Mass spectrometric characterization of urinary hydrafinil metabolites for routine doping control purposes.

Little information on the human metabolism and urinary elimination of hydrafinil (9-fluorenol) exists. In order to support preventive anti-doping activities concerning compounds such as hydrafinil, a pilot elimination study was conducted with three healthy male volunteers receiving a single oral dose of 50 mg of hydrafinil. Urine samples were collected prior to and up to 72-h post-administration and were subjected to both gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry, which allowed for the identification of the intact drug as well as Phase I and Phase II metabolites, primarily hydroxylated and/or glucuronidated or sulfo-conjugated hydrafinil. The identity of these metabolites was corroborated by high-resolution/high-accuracy tandem mass spectrometry, and the applicability of routine doping control workflows for the detection of hydrafinil and its main metabolites was assessed. Therefore, two findings of hydrafinil and its metabolites were recorded, which concerned out-of-competition doping control samples and, hence, were not pursued with confirmatory analyses. Yet, the initial testing procedure results indicate that hydrafinil might require consideration in sports drug testing programs to ensure its detection, if classified as prohibited by the World Anti-Doping Agency (WADA).

Toward a neuroprotective shift: Eight weeks of high intensity interval training reduces the neurotoxic kynurenine activity concurrently to impulsivity in emotionally impulsive humans - A randomized controlled trial.

BACKGROUND: Previous findings suggest that impulsivity is related to chronic low-grade inflammation. Inflammation is known to trigger the kynurenine pathway to a pathological level in various impulsivity-related disorders. Nonetheless, murine models and recent human studies have shown that physical exercise, in particular High Intensity Interval Training (HIIT), could counterbalance the negative effects of inflammation on the kynurenine pathway. AIM: This study evaluates the effects of eight weeks of HIIT versus an active control group on impulsivity levels and accompanying alterations of inflammatory-mediated changes of the kynurenine pathway in a sample of emotionally impulsive humans. METHODS: Participants were randomly allocated to either HIIT or stretching conditions (three trainings per week for eight weeks). Fitness level was evaluated via VO2peak values at the beginning at end of the intervention. Kynurenine metabolites, pro-inflammatory cytokines, and impulsivity levels were evaluated at T0, T4, and T8 weeks. Statistical analyses were performed using mixed models. RESULTS: Fifty-three participants were included in the modified Intention To Treat analysis (45 finished the intervention). The HIIT group (n = 28) largely increased the aerobic fitness of its participants and produced physiological changes while the stretching group (n = 25) did not. HIIT reduced interleukin 6 levels (small to moderate interaction) and reduced the activity of the neurotoxic branch of the kynurenine pathway (small to moderate interaction for KYNA/QA and KYN/QA) after eight weeks of training while the active control did not change. Both interventions were effective to decrease emotion-related impulsivity, however only the HIIT group decreased participants' emotion-unrelated levels. Changes in emotion-related and -unrelated impulsivity were moderately correlated to changes in KYNA/KYN. CONCLUSION: This study demonstrated that HIIT was able to switch the kynurenine pathway from its neurotoxic branch to its neuroprotective one. This shift was associated with a decrease in impulsivity. Based on these findings, future work may consider investigating more intensively the effect of HIIT on impulsivity-related disorders.

Exercise Diminishes Plasma Neurofilament Light Chain and Reroutes the Kynurenine Pathway in Multiple Sclerosis.

OBJECTIVE: To examine acute (single-bout) and training effects of high-intensity interval training (HIIT) vs standard exercise therapy (moderate continuous training [MCT]) on plasma neurofilament light chain (pNfL) and kynurenine (KYN) pathway of tryptophan degradation metabolites in persons with multiple sclerosis (pwMS). METHODS: Sixty-nine pwMS (Expanded Disability Status Scale score 3.0-6.0) were randomly assigned to a HIIT or an MCT group. Changes in pNfL and KYN pathway metabolites measured in blood plasma were assessed before, after, and 3 hours after the first training session as well as after the 3-week training intervention. RESULTS: Acute exercise reduced pNfL and increased the KYN pathway flux toward the neuroprotective kynurenic acid (KA). Changes in pNfL correlated positively with changes in KA and negatively with the quinolinic acid-to-KA ratio. HIIT consistently led to greater effects than MCT. Following the 3-week training intervention, the KYN pathway was activated in HIIT compared with MCT. CONCLUSION: Future studies and clinical assessments of pNfL should consider acute exercise as confounding factor for measurement reliability. Moreover, exercise-induced KYN pathway rerouting might mediate neuroprotection, potentially underlying the benefits in rehabilitation for pwMS. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that acute HIIT diminishes pNfL and increases KA levels, and 3 weeks of HIIT activate the KYN pathway in pwMS. TRIAL REGISTRATION INFORMATION: Clinical trial registration number: NCT03652519.

Detection of anti-SARS-CoV-2 antibodies in dried blood spots utilizing manual or automated spot extraction and electrochemiluminescence immunoassay (ECLIA).

Serological test methods to detect anti-SARS-CoV-2 antibodies represent a major measure to manage the pandemic caused by the coronavirus disease 2019 (COVID-19). In this communication, test results obtained from minimal-invasively collected dried blood spot (DBS) specimens, which can be sampled 'at home' without the need of medically trained personnel, are compared to conventionally collected venous blood samples. DBS samples were prepared for analysis either manually or by a card extraction robot, and electrochemiluminescence assay (ECLIA) characteristics, assay readout values as well as stability data covering a period of more than 200 days are provided. Constant anti-SARS-CoV-2 antibody readouts of quality control DBS were obtained over the entire test period using DBS specimens stored under dry and dark conditions. In addition, test results obtained from individuals tested twice within 10 months post-infection indicated a consistent presence of antibodies.

Effect of a Single Bout of Aerobic Exercise on Kynurenine Pathway Metabolites and Inflammatory Markers in Prostate Cancer Patients-A Pilot Randomized Controlled Trial.

The kynurenine (KYN) pathway gains growing research interest concerning the genesis, progression and therapy of solid tumors. Previous studies showed exercise-induced effects on metabolite levels along the KYN pathway. Modulations of the KYN pathway might be involved in the positive impact of exercise on prostate cancer progression and mortality. The objective of this trial was to investigate whether a single-physical exercise alters tryptophan (TRP) metabolism and related inflammatory markers in this population. We conducted a randomized controlled trial with 24 patients suffering from prostate cancer. While the control group remained inactive, the intervention group performed a 30-min aerobic exercise on a bicycle ergometer at 75% of individual VO2peak. Before (t0) and directly after the exercise intervention (t1) KYN, TRP, kynurenic acid, quinolinic acid as well as various inflammation markers (IL6, TNF-α, TGF-ß) were measured in blood serum. At baseline, the present sample showed robust correlations between TRP, KYN, quinolinic acid and inflammatory markers. Regarding the exercise intervention, interaction effects for TRP, the KYN/TRP ratio and TGF-ß were observed. The results show for the first time that acute physical exercise impacts TRP metabolism in prostate cancer patients. Moreover, baseline associations underline the relationship between inflammation and the KYN pathway in prostate cancer.

Analysis of cobalt for human sports drug testing purposes using ICP- and LC-ICP-MS.

Due to the current demands in the fight against manipulation of blood and blood components, commonly referred to as "blood doping" in sports drug testing, specific and sensitive detection methods enabling the detection of prohibited substances and methods of doping are required. Similar to illicit blood transfusions, erythropoiesis stimulating agents have been shown to be misused in sport, aiming at improving an athlete's aerobic capacity and endurance performance. Amongst other strategies, the administration of ionic cobalt (Co2+ ) can increase the number of erythrocytes by stimulating the endogenous erythropoietin (EPO) biosynthesis. Conversely, several organic Co-containing compounds such as cyanocobalamin (vitamin B12) are not prohibited in sports, and thus, an analytical differentiation of permitted and banned contributions to urinary Co-concentrations is desirable. An excretion study with daily applications of either 1 mg of CoCl2 or 1 mg of cyanocobalamin was conducted with 20 volunteers over a period of 14 consecutive days. Urine, plasma, and concentrated red blood cells were analyzed for their cobalt content. The samples were collected starting 7 days before the administration until 7 days after. Total Co concentrations were analyzed by using inductively coupled plasma mass spectrometry (ICP-MS), which yielded significantly elevated levels exclusively after inorganic cobalt intake. Furthermore, a liquid chromatography (LC)-ICP-MS approach was established and employed for the simultaneous determination of organically bound and inorganic cobalt by chromatographic separation within one single run. The analytical approach offers the option to further develop detection methods of illegal Co2+ supplementation in sport.

Elimination profiles of microdosed ostarine mimicking contaminated products ingestion.

The possibility of nutritional supplement contamination with minute amounts of the selective androgen receptor modulator (SARM) ostarine has become a major concern for athletes and result managing authorities. In case of an adverse analytical finding (AAF), affected athletes need to provide conclusive information, demonstrating that the test result originates from a contamination scenario rather than doping. The aim of this research project was to study the elimination profiles of microdosed ostarine and characterize the time-dependent urinary excretion of the drug and selected metabolites. Single- and multi-dose administration studies with 1, 10, and 50 µg of ostarine were conducted, and collected urine samples were analyzed by LC-MS/MS following solid-phase extraction or enzymatic hydrolysis combined with liquid-liquid extraction. In the post-administration samples, both the maximum urine concentrations/abundance ratios and detection times of ostarine and its phase-I and phase-II metabolites were found to correlate with the administered drug dose. With regard to the observed maximum levels of ostarine, the time points of peak urinary concentrations/abundance ratios, and detection windows, a high inter-individual variation was observed. However, the study demonstrated that a single oral dose of as little as 1 µg can be detected for up to 9 (5) days by monitoring ostarine (glucuronide), and hydroxylated metabolites (especially M1a) appear to offer a considerably shorter detection window. The obtained data on ostarine (metabolite) detection times and urinary concentrations following different administration schemes support the interpretation of AAFs, in particular when scenarios of proven supplement contamination are discussed and supplement administration protocols exist.

Detection of follistatin-based inhibitors of the TGF-ß signaling pathways in serum/plasma by means of LC-HRMS/MS and Western blotting.

Cytokines of the transforming growth factor beta (TGF-ß) superfamily such as myostatin and activin A are considered as key regulators of skeletal muscle mass. In vivo, their activity is controlled by different binding proteins such as follistatin (FST), whose interaction with the circulating growth factors prevents activation of the activin type II receptors. FST-based protein therapeutics are therefore not only promising drug candidates for the treatment of muscular diseases but also potential performance-enhancing agents in sports. Within this study, two complementary detection assays for FST-based inhibitors of the TGF-ß signaling pathways in doping control serum and plasma samples were developed by using both monomeric FST and dimeric FST-Fc fusion proteins as model compounds. The initial testing procedure is based on immunoaffinity purification, tryptic digestion, and LC-HRMS/MS, offering high specificity by targeting tryptic signature peptides of FST. As the glycoprotein is also produced endogenously, the confirmation method employs immunoaffinity purification, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and Western blotting in order to detect the intact proteins and differentiate synthetic FST-Fc constructs from naturally occurring FST isoforms. Both assays were found to be highly specific with an estimated detection limit of 10 ng/ml. Moreover, a commercial sandwich enzyme-linked immunosorbent assay was used to determine endogenous FST values. The detected FST serum levels of healthy volunteers were found below 5 ng/ml, which is in accordance with reference values from the literature and below the doping control detection methods' limit of detection (LOD). The presented assays expand the range of available tests for emerging doping agents, and the initial testing procedure can readily be modified to include further protein drugs.

Implementation of the HIF activator IOX-2 in routine doping controls - Pilot study data.

Early in 2020, racehorse doping cases revolved around the hypoxia-inducible factor (HIF) activator IOX-2. While the composition of IOX-2 has also been known and monitored in human doping controls for several years, the testing capability of routine sports drug testing methods was revisited for this newly surfaced doping agent. IOX-2 and the analytically well-established HIF activator roxadustat (FG-4592) share identical precursor/product ion pairs, enabling their co-detection in existing initial testing procedures in routine doping controls for the intact unconjugated analytes. In addition, hydroxylated IOX-2 and the corresponding glucuronic acid conjugates were identified as major metabolites in a microdose elimination study, contributing to enhanced initial testing and confirmation procedures.

Acute hypertrophic but not maximal strength loading transiently enhances the kynurenine pathway towards kynurenic acid.

PURPOSE: Due to distinct immuno- and neuro-modulatory properties, growing research interest focuses on exercise-induced alterations of the kynurenine (KYN) pathway in healthy and clinical populations. To date, knowledge about the impact of different acute strength exercise modalities on the KYN pathway is scarce. Therefore, we investigated the acute effects of hypertrophic (HYP) compared to maximal (MAX) strength loadings on the KYN pathway regulation. METHODS: Blood samples of twelve healthy males (mean age and weight: 23.5 ± 3.2 years; 77.5 ± 7.5 kg) were collected before (T0), immediately after (T1), and 1 h after completion (T2) of HYP (5 sets with 10 repetitions at 80% of 1RM) and MAX (15 sets with 1RM) loadings performed in a randomized cross-over design. Serum concentrations of tryptophan (TRP), KYN, kynurenic acid (KA), and quinolinic acid (QA) were assessed using high-performance liquid chromatography. RESULTS: The KA/KYN ratio increased from T0 to T1 (p = 0.01) and decreased from T1 to T2 (p = 0.011) in HYP, while it was maintained within MAX. Compared to MAX, serum concentrations of KA were greater in HYP at T1 (p = 0.014). Moreover, the QA/KA ratio was significantly lower in HYP than in MAX at T1 (p = 0.002). CONCLUSION: Acute HYP loading led to increases in the metabolic flux yielding KA, thereby possibly promoting immunosuppression and neuroprotection. Our findings emphasize the potential of acute HYP exercise as short-term modulator of KYN pathway downstream to KA in healthy males and need to be proven in other samples.

Exercise and the Kynurenine pathway: Current state of knowledge and results from a randomized cross-over study comparing acute effects of endurance and resistance training.

INTRODUCTION: The essential amino acid tryptophan (TRP) is primarily degraded through the kynurenine (KYN) pathway, which is dysregulated in several chronic diseases. KYN pathway metabolites have immune- and neuro-modulatory properties and are involved in th de novo synthesis of nicotinamide adenine dinucleotide (NAD+). Currently, little evidence exists demonstrating that physical exercise may influence this pathway. However, differences between acute and chronic stimuli as well as the influence of exercise modalities remain to be investigated. Here, we provide an overview of existing studies and present results of a randomized cross-over trial on acute effects of a single-bout of resistance and endurance exercise. METHODS: 24 healthy male adults conducted both an acute endurance exercise (EE) and resistance exercise (RE) session. Blood samples were collected before, immediately after and one hour after cessation of each exercise session. Outcomes comprised serum levels of TRP, KYN, kynurenic acid (KA), quinolinic acid (QA) and calculated ratios. Gene expression of the enzymes indoleamine 2,3 dioxygenase (IDO) 1 and kynurenine aminotransferase (KAT) 4 was measured in peripheral blood mononuclear cells (PBMCs). Moreover, serum concentrations of the potential KYN pathway mediators interleukin (IL)-6 and cortisol were determined. Finally, we investigated baseline correlations between immune cell subsets, potential mediators and initial KYN pathway activation outcomes. RESULTS: The KYN/TRP ratio correlated positively with IL-6 and CD56bright NK-cells and negatively with CD56dim NKcells. Expression of IDO1 in PBMCs correlated positively with IL-6, regulatory T-cells and CD56bright NK-cells, whereas negative correlations to cytotoxic T-cells and CD56dim NKcells were revealed. A significant time effect on KYN/TRP ratio was detected for RE. Regarding KA and KA/KYN ratio, an increase after exercise followed by a decrease at the follow- up measurement was revealed in EE. KAT4 expression also increased after exercise in EE. Moreover, elevated QA levels were observed after the EE session. CONCLUSIONS: In contrast to chronic exercise interventions, single-bouts of endurance exercise provoke acute alterations on KYN pathway outcomes in humans. Our results indicate that EE induces stronger alterations than RE. Enhanced conversion of KYN to both, KA and QA suggest a peripheral KYN clearance, thereby preventing pathological accumulation within the CNS. Future acute and chronic exercise studies are needed to examine the role of NAD+ synthesis starting with TRP and the interplay between KYN pathway activation and mid- to long-term immunological modulations.

Development of a mass spectrometry based detection method for the mitochondrion-derived peptide MOTS-c in plasma samples for doping control purposes.

RATIONALE: The mitochondrial open reading frame of 12S rRNA type-c (MOTS-c) peptide was recently discovered and described to control metabolic homeostasis through AMPK activation along with AICAR accumulation. Consequently, it appears advisable to monitor the potential use of synthetic MOTS-c in sports, and a detection method suitable for sports drug testing purposes is necessary. METHODS: For the detection of MOTS-c in doping control plasma samples, a test method employing liquid chromatography and mass spectrometry (LC/MS) was developed. Following optimization, the assay was comprehensively validated and additional parameters such as the (long-term) stability and in vitro metabolism of the peptide were evaluated. In order to determine endogenous MOTS-c reference limits, the results generated by LC/MS-based detection were compared with those obtained with a commercially available enzyme-linked immunosorbent assay (ELISA). RESULTS: The LC/MS-based test method was fully validated for quantitative results interpretation according to the World Anti-Doping Agency's International Standard for Laboratories (WADA's ISL). It was found to be specific and sensitive, enabling a lower limit of detection (LLOD) for hMOTS-c in plasma at 100 pg/mL. Following optimization, animal MOTS-c analogues and four metabolites as well as two oxidation products were implemented. However, endogenous levels of a reference population of 20 healthy subjects studied by ELISA experiments (45.9-218.5 ng/mL) could not be confirmed by LC/MS. CONCLUSIONS: A mass spectrometric detection assay for MOTS-c in human plasma samples was developed and successfully validated according to WADA's ISL, providing an additional tool for future doping control purposes. Besides MOTS-c, the assay also includes four in vitro derived metabolites and two oxidation products, which might further improve the traceability of the drug. The analytical approach was compared with a commercially available ELISA, and considerable differences in measured MOTS-c levels were observed.

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

With an increasing number of prohibited substances in doping controls, knowledge about their metabolism is crucial for efficient analysis. While for low molecular mass molecules, standard protocols for in vitro metabolism experiments are well established, the situation with peptidic drugs has been shown to be substantially more heterogeneous and complex. Two principle strategies aiming at simulating the metabolism of lower molecular mass peptides in vitro are presented within this study. The prohibited peptides ARA-290, GHRP-3, and Peforelin, with a to-date unknown metabolism, were chosen as model compounds for these experiments and metabolism after incubation with different blood specimens (EDTA-, heparin-, citrate-plasma, and serum) and exposure to recombinant amidase were investigated. The characterization of in vitro generated drug-derived peptidic analytes was accomplished by means of liquid chromatography coupled to high resolution mass spectrometry. Identification of the generated metabolites was ensured by dedicated high resolution product ion experiments after liquid chromatographic separation. While extensive exopeptidase-driven metabolism was observed for ARA-290 (with one main metabolite PyrEQLERALN), GHRP-3 and Peforelin were found to exhibit a considerable metabolic stability with a low tendency for deamidation only. Copyright © 2017 John Wiley & Sons, Ltd.

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.

In vitro metabolism studies on the selective androgen receptor modulator (SARM) LG121071 and its implementation into human doping controls using liquid chromatography-mass spectrometry.

LG121071 is a member of the tetrahydroquinolinone-based class of selective androgen receptor modulator (SARM) drug candidates. These nonsteroidal compounds are supposed to act as full anabolic agents with reduced androgenic properties. As SARMs provide an alternative to anabolic androgenic steroids, they represent an emerging class of potential doping substances abused by athletes for illicit performance enhancement. According to the World Anti-Doping Agency's regulations, SARMs are banned substances and part of the Prohibited List since 2008. In consideration of the increasing number of adverse analytical findings in doping controls caused by SARMs abuse, potential drug candidates such as LG121071 have been proactively investigated to enable a timely integration into routine testing procedures even though clinical trials are not yet complete. In the present approach, the collision-induced dissociation (CID) of LG121071 was characterized by means of electrospray ionization-high resolution/high accuracy mass spectrometry, MS(n), and isotope labeling experiments. Interestingly, the even-electron precursor ion [M + H](+) at m/z 297 was found to produce a radical cation at m/z 268 under CID conditions, violating the even-electron rule that commonly applies. For doping control purposes, metabolites were generated in vitro and a detection method for urine samples based on liquid chromatography-tandem mass spectrometry was established. The overall metabolic conversion of LG121071 was modest, yielding primarily mono-, bis- and trishydroxylated species. Notable, however, was the identification of a glucuronic acid conjugate of the intact drug, attributed to an N-glucuronide structure. The sample preparation procedure included the enzymatic hydrolysis of glucuronides prior to liquid-liquid extraction, allowing intact LG121071 to be measured, as well as the corresponding phase-I metabolites. The method was characterized concerning inter alia lower limit of detection (0.5 ng mL(-1) in urine), recovery (40%), and intra-/interday precision (2.3% to 11.7%) to assess its fitness for purpose. Prospectively, the assay can serve as detection method for LG121071 in drug testing and/or doping controls.