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

This research discusses a method to detect the illicit use of insulin and its synthetic counterparts in horse racing. The study introduces a way to recognize bovine, porcine, and human insulin along with synthetic analogues in horse plasma, in an effort to combat doping in equine sports.

Objective of the Research

• The primary aim of this study was to develop an efficient method to detect the unauthorised use of insulin and its synthetic analogues—Humalog and Novolog—in horse racing. This is crucial because insulin can be used to enhance the performance of horses by increasing muscle glycogen and muscle size.

Doping Agents

• The research focusses not only on human insulin and its synthetic versions but also explores their equivalents derived from other species. Bovine and porcine insulin have structural similarities to human insulin and can also be utilized as doping agents.

Detection Method

• The study presents a simultaneous detection process for bovine, porcine, and human insulins, including Humalog and Novolog. The process involves isolating insulins from equine plasma through immunoaffinity purification and centrifugal filtration. Subsequently, they are analyzed via nano-liquid chromatography-tandem mass spectrometry (LC/MS/MS).
• Each insulin type and their analogues are identified and validated by comparing their retention times and major product ions. The method enabled detection and confirmation of all exogenous insulins at a level of 0.05ng/mL.

Successful Identification

• The method successfully identified the presence of human insulin in plasma from horses up to four hours after administering a single low dose of Humulin R—which is a type of recombinant human insulin.
• Notably, this is the first time exogenous insulin has been identified in plasma samples from horses post-administration.