UPLC-MS/MS simultaneous quantification of urinary circadian rhythm hormones and related metabolites: Application to air traffic controllers

•The analytes of interest were efficiently eluted by an optimized solid phase extraction strategy.•Nine protential circadian rhythm hormones have been simultaneous monitorted using a SPE-UPLC-MS/MS methodin in human urine.•A clear correlation between melatonin metabolites and endogenous metabolites upstream and downstream of cortisol has been investigated in human urine. Civil aviation flight crew and civil aviation air traffic controllers are prone to circadian rhythm abnormalities, which can lead to a slew of other maladies. It could endanger people's health and provide a serious threat to the safety of civil aviation flights if it is not appropriately evaluated and addressed. Early detection of rhythm irregularities and prompt treatment for particular populations that are vulnerable to rhythm disorders are crucial for enhancing civil aviation safety. In general, monitoring of the classical circadian rhythm biomarkers (melatonin or cortisol) in plasma or saliva is an effective way to evaluate the rhythm status. Due to the challenging sample procedure and the trauma of plasma, urine sample testing has received an increasing amount of attention. While, urine circadian rhythm biomarkers have seldom been examined, and the relationship between urinary steroid hormones and melatonin is still poorly understood. In most cases, hormones are determined by immunoassays respectively, mainly enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). There are also reports describing the liquid chromatography with tandem mass spectrometry (LC-MS/MS) technique as a method of melatonin or few steroid hormones quantification, however, the simultaneous detection of multiple rhythmic hormones in human urine is rarely reported. For the quantification of the rhythmic hormones in human urine, an accurate approach using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was devised in this work. Nine endogenous hormones (melatonin, 6-hydroxymelatonin, 6-sulfatoxymelatonin, cortisol, corticosterone, cortisone, testosterone, epitestosterone and androsterone), in human overnight urine, were quantified after solid phase extraction (SPE). A reverse phase HSS C18 column was used for chromatographic separation with a 9-minute gradient elution and deuterated analogues of each analyte were applied as internal standards. This method was successfully applied to the analysis of 596 overnight urine samples (23:00–9:00) collected from 84 air traffic cont