternative indicators showing higher stability and reliability. However, there is short of research developing the method for simultaneous quantification of MEL, cortisol and their metabolites in hair.
OBJECTIVE: This study aimed to develop a method for the simultaneous quantification of F, MEL and their main metabolites (cortisone; N-acetyl-serotonin, NAS; 6-hydroxymelatonin, 6-O-MEL and 6-sulfatoxymelatonin, S-O-MEL) in human hair based on high-performance liquid chromatography tandem mass spectrometry method, and then explore the relationship between the biomarkers\' contents and sleep state.
METHODS: Analytes were extracted from 20-mg hair in 1 mL methanol at about 27°C, and then analyzed in a mobile phase of 95% methanol and 5% 5 mM ammonium acetate, and identified with an electrospray ionization source in positive ion mode. Hair samples closest to the scalp were collected from 65 undergraduates. Sleep state was measured based on participants\' scores of the Pittsburgh Sleep Quality Index, the Epworth Sleepiness Scale and the Morningness/Eveningness Questionnaire.
RESULTS: The method showed good linearity with the square of correlation coefficient > 0.99 at the ranges of 0.1-1000 pg/mg for MEL, 0.4-1000 pg/mg for NAS, 1.0-1000 pg/mg for 6-O-MEL, 1.0-1000 pg/mg for S-O-MEL, 0.5-1000 pg/mg for cortisol and 1.0-1000 pg/mg for cortisone. It showed the limit of detection ranged from 0.05 to 0.3 pg/mg and the limit of quantification ranged between 0.1 and 1.0 pg/mg for the six analytes. The inter- and intra-day coefficients of variation were < 20%. The compounds could be detected in natural hair samples except for S-O-MEL. The average concentration was 0.18 pg/mg for MEL, 3.5 pg/mg for NAS, 3.8 pg/mg for 6-O-MEL, 20.0 pg/mg for cortisone and 2.8 pg/mg for cortisol. The population analysis revealed that there was positive association between hair cortisone and sleep quality.
CONCLUSIONS: This study had developed an LC-MS/MS method for simultaneous quantification of MEL, NAS, 6-O-MEL, cortisone and cortisol in human hair. Hair cortisone might be a promising biomarker of long-term sleep state.