Development and evaluation of a liquid chromatography tandem mass spectrometry method for simultaneous determination of salivary melatonin, cortisol and testosterone
Introduction
Circadian disruption can have many possible health consequences [1], [2]. Laboratory studies of humans have shown that lack of synchronisation of circadian rhythms, measured by sleep and cortisol release, may cause a pre-diabetic change [3]. The internal balance of testosterone and cortisol has an effect on the risk of developing cardiovascular disease and type 2 diabetes [4]. In addition, the International Agency for Research on Cancer has classified shift work that involves circadian disruption as a probable human carcinogen [5]. Lack of coordination between circadian rhythms develops because different biological rhythms change with different speed [6]. Melatonin, cortisol and testosterone all have diurnal changes in concentrations [7], [8], [9], [10]. Thus, the combined changes in the concentrations of all three hormones can be used as a robust marker of circadian disruption. Touitou et al. used salivary melatonin and cortisol to measure circadian profiles in prepubertal boys, however they were not able to measure testosterone due to lack of sensitivity in the applied assay [10]. Circadian disruption may be specifically interesting in relation to night work or work without daylight. Phase shifts in the human circadian system in both cortisol and melatonin can be affected by daylight and has been shown to adapt to the length and intensity of light exposure [11]. Further, cortisol, and testosterone in saliva have been used to study the circadian disruption among construction workers with 12-h workdays and extended workweeks [12].
Melatonin is synthesized and secreted primarily by the pineal gland [13] and functions as a circadian pacemaker that synchronizes the internal hormonal environment to the light–dark cycle of the external environment and has importance for season variability [14]. Melatonin has been shown to be a good biomarker of circadian dysregulation [8] and therefore melatonin is often used in studies to evaluate the level of circadian disruption as a result of night or shift work. Cortisol is produced in the adrenal gland and is the principal marker for the activation of the hypothalamus–pituitary–adrenal (HPA) axis [15]. The HPA axis plays a central role in homeostatic processes and it is commonly thought to reflect attempts to adjust to daily pressures and joys [16] and is one of the major physiological stress responses in the body. Cortisol is used as a biomarker in many studies of physiological stress [17], [18], [19]. Cortisol has a characteristic and stabile diurnal rhythm with a rise 30 min after awakening [14] and is a good diurnal rhythm marker [20]. Harris et al. used cortisol as a marker of diurnal rhythm in a study of offshore shift workers [7]. Testosterone is an anabolic steroid produced in the testes in men and in the ovaries in females and in the adrenal glands in both sexes. There is a slight diurnal variation in both sexes and testosterone is highest in the morning after a good nights sleep [9]. Testosterone has been shown to be a good biomarker for adverse psychosocial working environment for both men and women [21].
Melatonin, cortisol and testosterone can be measured in blood [18], [22], [23], urine [24], [25], [26] and saliva [27], [28]. However saliva sampling is preferred since it has several advantages: It is noninvasive, painless and easy to perform [29]. Thereby multiple samples can easily be taken during the day in field studies where the participants in a study can be instructed in the sampling procedure at home and forward the samples by mail [30]. Several studies have shown that melatonin, cortisol and testosterone all have good correlations between saliva and serum levels of hormones [8], [31], [32].
Salivary hormones have, traditionally, been measured by immunochemistry based methods such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) [33], [34], [35], [36]. Examples of LODs using theses methods are: 1.29 pmol/L for melatonin (ELISA kit from DRG International Inc., NJ, USA), 0.8 nmol/L for cortisol (RIA kit from Origon Diagnostica, Espoo Finland) and 19.66 pmol/L for testosterone (ELISA kit from Abnova, Heidelberg, Germany). Although immunochemistry based methods are highly sensitive and also simple to use, they suffer inherently from a potential risk of cross-reactivity to structurally similar compounds [37]. In addition, RIA has been shown to be sensitive to both the collection procedure and the type of antibody used making it difficult to compare results obtained on different RIA kits [38].
When studying the working environment we primarily analyze samples from healthy adults. Thus, the variations in the concentrations of the hormones of interest are relatively small [39]. Hence it is crucial to use a precise and sensitive method making liquid chromatography tandem mass spectrometry the method of choice [40]. In addition, the time and resources spent on analyzing the hormones can be reduced dramatically by analyzing them in the same assay.
Several procedures for quantification of salivary hormones by LC–MS/MS have been reported previously [27], [28], [41], [42], [43], [44], [45], [46]. Turpeinen et al. proposed a simple liquid–liquid extraction (LLE) procedure where 0.1 mL saliva were extracted with dichloromethane and got a LOD for cortisol of 0.07 nmol/L [44]. Kataoka et al. got a slightly better LOD for cortisol (0.05 nmol/L) [46] by use of an online solid phase micro extraction procedure. Motoymama et al. [47] used column-switching semi-micro column liquid chromatographic system with three columns to attain large volume injection and online enrichment for the measurement of melatonin. 400 μL saliva was injected and the estimated the LOD was 10.7 pmol/L [47]. In another study, Eriksson et al. used solid phase extraction (SPE) on 1 mL saliva sample followed by evaporation to dryness and reconstitution in methanol–water yielding an estimated LOD for melatonin of 4.5 pmol/L [28]. Matusi et al. developed a method based on LLE followed by evaporation to dryness and reconstitution in acetonitrile for the simultaneous determination of salivary cortisol and testosterone obtaining LODs of 0.03 nmol/L and 17 pmol/L, respectively [27]. There are other examples of studies where two/several hormones are detected simultaneously in human saliva [27], [43], [48], however no procedure has been reported for the simultaneous determination of melatonin, cortisol and testosterone. Thus, the aim of the present study was to develop and evaluate a simple and robust method that facilitates simultaneous determination of all three hormones.
Section snippets
Chemicals
Melatonin (>97%), cortisol (>98%), testosterone (>99%), d-3-testosterone (>99%), d-4-cortisol (>99%) methanol (MeOH), ethyl acetate, formic acid and ammonium acetate were obtained from Sigma–Aldrich (St. Louis, MO). All solvents and additives were MS grade. d-4-melatonin (98%) was obtained from Qmx Laboratories (Essex, UK).
Preparation of standard solutions
Standard solutions were prepared from stock solutions in MeOH. For standard curves seven concentration levels were prepared for each hormone in 10% MeOH. Melatonin: 15.0–579.4
Sample preparation
We established a simple procedure by freezing the sample after LLE, making it possible to simply pour of the ethyl acetate layer as done by Matsui at al [27]. Use of LLE for sample purification yielded optimum speed and efficiency of the analysis and reduced the number of steps involved in sample preparation and thereby the number of possible sources of error. We found that reconstitution in 10% MeOH gave the best chromatographic separation. This procedure worked well for all three hormones and
Acknowledgements
Dorrit Meincke and Vivi Kofoed-Sørensen are acknowledged for skillful technical assistance. Jeanette Lindgaard Petersen is acknowledged for her work on the chromatographic conditions for cortisol.
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