Synthesis and characterization of the N‐terminal acetylated 17‐23 fragment of thymosin beta 4 identified in TB‐500, a product suspected to possess doping potential

Abstract

The formulation TB‐500 is suspected to be used as doping agent in sport. This work describes the detection and the identification of the N‐terminal acetylated 17‐23 fragment of human thymosin beta 4 (Ac‐LKKTETQ) in TB‐500 by means of high‐performance liquid chromatography/high resolution mass spectrometry using an Orbitrap Exactive benchtop mass spectrometer. Ac‐LKKTETQ was also synthesized by solid‐phase peptide synthesis, and an analytical strategy for detection in plasma and urine by high‐performance liquid chromatography/low resolution triple‐quadrupole mass spectrometry was suggested.

Development and validation of a liquid chromatography–mass spectrometry procedure after solid-phase extraction for detection of 19 doping peptides in human urine

“…Finally, novel, previously unknown macromolecules (i.e., TB500 17-23 fragment), with no current approval for human therapeutic use (i.e., agents under preclinical or clinical development, designer drugs, or compounds approved only for veterinary use), but illegally marketed (e.g., via Internet websites) [5] are included in section S0 ''Non-approved substances''. Different analytical approaches have already been developed and published to detect these compounds in nutritional supplements [6][7][8][9][10] and in doping control samples (either blood [11][12][13][14][15][16][17][18] or urine [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37]), employing immunological [26,[35][36][37], electrophoretic [16][17][18][19]27], or liquid chromatography-mass spectrometry techniques [6-15, 20-25, 28-34] or a combination of different analytical technologies. In general, the most effective analytical approach (combined sample pretreatment and the instrumental method) has to take into ...…”

In Vitro Metabolic Studies of REV-ERB Agonists SR9009 and SR9011

“…The potential interest as doping agents is clearly shown by their popularity in discussion forums on the Internet, where they are mentioned as the ultimate “exercise in a pill” compounds [8,9,10,11,12,13,14]. Although these REV-ERB agonists are currently still undergoing clinical evaluation and are therefore not approved for therapeutic use, distribution in black market products might be expected as observed before for designer steroids [15,16,17], peptides [18,19,20,21,22,23], several selective androgen receptor modulators (SARMs) [24,25,26] and peroxisome proliferator activated receptor δ (PPARδ) agonists e.g., GW501516 [26]. …”

Characterization of in vitro generated metabolites of selected peptides <2 kDa prohibited in sports

“…Peptides with a molecular mass below 2 kDa and assumed performance‐enhancing properties have been in the focus of sports drug testing for several years. Analytical procedures, which mainly utilize liquid chromatographic‐mass spectrometric (LC–MS) approaches, were readily established and adverse analytical findings have been reported worldwide by various different anti‐doping laboratories . In contrast to peptides or proteins >2 kDa, the sample preparation and analysis for lower molecular mass peptides can be handled similarly to the well‐known analysis of non‐peptidic target analytes (such as steroids, stimulants, etc.).…”