Faecal 11-ketoetiocholanolone measurement in Iberian red deer (Cervus elaphus hispanicus): validation of methodology using HPLC–MS/MS

Abstract

A cortisol metabolite, 11-ketoetiocholanolone (11-k), is widely used in monitoring stress in several vertebrates, and can be detected by immunoassay. However, these assays have certain limitations with respect to specificity. Also, differences in the excretion of faecal glucocorticoid metabolites (FGM) among species and even between sexes make validation necessary in each case. Therefore, our aims were, first, to develop and validate a high-pressure liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) methodology for monitoring 11-k in faeces of Iberian red deer (Cervus elaphus hispanicus), and second, to investigate the capability of our method to determine variations of this FGM in a longitudinal study. Third, and finally, we assessed the correspondence between faecal 11-k concentrations and plasma cortisol. An adrenocorticotropic hormone (ACTH) test was performed on six red deer stags translocated and kept in captivity for a week and faecal samples were collected twice a day. One single blood and faecal sample from another seven stags was also collected after 2 weeks in captivity. The results of the longitudinal study showed a first peak in 11-k 36 h after the ACTH test and handling, and a second peak at 120 h of being kept indoors. Maximum concentrations of 11-k ranged from 22.71 to 375.68 ng/g. In the second stag group, 11-k concentrations of 25.09 ± 20.53 ng/g had a correlation of r2 = 0.88 with the concentration of plasma cortisol, which was 54.6 ± 55.1 ng/mL. This technique is capable of detecting changes in the concentrations of faecal 11-k. The values determined have a good correlation with the cortisol concentration in blood, and we also detected differences in different individuals’ responses to the same stressors.

HPLC‐QTOF method for quantifying 11‐ketoetiocholanolone, a cortisol metabolite, in ruminants' feces: Optimization and validation

“…From a technical point of view, an important advantage of the method proposed was the high 11‐k fecal recoveries, which were around 40% greater than those obtained in the previous study carried out in our laboratory (Azorit et al., ). The time required for the fecal 11‐k extraction was also greatly reduced in comparison with the previous work mentioned.…”

“…The mobile phases and the elution gradient optimized in previous studies (Azorit et al, 2012) and described in the Supporting Information Appendix S1 were used.…”

“…Over the last few years specific LC‐MS/MS methods for analyzing steroids from several species in different body fluids and tissues have been developed (Hauser, Deschner, & Boesch, ; Miksik, Mikulikova, Pacha, Kucka, & Deyl, ; Shimada, Mitamura, & Higashi, ). Nevertheless, there are still few studies that have focused their attention on noninvasive FGC 11‐k determination using HPLC‐MS/MS (Azorit et al., ) and, of the few who have attempted it none have achieved either good accuracy or reproducibility (Weltring, Schaebs, Perry, & Deschner, ). There are two major difficulties (Kushnir et al., ), namely low concentrations, which call for high sensitivity, and the presence of numerous structurally similar metabolites, which thus demands a high degree of selectivity.…”

“…Such measurements preclude some of the above-mentioned problems as feces can be collected easily without disturbing the animal, allowing repeated sampling for monitoring purposes. Additionally, it enables longitudinal studies to be performed using the evaluation of long-term GC levels over a period of time (Azorit et al, 2012; Rettenbacher, Touma, El-Bahr, & Mostl, 2005) and can be used as a proxy for a daily average of circulating GC in plasma (Goymann, 2005;Palme, Fischer, Schildorfer, & Ismail, 1996).…”

“…Nevertheless, there are still few studies that have focused their attention on noninvasive FGC 11-k determination using HPLC-MS/MS (Azorit et al, 2012) and, of the few who have attempted it none have achieved either good accuracy or reproducibility (Weltring, Schaebs, Perry, & Deschner, 2012). There are two major difficulties (Kushnir et al, 2011), namely low concentrations, which call for high sensitivity, and the presence of numerous structurally similar metabolites, which thus demands a high degree of selectivity.…”