液相色谱-串联质谱检测血清褪黑素方法建立和临床应用研究

Liquid chromatography-tandem mass spectrometry(LC-MS/MS)based assay for the quantification of melatonin in human serum

目的:建立液相色谱-串联质谱(LC-MS/MS)定量检测人血清褪黑素的检测方法。方法:方法学评价研究。收集2022年2月至2023年3月就诊于复旦大学附属中山医院心理医学科门诊,182例初诊睡眠障碍患者[男56例,女162例,年龄(45.51±16.31)岁]以及182名表观健康人群[男性87名,女性95名,年龄(48.55±11.93)岁],检测两组人群血清褪黑素水平。使用液相色谱质谱系统,采用色谱柱(2.1 mm×100 mm,1.8μm)进行分离。柱温35℃,流动相为0.1%的甲酸水溶液和纯乙腈,流速为0.4 ml/min梯度洗脱。以褐黑素-d4作为内标建立方法,进行方法学预验证,评价特异性和选择性、基质效应、携带污染、重复性。随后进行性能验证,评价定量下限、线性、精密度、回收率、稀释一致性和血清样本稳定性等参数。结果:褪黑素的最低定量限为1 pg/ml,检测线性范围为1~500 pg/ml(r=0.999)。日间和批内不精密度的变异系数(CV)为3.07%~6.86%,均符合小于15%的要求,加标回收率结果为105.91%~116.30%。睡眠障碍组血清褪黑素的水平明显低于健康对照组[2.00(1.00,3.28)]比[8.35(4.28,14.80)]pg/ml,P<0.001。结论:新建立的血清褪黑素LC-MS/MS检测方法分析性能符合要求,可用于检测血清中褪黑素含量。

Objective The aim of this study was to develop and validate a new liquid chromatography-tandem mass spectrometry(LC-MS/MS)assay for the quantification of melatonin in human serum.Methods We describe the performance and validation of melatonin by LC-MS/MS.182 serum samples from the patients diagnosed with Sleep disturbance who visited the Department of Psychiatry at Zhongshan Hospital affiliated to Fudan University from February 2022 to March 2023(56 males,162 females,mean age[45.51±16.31]years),as well as 182 healthy individuals were included(87 males,95 females,mean age[48.55±11.93]years).The two groups were used to assess the application of serum melatonin levels as a diagnostic indicator for sleep disorders(SDs).The liquid chromatography mass spectrometry(LC-MS)system with an chromatography column(2.1×100 mm,1.8μm)was used for separation.The column temperature was set at 35℃,as well as the mobile phase consisting of a 0.1%formic acid aqueous solution and pure acetonitrile.The flowing rate was set at 0.4 ml/min for gradient elution.The LC-MS/MS method was validated according to guidance documents,including the following parameters:specificity,selectivity,matrix effect,carryover contamination and reproducibility,lower limit of measuring interval,linearity,precision,recovery rate,dilution consistency,and serum sample stability.Then,it was subsequently employed to profile melatonin changes in Sleep disturbance.Results The lower limit of quantification for melatonin was 1 pg/ml,and the linear range of detection was 1 pg/ml to 500 pg/ml(r=0.999).The intra-day and intra-batch precision,expressed as the coefficient of variation(CV),was within the range from 3.07%to 6.86%,which met the requirement of less than 15%.The recovery rate of the spiked samples ranged from 105.91%to 116.30%.The level of serum melatonin in the sleep disturbance group was significantly lower than that in the healthy control group([2.00(1.00,3.28)]vs[8.35(4.28,14.80)]pg/ml,P<0.001).Conclusions The LC-MS/MS method we developed for the quantification of melatonin is clinical practicable.