Confirmatory analysis of etanercept in equine plasma by LC-MS for doping control.

The research focuses on detecting a protein-based drug called Etanercept, which is used for treating human patients with autoimmune diseases, in horse racing, using a method called liquid chromatography-mass spectrometry (LC-MS).

Overview

• The study is aimed at detecting the use of a protein-based drug called Etanercept in horse racing using a method known as liquid chromatography-mass spectrometry (LC-MS).
• Etanercept is usually used to treat people suffering from rheumatoid arthritis and other autoimmune diseases by inhibiting tumor necrosis factor alpha.

Etanercept Extraction and LC-MS Process

• The researchers extracted Etanercept from horse plasma using an anti-human IgG antibody attached to magnetic beads.
• The extracted Etanercept was then reduced, alkylated, and digested by an enzyme called trypsin.
• The researchers used three specific tryptic peptides from the digested Etanercept to confirm its presence.

Results & Implications

• The limit of detection of Etanercept through this method was 5 ng/mL, and the limit of confirmation was 10 ng/mL.
• The results were confirmed using BLAST and SEQUEST searches, which revealed the method’s specificity for Etanercept detection.
• The SEQUEST searches also discovered an unexpected unique specificity of product ion spectrum of IgG1 T27 with only a single product ion for the identification of etanercept—something not reported earlier.
• The method was successful in examining post-administration plasma samples from horses. The drug was detected up to 11 days post administration.
• This method can prove helpful for confirming the presence of Etanercept and other drugs composed of human IgG1 in horse drug testing.