Growth hormone abuse in the horse: preliminary assessment of a mass spectrometric procedure for IGF‐1 identification and quantitation

Abstract

Previous studies have shown that insulin-like growth factor 1 (IGF-1) is a promising marker for the detection of growth hormone (GH) abuse in the horse. The significant increases observed with GH administration in comparison to natural levels imply the possibility of setting a threshold level for IGF-1 that would be indicative of GH abuse. Although an immunoradiometric assay (IRMA) has been identified as a reliable screening method, a more specific IGF-1 quantification method needs to be developed for the prosecution of GH abuse by horseracing authorities. This study describes such an HPLC electrospray mass spectrometry (LC/ESI-MS) method that was developed and then assessed for the specific analysis of IGF-1 at the low levels encountered in serum. The structural identity of IGF-1 was confirmed by endoproteinase Asp-N digestion followed by LC/MS and LC/MS/MS characterisation. This was followed by quantification of IGF-1 as the intact molecule against an internal standard.

Metabolism of Anabolic Steroids and Their Relevance to Drug Detection in Horseracing

“…The use of biomarker approaches is highly attractive and initial work carried out in detection of growth promoters in cattle appears promising [113]. Initial investgations into their application to equine sport have been carried out [114][115][116][117][118][119][120]. Similarly, the use of receptor assays has been extensively researched for application to meat residue ana lysis and also shows promise for future use in sports testing [91,121,122].…”

Laboratory issues in the implementation of the marker method

“…Popot and co-workers [12][13][14] used immunoaffinity purification after acid displacement ethanol extraction and then analysis of the intact protein using R 3 IGF-1 that has an arginine instead of glutamic acid at position 3 as the internal standard. de Kock et al [15] chose to perform a protein digest using endoproteinase Asp-N, rather than tryptic digest that they had found to be inefficient, followed by reduction of the free thiols and LC tandem MS assay of the digest using R 3 IGF-1 treated in the same way as the internal standard. Kirsch et al [16] claimed to have used tryptic digests effectively to quantify both IGF-I and also IGFBP-3 in serum using heavy isotopes of suitable fragments as internal standards with a triple quadrupole mass spectrometer.…”

Analytical procedures for quantification of peptides in pharmaceutical research by liquid chromatography?mass spectrometry

“…as drugs for therapeutic treatment [1,2,3,4,5,6,7,8,9], as disease markers in human and veterinary clinical diagnosis [10,11,12,13,14,15,16], as antimicrobial animal feed additives [17], and as analytes for protein identification in proteome research [18,19]. In pharmaceutical research quantitative assays are required for some essential studies; investigations of peptide stability in biological matrices are of great importance for clarification of metabolism and enzyme kinetics [18,20,21].…”