Abstract
There is a growing interest concerning the relevance of salivary cortisone levels in stress-related research. However, studies investigating morning patterns and day-to-day variability of cortisone versus cortisol levels are lacking. Cortisol and cortisone analysis by liquid chromatography-tandem mass spectroscopy (LC-MS/MS) has been widely used for routine laboratory measurements in the last years. The aim of this study was to develop an ultra-performance LC-MS/MS method for the simultaneous quantification of salivary cortisol and cortisone levels for assessing the temporal variability of these hormones. Saliva samples were collected from 18 healthy volunteers at 0, 15, and 30 min after awakening on each day for 1 week and analysed with the newly developed method. We used a novel atmospheric pressure ionization source, which resulted in high sensitivity and specificity for both cortisol and cortisone as well as higher peak values and signal-to-noise ratio as compared with the electrospray ionization source. Cortisone showed similar morning patterns as cortisol: a 25% and 49% increase in levels at 15 and 30 min after awakening, respectively. Most cortisone indices showed somewhat lower day-to-day variability and were less affected by state-related covariates. We recommend further exploration of the potential of salivary cortisone as a biomarker in stress-related research.
Highlights
Cortisol is a steroid hormone produced by the hypothalamic-pituitary-adrenal (HPA) axis, one of the main components in individuals’ response to stressors
ultra-performance LC (UPLC)-MS/MS method was developed by optimising the following segments: 1) MS conditions, 2) chromatographic conditions, and 3) sample preparation
Use of electrospray ionisation (ESI) source resulted in higher limit of detection (LOD) (100 pg/mL for cortisol and 500 pg/mL for cortisone) and limit of quantification (LOQ) (500 pg/mL for cortisol and 1 ng/mL for cortisone) compared to those obtained using the new UniSpray ionization (USI) source (LOD = 5 pg/mL for both compounds, LOQ = 10 pg/mL for cortisol and LOQ = 50 pg/mL for cortisone)
Summary
Cortisol is a steroid hormone produced by the hypothalamic-pituitary-adrenal (HPA) axis, one of the main components in individuals’ response to stressors. Salivary CAR assessment has www.nature.com/scientificreports several advantages because of its simplicity and non-invasiveness, which allows for easy self-performed collection of samples at multiple times during the day It is the sampling method of choice in epidemiological studies[9]. There are no established norms for CAR, for neither salivary cortisol level immediately after awakening nor peak levels released 30 min after awakening The reason for this is mainly the relatively high day-to-day variability of CAR10 as well as numerous confounding factors, such as age, sex, awakening time, effect of light, weekday versus weekend collection and participant adherence[11]. There is a number of developed methods for assessing salivary cortisol level with LC-MS/MS in the literature, mostly using the electrospray ionisation (ESI) source[13,18]
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