Abstract：
Previous liquid chromatography/mass spectrometry (LC/MS) methods for the detection of insulin and other similar peptide hormones in equine plasma relied on the use of antibody affinity extraction. As a result, these methods were not suitable for routine high-throughput analysis. A solid-phase extraction (SPE) method incorporating size exclusion as well as reversed-phase interactions allows the selective extraction of peptide hormones such as adrenocorticotropic hormone (ACTH), insulin and their synthetic analogues from equine plasma with approximately 80% extraction efficiencies. This extraction was combined with on-column derivatisation with acetic anhydride, followed by tryptic digestion and analysis by micro-LC/MSMS for high-sensitivity peptide hormone detection. The analysis of tryptic peptides provides greater sensitivity and more robust chromatography compared with the analysis of intact insulin and ACTH. For quantitative analysis, isotopically labelled internal standards of target peptides can be prepared in the laboratory through the use of deuterated acetic anhydride. The utility of the method was assessed for the analysis of ACTH and insulin in samples from horses suffering from pituitary pars intermedia dysfunction (PPID).
摘要：
先前用于检测马血浆中胰岛素和其他类似肽激素的液相色谱/质谱（LC/MS）方法依赖于使用抗体亲和提取。因此,这些方法不适用于常规高通量分析.结合了尺寸排阻和反相相互作用的固相萃取（SPE）方法可以选择性提取肽类激素，例如促肾上腺皮质激素（ACTH）。从马血浆中提取胰岛素及其合成类似物，提取效率约为80％。该萃取与乙酸酐的柱上衍生化相结合，然后通过胰蛋白酶消化和微量LC/MSMS分析用于高灵敏度肽激素检测。与完整胰岛素和ACTH的分析相比，胰蛋白酶肽的分析提供了更高的灵敏度和更强大的色谱。为了进行定量分析，同位素标记的目标肽的内标可以在实验室中通过使用氘代乙酸酐来制备。评估了该方法的实用性，用于分析患有垂体中间部功能障碍（PPID）的马的样品中的ACTH和胰岛素。
