Simultaneous Determination Of Melatonin And Related Hormones In Hair With Liquid Chromatography-tandem Mass Spectrometry

Long-term sleep state can have a direct impact on individual’s physical and mental health.Melatonin(MEL)and cortisol(F)with circadian rhythms are often deemed to be potential biomarkers of sleep state.However,MEL and F showed relatively higher variations due to their rapid metabolisms.The main metabolites of MEL and F,N-acetylserotonin(NAS),6-hydroxymelatonin(6-O-MEL),6-sulfatoxymelatonin(S-O-MEL)and cortisone(E)could become their alternative indicators because of the higher stability and reliability.In addition,the contents of the above compounds in the hair matrix are clearly more suitable for the assessment of long-term sleep state than fluid biomatrices.To date,there is short of the method for simultaneous quantification of MEL,F and their metabolites in hair.Therefore,the study aimed to develop a LC-MS/MS method for the simultaneous determination of these six substances in human hair and then investigate the correlation between the contents of these compounds in hair and long-term sleep state.In this study,the six compounds were extracted from 20-mg hair in 1 m L methanol at room temperature for 24 h,and then separated in a mobile phase with 95 %methanol and 5 % water containing 5 m M ammonium acetate.They were ionized in electrospray ionization(ESI)source in positive ion mode on LC-MS/MS system and identified in multiple reaction monitoring mode.The method showed good performances at the detection limits ranged from 0.05-0.3 pg/mg and the quantification limits ranged from 0.1-1.0 pg/mg and good linearity over a wide range with the square of correlation coefficients > 0.99.The method showed the inter-day intra-day coefficients of variation,recovery and stability met the validation requirements of the detection of the substances in hair.Sixty-five undergraduate students recruited in Shenzhen University self-reported their long-term sleep state with the Pittsburgh sleep quality index,Epworth sleepiness scale and morningness/eveningness questionnaire.The population analysis revealed that all compounds were detected in the natural hair samples except S-O-MEL,showing the mean concentration at 0.18 pg/mg for MEL,3.5 pg/mg for NAS,3.8 pg/mg for6-O-MEL,20.0 pg/mg for cortisone and 2.8 pg/mg for cortisol.There was a significantly positive correlation of cortisone with Pittsburgh sleep quality index.This suggests that hair cortisone might be a promising biomarker of long-term sleep state.