Comparison of various in vitro model systems of the metabolism of synthetic doping peptides: Proteolytic enzymes, human blood serum, liver and kidney microsomes and liver S9 fraction

Annual banned‐substance review: Analytical approaches in human sports drug testing

“…In the light of their non‐natural compositions and largely common core structures (relevant for growth hormone secretagogue receptor 1a activation), target testing for these substances has been a viable means to uncover their presence in sports drug‐testing samples. This approach however necessitates a knowledge of the compounds’ biotransformation, and the metabolic fate of GHRP‐1, ‐2, ‐4, ‐5, and ‐6 as well as alexamorelin, hexarelin, and iparmorelin the intricacies of which have been revisited by Zvereva et al Using different in vitro models (including human kidney and liver microsomes, human serum, carboxypeptidases M and B, etc.) various metabolites were generated and characterized by nanoLC‐HRMS, confirming earlier findings of in vitro and in vivo studies and, thus, the utility of in vitro simulations for a variety of metabolic degradation processes.…”

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.).…”

“…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. [1][2][3][4][5][6][7] 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.). Urine analysis represents the most common strategy in doping controls, thus, the metabolism of the administered compound must be taken into consideration when new assays are developed.…”

“…[4,8] Metabolism studies for lower molecular mass performanceenhancing peptides is not entirely new and several earlier studies yielded considerable results lately. [4,7,8,11] Besides the simple incubation with serum or plasma, also classical approaches with human liver microsomes (HLM) or respective enzymes were successfully applied. Separation and detection was usually achieved by means of LC-MS.…”

Determination of GnRH and its synthetic analogues' abuse in doping control: Small bioactive peptide UPLC–MS/MS method extension by addition of in vitro and in vivo metabolism data; evaluation of LH and steroid profile parameter fluctuations as suitable biomarkers

“…Thus, some methods for anti-doping analysis of GnRH, 15 its 3 synthetic analogues (buserelin, triptorelin, leuprolide), 16 and other peptides, such as GHRPs [16][17][18][19][20][21] and TB-500 22 in urine were developed. As was shown in various in vivo 17,[23][24][25] and in vitro 20,24,[26][27][28] studies, prohibited substances metabolites identification is of considerable importance, so some of these methods include not only the parent substance but also target metabolites.…”