Comprehensive insights into the formation of metabolites of the ghrelin mimetics capromorelin, macimorelin and tabimorelin as potential markers for doping control purposes.

Analytical methods to determine the potential misuse of the ghrelin mimetics capromorelin (CP-424,391), macimorelin (macrilen, EP-01572) and tabimorelin (NN703) in sports were developed. Therefore, different extraction strategies, i.e. solid-phase extraction, protein precipitation, as well as a "dilute-and-inject" approach, from urine and EDTA-plasma were assessed and comprehensive in vitro/in vivo experiments were conducted, enabling the identification of reliable target analytes by means of high resolution mass spectrometry. The drugs' biotransformation led to the preliminary identification of 51 metabolites of capromorelin, 12 metabolites of macimorelin and 13 metabolites of tabimorelin. Seven major metabolites detected in rat urine samples collected post-administration of 0.5-1.0 mg of a single oral dose underwent in-depth characterization, facilitating their implementation into future confirmatory test methods. In particular, two macimorelin metabolites exhibiting considerable abundances in post-administration rat urine samples were detected, which might contribute to an improved sensitivity, specificity, and detection window in case of human sports drug testing programs. Further, the intact drugs were implemented into World Anti-Doping Agency-compliant initial testing (limits of detection 0.02-0.60 ng/ml) and confirmation procedures (limits of identification 0.18-0.89 ng/ml) for human urine and blood matrices. The obtained results allow extension of the test spectrum of doping agents in multitarget screening assays for growth hormone-releasing factors from human urine.

Discovery of urinary biomarkers to discriminate between exogenous and semi-endogenous thiouracil in cattle: A parallel-like randomized design.

In the European Union, the use of thyreostats for animal fattening purposes has been banned and monitoring plans have been established to detect potential abuse. However, this is not always straightforward as thyreostats such as thiouracil may also have a semi-endogenous origin. Therefore, this study aimed at defining urinary metabolites, which may aid in defining the origin of detected thiouracil. Hereto, a parallel-like randomized in vivo study was conducted in which calves (n = 8) and cows (n = 8) were subjected to either a control treatment, rapeseed-enriched diet to induce semi-endogenous formation, or thiouracil treatment. Urine samples (n = 330) were assessed through metabolic fingerprinting, employing liquid-chromatography and Q-ExactiveTM Orbitrap mass spectrometry. Urinary fingerprints comprised up to 40,000 features whereby multivariate discriminant analysis was able to point out significant metabolome differences between treatments (Q2(Y) ≥ 0.873). Using the validated models, a total of twelve metabolites (including thiouracil) were assigned marker potential. Combining these markers into age-dependent biomarker panels rendered a tool by which sample classification could be improved in comparison with thiouracil-based thresholds, and this during on-going thiouracil treatment (specificities ≥ 95.2% and sensitivities ≥ 85.7%), post-treatment (sensitivities ≥ 80% for ≥ 24 h after last administration), and simulated low-dose thiouracil treatment (exogenous thiouracil below 30 ng µL-1). Moreover, the metabolic relevance of revealed markers was supported by the suggested identities, for which a structural link with thiouracil could be determined in most cases. The proposed biomarker panels may contribute to a more justified decision-making in monitoring thiouracil abuse.

Pharmacokinetic and urinary profiling reveals the prednisolone/cortisol ratio as a valid biomarker for prednisolone administration.

BACKGROUND: In Europe, synthetic corticosteroids are not allowed in animal breeding for growth-promoting purposes. Nevertheless, a high prevalence of non-compliant urine samples was recently reported for prednisolone, however, without any indication of unauthorized use. Within this context, 20ß-dihydroprednisolone and the prednisolone/cortisol ratio have been suggested as potential tools to discriminate between exogenous and endogenous urinary prednisolone. In this study, the validity of these strategies was verified by investigating the plasma pharmacokinetic and urinary excretion profiles of relevant glucocorticoids in bovines, subjected to exogenous prednisolone treatment or tetracosactide hexaacetate administration to induce endogenous prednisolone formation. Bovine urine and plasma samples were analysed by liquid chromatography and mass spectrometry. RESULTS: Based on the plasma pharmacokinetics and urinary profiles, 20ß-dihydroprednisolone was confirmed as the main prednisolone-derived metabolite, being detected in the biological fluids of all 12 bovines (plasma AUC0-inf of 121 h µg L-1 and urinary concentration > 0.695 µg L-1). However, this metabolite enclosed no potential as discriminative marker as no significant concentration differences were observed upon exogenous prednisolone treatment or tetracosactide hexaacetate administration under all experimental conditions. As a second marker tool, the prednisolone/cortisol ratios were assessed along the various treatments, taking into account that endogenous prednisolone formation involves the hypothalamic-pituitary-adrenal axis and is associated with an increased cortisol secretion. Significantly lower ratios were observed in case of endogenous prednisolone formation (i.e. ratios ranging from 0.00379 to 0.129) compared to the exogenous prednisolone treatment (i.e. ratios ranging from 0.0603 to 36.9). On the basis of these findings, a discriminative threshold of 0.260 was proposed, which allowed classification of urine samples according to prednisolone origin with a sensitivity of 94.2% and specificity of 99.0%. CONCLUSION: The prednisolone/cortisol ratio was affirmed as an expedient strategy to discriminate between endogenous and exogenous prednisolone in urine. Although the suggested threshold value was associated with high specificity and sensitivity, a large-scale study with varying experimental conditions is designated to optimize this value.

Determination of LongR3-IGF-I, R3-IGF-I, Des1-3 IGF-I and their metabolites in human plasma samples by means of LC-MS.

According to the regulations of the World Anti-Doping Agency (WADA), growth promoting peptides such as the insulin-like growth factor-I (IGF-I) and its synthetic analogues belong to the class of prohibited compounds. While several assays to quantify endogenous IGF-I have been established, the potential misuse of synthetic analogues such as LongR3-IGF-I, R3-IGF-I and Des1-3-IGF-I remains a challenge and superior pharmacokinetic properties have been described for these analogues. Within the present study, it was demonstrated that the target peptides can be successfully detected in plasma samples by means of magnetic beads-based immunoaffinity purification and subsequent nanoscale liquid chromatographic separation with high resolution mass spectrometric detection. Noteworthy, the usage of a specific antibody for LongR3-IGF-I enables the determination in low ng/mL levels despite the presence of an enormous excess of endogenous human IGF-I. In addition, different metabolism studies (in-vitro and in-vivo) were performed using sophisticated strategies such as incubation with skin tissue microsomes, degradation in biological fluids (for all analogues), and administration to rats (for LongR3-IGF-I). Herewith, several C-and N-terminally truncated metabolites were identified and their relevancy was additionally confirmed by in-vivo experiments with rodents. Especially for LongR3-IGF-I, a metabolite ((Des1-11)-LongR3-IGF-I) was identified that prolonged the detectability in-vivo by a factor of approximately 2. The method was validated for qualitative interpretation considering the parameters specificity, identification capability, recovery (26-60%), limit of detection (0.5ng/mL), imprecision (<25%), linearity, stability, and matrix effects. A stable isotope labelled (15N)-IGF-I was used as internal standard to control all sample preparation steps.

Identification and characterization of in vitro and in vivo generated metabolites of the adiponectin receptor agonists AdipoRon and 112254.

Peroxisome proliferator-activated receptors (PPARs), peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), sirtuin 1 (SIRT1) and adenosine monophosphate-activated protein kinase (AMPK) are regulators of transcriptional processes and effects of exercise and pseudo-exercise situations. Compounds occasionally referred to as endurance exercise mimetics such as AdipoRon and 112254, both adiponectin receptor agonists, can be used to simulate the physiology of endurance exercise via pathways including these transcriptional regulators. Adiponectin supports fatty acid utilization and triglyceride-content reduction in cells and increases both the mitochondrial biogenesis and the oxidative metabolism in muscle cells. In routine doping control analysis, knowledge about phase-I and -II metabolic products of target analytes is essential. Hence, in vitro- and in vivo-metabolism experiments are frequently employed tools in preventive doping research to determine potential urinary metabolites for sports drug testing purposes, especially concerning new, (yet) unapproved compounds. In the present study, in vitro assays were conducted using human liver microsomal and S9 fractions, and rat in vivo experiments were performed using both AdipoRon and 112254. For AdipoRon, obtained samples were analyzed using liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry with both electrospray ionization or atmospheric-pressure chemical ionization techniques. Overall, more than five phase I-metabolites were found in vitro and in vivo, including particularly monohydroxylated and hydrogenated species. No phase II-metabolites were found in vitro; conversely, signals suggesting the presence of glucuronic acid or other conjugates in samples collected from in vivo experiment were observed, the structures of which were however not conclusively identified. Also for 112254, several phase-I metabolites were found in vitro, e.g. monohydroxylated and demethylated species. Here, no phase II-metabolites were observed neither using in vitro nor in vivo samples. Based on the generated data, the implementation of metabolites and unmodified drug candidates into routine doping control protocols is deemed warranted for comprehensive sports drug testing programs until human elimination study data are available.

Qualitative identification of growth hormone-releasing hormones in human plasma by means of immunoaffinity purification and LC-HRMS/MS.

The use of growth hormone-releasing hormones (GHRHs) is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA). The aim of the present study was to develop a method for the simultaneous detection of four different GHRHs and respective metabolites from human plasma by means of immunoaffinity purification and subsequent nano-ultrahigh performance liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry. The target analytes included Geref (Sermorelin), CJC-1293, CJC-1295, and Egrifta (Tesamorelin) as well as two metabolites of Geref and CJC-1293, which were captured from plasma samples using a polyclonal GHRH antibody in concert with protein A/G monolithic MSIA™ D.A.R.T.'S® (Disposable Automation Research Tips) prior to separation and detection. The method was fully validated and found to be fit for purpose considering the parameters specificity, linearity, recovery (19-37%), lower limit of detection (<50 pg/mL), imprecision (<20%), and ion suppression/enhancement effects. The analytes' stability and metabolism were elucidated using in vitro and in vivo approaches. EDTA blood samples were collected from rats 2, 4, and 8 h after intravenous administration of GHRH (one compound per test animal). All intact substances were detected for at least 4 h but no anticipated metabolite was confirmed in laboratory rodents' samples; conversely, a Geref metabolite (GHRH3-29) was found in a human plasma sample collected after subcutaneous injection of the drug to a healthy male volunteer. The obtained results demonstrate that GHRHs are successfully detected in plasma using an immunoaffinity-mass spectrometry-based method, which can be applied to sports drug testing samples. Further studies are however required and warranted to account for potential species-related differences in metabolism and elimination of the target analytes.

Expanded test method for peptides >2 kDa employing immunoaffinity purification and LC-HRMS/MS.

Bioactive peptides with an approximate molecular mass of 2-12 kDa are of considerable relevance in sports drug testing. Such peptides have been used to manipulate several potential performance-enhancing processes in the athlete's body and include for example growth hormone releasing hormones (sermorelin, CJC-1293, CJC-1295, tesamorelin), synthetic/animal insulins (lispro, aspart, glulisine, glargine, detemir, degludec, bovine and porcine insulin), synthetic ACTH (synacthen), synthetic IGF-I (longR(3) -IGF-I) and mechano growth factors (human MGF, modified human MGF, 'full-length' MGF). A combined initial test method using one analytical procedure is a desirable tool in doping controls and related disciplines as requests for higher sample throughput with utmost comprehensiveness preferably at reduced costs are constantly issued. An approach modified from an earlier assay proved fit-for-purpose employing pre-concentration of all target analytes by means of ultrafiltration, immunoaffinity purification with coated paramagnetic beads, nano-ultra high performance liquid chromatography (UHPLC) separation, and subsequent detection by means of high resolution tandem mass spectrometry. The method was shown to be applicable to blood and urine samples, which represent the most common doping control specimens. The method was validated considering the parameters specificity, recovery (11-69%), linearity, imprecision (<25%), limit of detection (5-100 pg in urine, 0.1-2 ng in plasma), and ion suppression. The analysis of administration study samples for insulin degludec, detemir, aspart, and synacthen provided the essential data for the proof-of-principle of the method.