The detection of ACTH and insulin in equine plasma by solid-phase extraction and micro-flow LC/MSMS.

IF 2.6 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Drug Testing and Analysis Pub Date : 2024-07-08 DOI:10.1002/dta.3762

Rohan Steel, Mark Timms, Nicholas Bamford, Robert Spence, Martin Sillence

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引用次数: 0

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).

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通过固相萃取和微流 LC/MS 检测马血浆中的促肾上腺皮质激素和胰岛素。

以前检测马血浆中胰岛素和其他类似肽类激素的液相色谱/质谱（LC/MS）方法依赖于抗体亲和提取。因此，这些方法不适合常规高通量分析。固相萃取（SPE）方法结合了尺寸排阻和反相作用，可以从马血浆中选择性地萃取肽类激素，如促肾上腺皮质激素（ACTH）、胰岛素及其合成类似物，萃取效率约为 80%。这种萃取方法结合了醋酸酐柱上衍生化、胰蛋白酶消化和微量液相色谱/质谱分析，可用于高灵敏度的肽类激素检测。与分析完整的胰岛素和促肾上腺皮质激素相比，分析胰蛋白酶肽具有更高的灵敏度和更稳健的色谱法。为了进行定量分析，可使用氘化乙酸酐在实验室中制备目标肽的同位素标记内标。在分析垂体中叶旁功能障碍（PPID）马匹样本中的促肾上腺皮质激素和胰岛素时，对该方法的实用性进行了评估。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Drug Testing and Analysis BIOCHEMICAL RESEARCH METHODS-CHEMISTRY, ANALYTICAL

CiteScore

5.90

自引率

24.10%

发文量

191

审稿时长

2.3 months

期刊介绍： As the incidence of drugs escalates in 21st century living, their detection and analysis have become increasingly important. Sport, the workplace, crime investigation, homeland security, the pharmaceutical industry and the environment are just some of the high profile arenas in which analytical testing has provided an important investigative tool for uncovering the presence of extraneous substances. In addition to the usual publishing fare of primary research articles, case reports and letters, Drug Testing and Analysis offers a unique combination of; ‘How to’ material such as ‘Tutorials’ and ‘Reviews’, Speculative pieces (‘Commentaries’ and ‘Perspectives'', providing a broader scientific and social context to the aspects of analytical testing), ‘Annual banned substance reviews’ (delivering a critical evaluation of the methods used in the characterization of established and newly outlawed compounds). Rather than focus on the application of a single technique, Drug Testing and Analysis employs a unique multidisciplinary approach to the field of controversial compound determination. Papers discussing chromatography, mass spectrometry, immunological approaches, 1D/2D gel electrophoresis, to name just a few select methods, are welcomed where their application is related to any of the six key topics listed below.