Wild animals are faced with a broad range of environmental stressors and research is needed to better understand their effect on populations. Hormone analysis based on enzyme immunoassays (EIAs) can provide valuable information on adrenocortical activity (stress), and assessment of cortisol in hair may allow the quantification of cortisol production. To validate hair hormone analysis, we compared two EIAs based on antibodies against cortisol-3-CMO-BSA and cortisol-21-HS-BSA for hair glucocorticoid (hGC) measurements in Egyptian mongoose, Iberian lynx, Alpine marmot, Asiatic black bear, spotted hyena and cheetah, with results obtained by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) measurements. Both EIAs were also characterized by HPLC immunograms. Our results revealed that the cortisol-21-HS EIA measured 2.3- to 12-fold higher hGC concentrations than the cortisol-3-CMO assay. In dependence of the species, high-performance liquid chromatography (HPLC) immunograms showed that up to 70% of immunoreactivities determined by the cortisol-21-HS constituted of unknown unpolar compounds leading to an overestimation of hGC. The cortisol-3-CMO EIA expressed a better specificity, with 32.1–67.4% of immunoreactivity represented by cortisol and cortisone. The LC-MS/MS analyses (gold standard) revealed that the cortisol-3-CMO EIA also resulted in an (up to 3-fold) overestimation of hGC, but EIA results were correlated with LC-MS/MS in the mongoose, the lynx, the spotted hyena and the marmot. No correlation was obtained for Asiatic black bears. As a result of our study, we strongly recommend to test any cortisol EIA for its specificity towards extracted hair components. In all analyzed species, except the Asiatic black bear, cortisone and cortisol were simultaneously present in hair extracts; consequently, an appropriate EIA should cross-react to these two glucocorticoid hormones and express negligible affinity towards substances with less polarity than corticosterone. Choosing the wrong EIA for hGC analyses may lead to overestimations of hGC or—in the worst case—to results that do not mirror real adrenocortical activity.

中文翻译：

---

不同哺乳动物物种的毛发皮质醇分析：选择错误的检测方法可能会导致错误的结果

野生动物面临着广泛的环境压力，需要进行研究以更好地了解它们对种群的影响。基于酶免疫分析 (EIA) 的激素分析可以提供有关肾上腺皮质活动（压力）的有价值的信息，并且对头发中皮质醇的评估可以定量皮质醇的产生。为了验证毛发激素分析，我们比较了两种基于皮质醇-3-CMO-BSA 和皮质醇-21-HS-BSA 抗体的 EIA，用于埃及猫鼬、伊比利亚山猫、高山土拨鼠、亚洲黑熊、斑点鬣狗和猎豹，结果通过液相色谱结合串联质谱 (LC-MS/MS) 测量获得。两种 EIA 还通过 HPLC 免疫图进行了表征。我们的结果显示，cortisol-21-HS EIA 测得的 hGC 浓度比 cortisol-3-CMO 测定高 2.3 至 12 倍。根据物种的不同，高效液相色谱 (HPLC) 免疫图显示，由 cortisol-21-HS 测定的高达 70% 的免疫反应性由未知的非极性化合物组成，导致 hGC 的高估。cortisol-3-CMO EIA 表现出更好的特异性，皮质醇和可的松代表了 32.1-67.4% 的免疫反应性。LC-MS/MS 分析（金标准）显示皮质醇-3-CMO EIA 也导致 hGC 高估（高达 3 倍），但 EIA 结果与猫鼬中的 LC-MS/MS 相关，山猫、斑鬣狗和土拨鼠。没有获得亚洲黑熊的相关性。根据我们的研究结果，我们强烈建议测试任何皮质醇 EIA，以确定其对提取的头发成分的特异性。在所有分析的物种中，除亚洲黑熊外，可的松和皮质醇同时存在于毛发提取物中；因此，适当的 EIA 应与这两种糖皮质激素发生交叉反应，并对极性小于皮质酮的物质表现出可忽略不计的亲和力。为 hGC 分析选择错误的 EIA 可能会导致 hGC 的高估，或者在最坏的情况下导致结果不反映真实的肾上腺皮质活动。