Probing for peptidic drugs (2-10 kDa) in doping control blood samples.
This study investigated a method for detecting a broad range of peptide-based doping agents (molecular mass 2–10 kDa) in blood samples collected for anti-doping control purposes. Researchers developed a simplified, generic sample preparation workflow using mixed-mode solid-phase extraction (SPE), coupled with liquid chromatography and high-resolution mass spectrometry (HRMS; resolution >100,000 FWHM) as an initial testing procedure. The target analytes included multiple insulin variants (human and synthetic analogues such as lispro, aspart, glulisine, detemir, glargine, and others), growth hormone–releasing hormones (sermorelin, CJC-1295, tesamorelin), insulin-like growth factors (Long-R3-IGF-I, R3-IGF-I, Des1-3-IGF-I), and mechano growth factors. The study demonstrated that the method met WADA's Technical Document 2022 (TD2022 MRPL) requirements for minimum required performance levels. Proof-of-principle was shown using real post-administration blood samples from subjects treated with synthetic insulin analogues. A key advantage noted was that blood, unlike urine, contains intact peptide hormones at relatively higher concentrations, simplifying detection. Limitations include the study's primarily analytical/methodological scope and the small number of post-administration samples used for validation.
Why this grade: This is a methodological/analytical validation study using a small number of human post-administration samples solely to demonstrate proof-of-principle for a detection technique, not a clinical or interventional trial assessing efficacy or safety outcomes.
Bioactive peptides with a molecular mass between 2 and 10 kDa represent an important class of substances banned in elite sports, which has been recognized with an increasing number and variety of substances by anti-doping organizations. Also, the annually renewed list of prohibited substances of the World Anti-Doping Agency (WADA) explicitly mentions more and more of these peptides, and efficient testing procedures are required. Even under simplified sample preparation conditions, liquid chromatography coupled to high-resolution mass spectrometry (with resolution properties > 100,000 full width at half maximum) offers suitable conditions for this task and can therefore be used as an initial testing procedure. In contrast to urine, blood analysis essentially relies on the detection of intact peptide hormones, and the expected concentrations are commonly higher in blood samples than in urine. This facilitates the analysis, and a generic sample preparation by means of mixed-mode solid-phase extraction could be realized in this study. Co-extraction and analysis of several different peptides such as insulins (human, lispro, aspart, glulisine, tresiba, detemir, glargine, bovine insulin and porcine insulin), growth hormone releasing hormones (sermorelin, CJC-1295 and tesamorelin), insulin-like growth factors (long-R 3 -IGF-I, R 3 -IGF-I and Des 1-3 -IGF-I) and mechano growth factors (human MGF and MGF-Goldspink) with criteria that fulfil the requirements of the WADA documents (TD2022 MRPL) for doping controls. The proof of principle was shown by the analysis of post administration samples after treatment with synthetic insulin analogues.
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