Determination of urinary cortisol, cortisone and 6-sulfatoxymelatonin using dilute and shoot ultra-high pressure liquid chromatography–tandem mass spectrometry

•UHPLC–MS/MS method was developed for analysis of melatonin and cortisol.•We report fast and sensitive dilute and shoot method.•Our method allows for simultaneous analysis of both robust circadian markers.•The total run time of 6 min with compounds eluting within 3 min was obtained.•Method feasible for large multi-scale studies with high throughput.

Human sleep is a natural part of every individual's life. Clear relationship between sleep and endocrine system has been already established. In particular, melatonin and cortisol are known to affect and regulate sleep/wake patterns. Here we report the development of an ultra-high pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for simultaneous measurement of 6-sulfatoxymelatonin (MT6s), cortisol and cortisone in urine. A separate method was developed for measurement of creatinine in urine. These levels were used to normalise the levels of analytes. First void morning urine samples were collected from 24 healthy volunteers. Samples were diluted 1:1 in water prior to injection onto reversed-phase C18 column and analysed using UHPLC–MS/MS method. Linear calibrations were obtained for all analytes with correlation coefficient in the range 0.998–0.999. The observed concentration was found to be in the range 92–105% for cortisol, 92–107% for cortisone and between 93 and 120% for MT6s of the reference levels. The total run time of 6 min with all peaks of interest eluting within 3 min was obtained. This demonstrates the feasibility of utilising the method for large multi-scale studies, where high throughput is required for studying the circadian rhythm of melatonin and cortisol secretion. These hormones play significant role in circadian rhythm and sleep/wake cycle; therefore it is important to monitor the levels of these endocrine markers in individuals suffering from sleep disorders. It is also beneficial with clinical applications to analyse melatonin and cortisol simultaneously in order to assess their interrelationships of these substances, such as their effect on diurnal rhythm and sleep.