Determination of testosterone and corticosterone in feathers using liquid chromatography-mass spectrometry
Introduction
In living organisms ranging from fungi to humans, many structural and functional properties are determined by steroid hormones, which have effects at almost all levels of biological organization [1]. Therefore, the function and effects of steroid hormones are of relevance to veterinary practice, research, conservation biology, farming, zoos and many other activities connected with both wildlife and domestic animals. Several studies have focused on the measurement of stress hormones or sex hormones in birds [[2], [3], [4], [5], [6]]. Such data can offer valuable information about stress levels and the condition of an individual bird [5]. These hormones are responsible for the regulation of many physiological traits, including individual fitness, survival, ornament expression, and reproduction [7]. Contrariwise, hormonal levels can be affected by environmental conditions and different social interactions [8]. In these studies, the concentration of hormones in blood plasma is usually measured. Analyses of plasma samples give information about the concentration of hormones and their metabolites at the time of capture. The concentration of corticosterone (CORT), for example, culminates within 3 min after stress reaction [9]. Hormones are gradually accumulated during feather growth. Therefore, the concentration of testosterone (TEST) in feathers can provide valuable information about the condition of an individual bird over a longer period of time. Feather keratin, due to its slow growth and other attributes, has limited application for detecting temporal variations in steroid content. [10,11].
Using feathers as a research material in studies which need to track stress and sex hormone concentrations over time have many benefits. Hormones in feathers are resistant to cold and heat [12], which allows the easy and inexpensive storage of feather material. Since samples are stable for a long time, feather sampling can even be performed from museum exhibits [13]. The other advantage is that feathers are also processed with a high degree of biomedical safety, in contrast to blood, for example, which carries a much higher risk of the transmission of disease [14].
In this study we used a limited amount of feather material from small bodied birds. In general, it is only possible to obtain a small quantity of sample, usually less than 25 mg. For the measurement of hormones in such a small sample it is necessary to have a highly selective and sensitive method of quantitative analysis. Current analytical methods meet most requirements such as high sensitivity, high throughput, ease of use, precision, accuracy and automation. However, other requirements such as efficient sample preparation, decreases in the limits of detection and limits of quantification (LOD/LOQ), and improving the specificity of detection are still under active research and development depending on the properties of the matrix [15]. Regarding steroid hormones in feathers, methods based on radioimmunoassay (RIA) are typically used to determine their concentration in extracts from feathers [5,11,16,17]. Enzyme immunoassay (EIA) has also been used several times for these purposes [18,19]. Liquid chromatography-tandem mass spectrometry (LC–MS/MS) has been used somewhat less to detect and quantify TEST and CORT in feathers [6]. However, CORT is often not detected in all feather samples [20]. A possible cause could be the low concentrations of CORT in some samples that are under the limit of quantification (LOQ) of the method used. Another issue is the very large range of quantities reported in different studies, independent of the technique used (RIA, EIA or HPLC). CORT quantities varied from 0.1 pg of hormone/mm of feather to more than 130 pg/mm [13,21]. The effect of feather type also contributes to the different levels of hormones analyzed in different kinds of feathers, these depending on the different moulting times in different species [5]. However, a large range of hormone concentrations can also be found within one species. Specifically, the amount of hormones in the Tree swallow ranged between 0.5 and 10 pg per millimeter of feather [22]. In addition, some authors discuss an effect called “the effect of small sample artefact” associated with the use of feather samples with different sample weights, according to which samples of lower mass appear to exhibit relatively higher hormone concentrations [23].
In our study we applied liquid chromatography coupled with tandem mass spectrometry to determine levels of CORT and TEST in feathers, an approach that was previously shown to reduce the problem of cross-reactivity commonly encountered with RIA or EIA [6]. Due to the small amount of feather samples and also in order to achieve the lowest levels of LOD/LOQ, we included the derivatization of hormones as special steps in our analyses. The derivatization of target compounds during sample preparation can substantially improve the selectivity of chromatographic separation, and the specificity and sensitivity of the detection of analytes [15]. In our study, a new derivatization procedure was used to detect and quantify TEST and CORT in extracts from bird feathers. Hormones in extract from feathers were chemically derivatized using O-(3-trimethylammoniumpropyl) hydroxylamine bromide (QAO Reagent, Amplifex™ Keto reagent, AB SCIEX, Framingham, MA) before final HPLC-ESI-MS/MS analysis. This derivatization procedure was primarily developed for the determination of hormones in body fluids [24] and had not been used previously for the determination of hormones in feathers.
The main aim of our study was to test the conditions of sample preparation, the conditions of hormone extraction (kind of extraction solvent, time of extraction), the solid phase extraction condition, and the reaction conditions of keto-derivatization on the sensitivity of LC–ESI–MS/MS analyses of TEST and CORT. The main reason was the limited availability of information on the effects exerted by individual parameters on the results of analyses. The use of the improved method is demonstrated on the LC–ESI–MS/MS analysis of steroids in of different species.
Section snippets
Chemicals and reagents
Analytical standards (Sigma-Aldrich, Germany): Testosterone (Vetranal™), Corticosterone (Vetranal™). Internal standards (C/D/N Isotops Inc., Pointe Claire, QC, Canada): 4-Pregnen-11β,21-diol-3,20-dione-2,2,4,6,6,17α,21,21-d8 (corticosterone-d8), testosterone-16,16,17-d3. Solvents: methanol (MeOH, 99.9%), acetonitrile (ACN; 99.9%) (Fluka, Sigma-Aldrich, Germany), acetone (99.8%), dichloromethane (DCM, 99.8%) (LAB-SCAN, Poland), diethyl ether (PENTA, Czech Republic). QAO reagent: (O
Chromatographic analyses of testosterone and corticosterone derivatives
The derivatization reaction takes place on keto-groups of steroid hormones The result is the formation of two steric forms of derivative for each keto-group. This means two steric forms for testosterone and four steric forms for corticosterone. All steric forms may be separated depending on the selectivity of the chromatographic system used. Depending on the ratio of acetonitrile to water in the mobile phase, one or two chromatographic peaks can be obtained for two TEST derivatives and one to
Conclusion
The aim of our study was to verify appropriate conditions for the extraction of TEST and CORT from feathers of small birds and to optimize reaction conditions for the determination of TEST and CORT in extract obtained after the pre-column keto-derivatization of hormones.
Due to the limited information available in published papers on the effects of individual conditions of sample preparation on the analysis of hormones in feather samples, the effects of the type of extraction solvent, the
Acknowledgement
This work was financially supported by the Grant Agency of the Czech Republic Project (GA17-24782S). In addition, support provided by the RECETOX Research Infrastructure (LM2015051 and CZ.02.1.01/0.0/0.0/16_013/0001761) is acknowledged.
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