Abstract
In the context of this thesis, detection methods for banned substances of peptidic and pseudopeptidic nature were developed from urine, blood and dried blood spots (DBS). In addition, metabolism studies were conducted to study the biotransformation of three new ghrelin mimetics.
The results were published as three research articles in different international peer-reviewed scientific journals. These articles report on the development of liquid chro-matography-mass spectrometry-based detection methods and in vitro/in vivo metabo-lism studies. The target analytes of these methods which were proteins or peptides and pseudo-peptides were expected to be misused by athletes due to their potential performance-enhancing effects. Therefore, they are part of the World Anti-Doping Agency's (WADA) Prohibited List. In detail, these were first, sotatercept and luspater-cept, two Fc-fusion proteins, and bimagrumab, a therapeutic antibody; second, a total of 46 lower molecular mass peptides and pseudo-peptides (< 2 kDa), including growth hormone-releasing factors, luteinizing hormone releasing hormones, antidiuretic hor-mones, and other banned substances including representative metabolites, and third, three ghrelin mimetics, namely capromorelin, macimorelin and tabimorelin. Further-more, both, the conventional biological matrices urine and blood and the upcoming alternative matrix DBS, were considered for method development. The detection of pharmacologically relevant drug levels from a small volume of DBS was successfully achieved by using efficient sample preparation protocols and state-of-the-art analytical instrumentation. Both, manual and automated sample preparation strategies were applied within the presented studies. The analytes were extracted and purified from DBS in aqueous buffer using ultrasonication and subsequent immunoaffinity purifica-tion or by robot-assisted aqueous flow-through desorption followed by solid phase ex-traction (SPE). For the ghrelin mimetics, manual SPE or a “dilute-and-inject” approach was applied for urine sample preparation. Purification of analytes from blood plasma was achieved by protein precipitation with methanol. The analytical methods were de-signed as initial testing procedures and/or confirmation procedures, and were optimized and fully validated according to the current version of the WADA guidelines. Finally, as a proof-of-concept, authentic human samples were analyzed, provided that the application of the drugs were ethically justifiable.
ABSTRACT
170
This thesis presents an important contribution to the detection of doping-relevant (pseudo-)peptides and proteins from DBS. The results could conduce to the establish-ment of DBS as an alternative matrix complementary to the conventional matrices blood and urine in future doping control scenarios. With regard to the upcoming Olympic and Paralympic Winter Games in Beijing, China (2022), where DBS will be used as official matrix for the first time, the analytical spectrum for DBS analysis was successfully extended by these substances. In this context, DBS proved to be a suit-able matrix for multi-target screening approaches, also enabling retrospective data analysis. The new methods for the detection of ghrelin mimetics will henceforth extend the multiplexed analytical routine screenings of growth hormone releasing factors from human blood and urine. Moreover, important information about the metabolites of these pseudopeptidic substances (with suspected short plasma half-life) were ob-tained, which could further improve the specificity and sensitivity of analytical detection methods for future human doping controls.
The results were published as three research articles in different international peer-reviewed scientific journals. These articles report on the development of liquid chro-matography-mass spectrometry-based detection methods and in vitro/in vivo metabo-lism studies. The target analytes of these methods which were proteins or peptides and pseudo-peptides were expected to be misused by athletes due to their potential performance-enhancing effects. Therefore, they are part of the World Anti-Doping Agency's (WADA) Prohibited List. In detail, these were first, sotatercept and luspater-cept, two Fc-fusion proteins, and bimagrumab, a therapeutic antibody; second, a total of 46 lower molecular mass peptides and pseudo-peptides (< 2 kDa), including growth hormone-releasing factors, luteinizing hormone releasing hormones, antidiuretic hor-mones, and other banned substances including representative metabolites, and third, three ghrelin mimetics, namely capromorelin, macimorelin and tabimorelin. Further-more, both, the conventional biological matrices urine and blood and the upcoming alternative matrix DBS, were considered for method development. The detection of pharmacologically relevant drug levels from a small volume of DBS was successfully achieved by using efficient sample preparation protocols and state-of-the-art analytical instrumentation. Both, manual and automated sample preparation strategies were applied within the presented studies. The analytes were extracted and purified from DBS in aqueous buffer using ultrasonication and subsequent immunoaffinity purifica-tion or by robot-assisted aqueous flow-through desorption followed by solid phase ex-traction (SPE). For the ghrelin mimetics, manual SPE or a “dilute-and-inject” approach was applied for urine sample preparation. Purification of analytes from blood plasma was achieved by protein precipitation with methanol. The analytical methods were de-signed as initial testing procedures and/or confirmation procedures, and were optimized and fully validated according to the current version of the WADA guidelines. Finally, as a proof-of-concept, authentic human samples were analyzed, provided that the application of the drugs were ethically justifiable.
ABSTRACT
170
This thesis presents an important contribution to the detection of doping-relevant (pseudo-)peptides and proteins from DBS. The results could conduce to the establish-ment of DBS as an alternative matrix complementary to the conventional matrices blood and urine in future doping control scenarios. With regard to the upcoming Olympic and Paralympic Winter Games in Beijing, China (2022), where DBS will be used as official matrix for the first time, the analytical spectrum for DBS analysis was successfully extended by these substances. In this context, DBS proved to be a suit-able matrix for multi-target screening approaches, also enabling retrospective data analysis. The new methods for the detection of ghrelin mimetics will henceforth extend the multiplexed analytical routine screenings of growth hormone releasing factors from human blood and urine. Moreover, important information about the metabolites of these pseudopeptidic substances (with suspected short plasma half-life) were ob-tained, which could further improve the specificity and sensitivity of analytical detection methods for future human doping controls.
| Original language | German |
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| Place of Publication | Köln |
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| Publisher | Deutsche Sporthochschule Köln |
| Number of pages | 185 |
| Publication status | Published - 2022 |