Quantitative determination of the metabolite Ac-Tβ1-14 in in-vitro and urine of rats treated with Thymosin β4 by LC Orbitrap HR-MS/MS

Abstract

Thymosin β4 (Tβ4) was reported to exert beneficial bioactivities such as tissue repair, anti-inflammation, and reduced scar formation. It is listed as a prohibited substance in sports by the World Anti-Doping Agency. However, no metabolism studies of Tβ4 were reported yet. Previously, our lab reported in an in-vitro experiment that a total of 13 metabolites were found by using multiple enzymes, and six metabolites (Ac-Tβ31-43, Ac-Tβ1-11, Ac-Tβ17-43, Ac-Tβ1-14, Ac-Tβ1-15, and Ac-Tβ1-17) were confirmed by comparing with the synthetic standards. This study aimed to validate the metabolite analysis in rats urine and to develop a method for quantifying the metabolites. Among the six metabolites mentioned above, Ac-Tβ1-14 was a common metabolite in the pure enzyme and microsome systems, huvec cells, and rat urine samples of rats intraperitoneally treated with Ac-Tβ4. A method for detecting and quantifying Ac-Tβ1-14 was developed and validated using Q-Exactive orbitrap mass spectrometry. LOD and LOQ of the Ac-Tβ1-14 were determined to be 0.19 and 0.58 ng/ml and showed good linearity (r2 = 0.9998). Among 3 in-vitro systems, LAP produces the highest amount of Ac-Tβ1-14, compared to HKM and huvec cells. The metabolite Ac-Tβ1-14 was quantitatively determined by 48 hr in rats, with the highest concentration occurring between 0-6 hr. These results suggest that the metabolite should be a good tool for detecting Ac-Tβ1-14 in urine, a potential biomarker for screening the parent Tβ4 in doping tests.