Insulins in equine urine: qualitative analysis by immunoaffinity purification and liquid chromatography/tandem mass spectrometry for doping control purposes in horse-racing.

This research analyses the presence of different types of insulin in horse urine in relation to doping control in horse-racing. It utilises methods like solid-phase extraction, immunoaffinity chromatography, and tandem mass spectrometry to identify insulin variants in the urine samples.

Background

• Insulin, a peptide hormone with two peptide chains, is produced in different forms (long-, intermediate-, and rapid-acting) for improved pharmacokinetic action in diabetes treatment. The B-chain’s C-terminal end of insulin plays a significant role.
• The rarity of equine type I diabetes suggests that use of insulin therapeutically in racing horses is rare. However, the misuse of insulin preparations as performance-enhancing substances is reported among elite human athletes.
• Insulin can increase muscle glycogen, beneficial prior to sports events, and contribute to a chalonic action—increasing muscle size by inhibiting protein breakdown.

Purpose and Methods

• The study aims to identify urinary insulin in equine samples and distinguish between equine insulin, human insulin, and the fast-acting recombinant insulin variants.
• The researchers used solid-phase extraction (SPE) to purify the urine samples and immunoaffinity chromatography (IAC) to separate the different types of insulin. Following purification, they used microbore liquid chromatography (LC) and tandem mass spectrometry (MS/MS) for analysis.

Findings

• The method allowed for the differentiation of endogenously produced equine insulin, its DesB30 metabolite, human insulin, and recombinant insulin analogs.
• The assay permitted the assignment of individual product ions, particularly those coming from the modified C-termini of B-chains. These findings suggest the presence of different types of insulin can be detected and differentiated in horse urine, which is significant for doping control in horse-racing.