The aim of the present study was to measure salivary cortisol concentrations of horses before and after hypothalamic-pituitary-adrenal (HPA) axis stimulation by means of liquid chromatography-tandem-mass spectrometry (LC-MS/MS) and an immunoassay (cELISA) for method comparison. Nine clinically healthy horses participated in the study. An ACTH stimulation test was performed. Saliva samples were collected before (T0) and 60 (T60) min after intravenous injection of 1 μg/kg BW synthetic ACTH1-24. LC-MS/MS was assessed for the determination of equine salivary cortisol. The results of these measurements were then compared to the results obtained by a cELISA, which has previously been validated for use in horses. The Pearson correlation coefficient was calculated and showed no correlation at T0 (r = −0.2452; P = 0.5249) and significantly correlated results at T60 (r = 0.8334; P = 0.0053). Bland-Altman-Plots of T60 revealed that immunoassay measurements led to higher outcome values than LC-MS/MS. On average, immunoassay results were 2.3 times higher. Poor agreement between both methods at T0 is potentially a consequence of inaccuracy in the very low measuring range of the immunoassay, and to a smaller extent, structurally similar cross-reacting agents and matrix effects, which might bias the results. Overestimation of immunoassay results at T60 might be due to different standardization of both methods, non-avoidable matrix effects on the antigen-antibody interaction in the ELISA, and possibly cross-reactions of other steroids. While immunoassay measurements of equine salivary cortisol yielded higher but reasonably correlated results for elevated cortisol concentrations after stimulation of the HPA axis, LC-MS/MS provided more accurate results, particularly for baseline cortisol concentrations close to the limit of detection of the ELISA.

本研究的目的是通过液相色谱-串联质谱（LC-MS / MS）和免疫测定（cELISA）测量下丘脑-垂体-肾上腺（HPA）轴刺激前后马的唾液皮质醇浓度用于方法比较。九名临床健康的马参加了这项研究。进行了ACTH刺激测试。静脉注射1μg/ kg BW合成ACTH1-24之前（T0）和60（T60）分钟之前收集唾液样品。评估LC-MS / MS用于测定马唾液皮质醇。然后将这些测量结果与通过cELISA获得的结果进行比较，该cELISA先前已被验证可用于马匹。计算了皮尔逊相关系数，并且在T0时没有相关性（r = -0.2452；P= 0.5249）和T60时的显着相关结果（r = 0.8334；P= 0.0053）。T60的Bland-Altman-Plots显示，与LC-MS / MS相比，免疫分析的测量结果值更高。平均而言，免疫分析结果高出2.3倍。两种方法在T0时的一致性差可能是由于免疫测定的非常低的测量范围内的准确性所致，并且在较小程度上还存在结构上相似的交叉反应剂和基质效应，这可能会使结果产生偏差。T60时免疫测定结果的高估可能是由于两种方法的标准化不同，ELISA中抗原-抗体相互作用不可避免的基质作用以及其他类固醇的交叉反应所致。虽然在刺激HPA轴后，马唾液皮质醇的免疫测定结果得出较高但合理相关的结果，