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

Author:

Steel Rohan1ORCID,Timms Mark1,Bamford Nicholas2ORCID,Spence Robert3ORCID,Sillence Martin3

Affiliation:

1. Biological Research Unit Racing Analytical Services Ltd Flemington Australia

2. Veterinary Biosciences Melbourne University Parkville Australia

3. Department of Biological and Environmental Sciences Queensland University of Technology Brisbane Australia

Abstract

AbstractPrevious 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).

Funder

Australian Research Council

Publisher

Wiley

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