Evaluation of long-term 11-oxoaetiocholanolone stability in red deer faecal samples under different storage conditions

Abstract

Faecal cortisol metabolite (FCM) analysis is a reliable non-invasive method used in field endocrinology studies to assess levels of stress in animals. It is known that weather and, above all, humidity, can affect FCM concentrations in faecal samples. In addition, the prolonged storage of samples and delay in their analysis may alter metabolite concentrations. Intrinsic factors such as the heterogeneous distribution of FCMs within scats may likewise cause intra-sample variation. All of these sources of variation in FCM concentrations need to be addressed if we are to interpret results correctly. The aim of this study was to assess the effects of lyophilisation and storage temperature on the long-term stability of 11-oxoaetiocholanolone (11-o) in red deer (Cervus elaphus) faecal samples. After pre-cleaning with hexane and extraction with methanol, 11-o levels were analysed using high-performance liquid chromatography coupled with tandem mass spectrometry HPLC-MS/MS at 1, 2, 4, 6, 8, 16 and 32 weeks post-collection. We used linear mixed models to explore the effects of temperature and storage time on concentrations of faecal 11-o in wet and dry samples. Our results showed significant variations in 11-o concentrations in wet faecal samples over time and at different storage temperatures. By contrast, the 11-o values of dry samples were more stable in terms of storage temperatures. Lyophilising red deer faecal samples and storage at − 80 °C guarantees the stability of 11-o for several months.