Doping control analysis of TB-500, a synthetic version of an active region of thymosin β4, in equine urine and plasma by liquid chromatography–mass spectrometry

Abstract A veterinary preparation known as TB-500 and containing a synthetic version of the naturally occurring peptide LKKTETQ has emerged. The peptide segment 17 LKKTETQ 23 is the active site within the protein thymosin β 4 responsible for actin binding, cell migration and wound healing. The key ingredient of TB-500 is the peptide LKKTETQ with artificial acetylation of the N-terminus. TB-500 is claimed to promote endothelial cell differentiation, angiogenesis in dermal tissues, keratinocyte migration, collagen deposition and decrease inflammation. In order to control the misuse of TB-500 in equine sports, a method to definitely identify its prior use in horses is required. This study describes a method for the simultaneous detection of N-acetylated LKKTETQ and its metabolites in equine urine and plasma samples. The possible metabolites of N-acetylated LKKTETQ were first identified from in vitro studies. The parent peptide and its metabolites were isolated from equine urine or plasma by solid-phase extraction using ion-exchange cartridges, and analysed by liquid chromatography–mass spectrometry (LC/MS). These analytes were identified according to their LC retention times and relative abundances of the major product ions. The peptide N-acetylated LKKTETQ could be detected and confirmed at 0.02 ng/mL in equine plasma and 0.01 ng/mL in equine urine. This method was successful in confirming the presence of N-acetylated LKKTETQ and its metabolites in equine urine and plasma collected from horses administered with a single dose of TB-500 (containing 10 mg of N-acetylated LKKTETQ). To our knowledge, this is the first identification of TB-500 and its metabolites in post-administration samples from horses.