Feathers gradually accumulate hormones and reflect long-term average plasma steroid levels during their growth. Feather hormone levels thus provide for the measurement of plasma hormones concentrations integrated over a period of several days or weeks. In this study, we focused on the development of a method to determine testosterone (TEST) and corticosterone (CORT) levels in extracts from feathers of small bodied birds with a limited amount of feathers available per individual. For this purpose, the method had to be verified for a small weight of samples. The present study describes the effect of the conditions of sample preparation and keto-derivatisation on the sensitivity of the LC-ESI-MS/MS analysis of TEST and CORT. Generally, chemical derivatization improves the sensitivity and selectivity of LC-MS/MS analysis. It can be used particularly in situations when the total amount of collected sample is limited (such as in our studies). Both the conditions of feather sample preparation (the selection of the extraction solvent, the time of extraction, and the conditions of solid phase extraction) and the reaction conditions affecting the formation of keto-derivatives (such as reaction temperature and reaction time) were tested. Methanol as the extraction solvent, 8 h as the extraction time, 50 °C as the reaction temperature of derivatization, and 90 min as the reaction time of derivatization are the most suitable conditions in terms of achieving a high sensitivity of analyses. Calibration curves are linear, at least in the range 25-2500 pg mL-1, which is usually found in feather extracts. The limit of detection (LOD) for TEST and CORT was 1.0 and 0.3 pg per mL, respectively. The limit of quantification (LOQ) for TEST and CORT was 3.3 and 1.0 pg per mL, respectively. The optimized procedure was successfully applied for the analysis of TEST and CORT in real feather samples. The method could be used in a variety of research direction including wildlife, agricultural or veterinary studies.

Department of Botany and Zoology Masaryk University Kotlářská 2 611 37 Brno Czech Republic

Department of Zoology Charles University Viničná 1594 7 128 00 Praha Czech Republic

Institute of Vertebrate Biology Academy of Sciences Studenec 122 675 02 Koněšín Czech Republic

Research Centre for Toxic Compounds in the Environment Masaryk University Kamenice 753 5 625 00 Brno Czech Republic

References provided by Crossref.org