Development of analytical methods for TB-500 and its metabolites by LC-MS/MS

Abstract

Ac-LKKTETQ (TB-500) is a small peptide processed from the active site of thymosin β4. Like parent thymosin β4, TB-500 has tissue regeneration, anti-inflammation, and fast repair capabilities. TB-500 misuse was found in equine sports and was reported to be used illegally by athletes. Therefore, this study aimed to discover metabolites of TB-500 peptides in in-vivo (rats) and in various in-vitro systems to detect in-vivo time kinetic in urine and to develop a SPE quantitative method for TB-500 and its metabolites. In in-vitro, we found 4 metabolites which were Ac-LKKTE (m/z 330), Ac-LKK (m/z 215), Ac-LK (m/z 302), and Ac-L (m/z 174). In rats, we found 5 metabolites which are Ac-LKKTET (m/z 381), Ac-LKKTE (m/z 330), Ac- LKK (m/z 215), and Ac-LK (m/z 302), Ac-L (m/z 174). Parent and 6 metabolites were synthesized commercially and identified for their structure. All metabolite structures were confirmed. Later we quantitatively measured metabolites in urine in a time kinetic manner (0~96 hr). We observed that Ac-LK is the highest concentration in urine, and the metabolite Ac-LKK in urine samples was detected until 72 hours. In this study, we developed an analytical method to quantify TB-500 and its metabolites generated by in-vitro and in-vivo systems.