Doping control analysis of insulin and its analogues in equine plasma by liquid chromatography–tandem mass spectrometry

Abstract

Insulin administration can increase muscle glycogen by utilising hyperinsulinaemic clamps prior to sports events or during the recovery phases, and increase muscle size by its chalonic action to inhibit protein breakdown. In order to control insulin abuse in equine sports, a method to detect effectively the use of insulins in horses would be required. Besides the readily available human insulin and its synthetic analogues, structurally similar insulins from other species can also be used as doping agents. This study describes a method for the simultaneous detection of bovine, porcine and human insulins, as well as the synthetic analogues Humalog (Lilly) and Novolog (Novo Nordisk) in equine plasma. Insulins were isolated from equine plasma by immunoaffinity purification, followed by centrifugal filtration, and analysed by nano-liquid chromatography-tandem mass spectrometry (LC/MS/MS). Insulin and analogues were detected and confirmed by comparing their retention times and major product ions. All five insulins (human insulin, Humalog, Novolog, bovine insulin and porcine insulin), which are exogenous in the horse, could be detected and confirmed at 0.05ng/mL. This method was successful in confirming the presence of human insulin in plasma collected from horses up to 4h after having been administered a single low dose of recombinant human insulin (Humulin R, Eli Lilly). To our knowledge, this is the first identification of exogenous insulin from post-administration horse plasma samples.

Comparison of two methods for measurement of equine insulin

“…However, this assay consistently underestimates equine insulin concentrations when compared with LC-MS. 12 Although mammalian insulins demonstrate substantial homology, equine insulin does differ from porcine, human, and bovine insulin by 1-3 amino acids. 5 These substitutions may affect insulin binding due to different secondary, tertiary, or quaternary protein structure. 1,4 Depending on the amino acid sequence recognized by antibodies in different immunoassays, available assays may vary in detection of equine insulin.…”

Quantification of Alefacept, an immunosuppressive fusion protein in human plasma using a protein analogue internal standard, trypsin cleaved signature peptides and liquid chromatography tandem mass spectrometry

“…Current methods for protein quantification by LC/MS have employed antibody-based purification through molecular recognition of target proteins and/or peptides in addition to immunodepletion of major background proteins [17,18,21]. Two-dimensional solid phase extractions have been used to reach low ng/mL levels and achieve precise and accurate results without the use of antibodies [27].…”

“…The drawbacks associated with this technique include expensive kits, possible imprecision, minimal volume applied, and minimal column life (<200 samples) [16]. Immunopurification of the target protein can also be accomplished and has also been shown to yield lower limits of detection [17][18][19][20][21]. However, the requirement for antibodies for immunopurification makes this technique less desirable.…”

Strategies for improving sensitivity and selectivity for the quantitation of biotherapeutics in biological matrix using LC-MS/MS

“…IA purification of the protein target is useful for both native and therapeutic proteins. For example, Ho et al [37] reported the simultaneous detection of five exogenous insulin in equine plasma via IA purification LC-MS analysis. Lopez et al [38] demonstrated the feasibility of immunopurification of the parathyroid hormones and the analysis of the same by MALDI-TOF.…”