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Rationale: Anti-SARS-CoV-2 antibody responses elicited by infection or vaccination vary among individuals and over time. Their knowledge is of particular importance in, amongst others, elite sport. Can dried blood spots serve as a minimally-invasive, low-cost, decentralized tool to monitor the quantitative antibody response and thus represent an alternative to full blood tests?Methods: Cross-validation of dried blood and venous blood samples of 27 individuals post-infection and 96 individuals post-vaccination, longitudinal antibody monitoring of 27 individuals after vaccination using different vaccines and vaccine schedules and detection of seropositive individuals in a cohort of 557 people using self-collected DBS (dried blood spots) and two commercial immunoassays.Findings: Plasma and DBS values were highly correlated allowing for extrapolation of plasma values from DBS using a factor of a least 10 following the presented procedure. Capillary volumetric sampling and self-sampling produced reliable results. After vaccination, participants showed heterogenous antibody responses but a consistent increase after the second dose. DBS allowed for the analysis of a huge sample volume in a timely manner by limited laboratory personnel.Discussion: DBS offer the possibility of infection and vaccination traceability of individuals and cohorts via minimally-invasive self-sampling. This way, they allow to screen and monitor the presence and evolution of anti-SARS-CoV-2 antibodies in a qualitative and quantitative manner. Using two commercial, automated assays enables large-scale and frequent testing, global implementation and comparability of results.Key Words: Coronavirus, Pandemic, Diagnostics, Immunity, DBS

For decades, blood testing has been an integral part of routine doping controls. The breadth of information contained in blood samples has become considerably more accessible for anti-doping purposes over the last 10 years through technological advancements regarding analytical instrumentation as well as enhanced sample collection systems. Particularly, microsampling of whole blood and serum, for instance as dried blood spots (DBS), has opened new avenues in sports drug testing and substantially increased the availability and cost-effectiveness of doping control specimens. Thus, microvolume blood specimens possess the potential to improve monitoring of blood hormone and drug levels, support evaluation of circulating drug concentrations in competition, and enhance the stability of labile markers and target analytes in blood passport analyses as well as peptide hormone and steroid ester detection. Further, the availability of the fraction of lysed erythrocytes for anti-doping purposes warrants additional investigation, considering the sequestering capability of red blood cells (RBCs) for certain substances, as a complementary approach in support of the clean sport. [ABSTRACT FROM AUTHOR]

Objectives: The mechanism by which methotrexate (MTX) improves glucose homeostasis in patients with rheumatoid (RA) and psoriatic arthritis (PsA) remains undetermined. Animal studies indicate a role for intracellular accumulation of 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl 5'-monophosphate (ZMP) but this has not been directly demonstrated in humans. We explored whether accumulation of ZMP is associated with improvements in glucose homeostasis during MTX therapy.Method: MTX-naïve, non-diabetic RA (n = 16) and PsA (n = 10) patients received uninterrupted MTX treatment for 6 months. To evaluate whether ZMP accumulated during MTX therapy, we measured the concentration of ZMP in erythrocytes and the concentration of its dephosphorylated derivative 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) in urine using liquid chromatography mass spectrometry (LC-MS/MS). To assess glucose homeostasis, we determined the concentration of glycated haemoglobin (HbA1c) and homeostasis model assessment of insulin resistance [HOMA-IR: fasting glucose (mmol/L) × fasting insulin (μU/mL)/22.5].Results: Erythrocyte ZMP and urinary AICAR concentrations did not increase during 6 months of MTX therapy. HbA1c concentration was reduced from 5.80 ± 0.29% at baseline to 5.51 ± 0.32% at 6 months (p < 0.001), while HOMA-IR remained unaltered. Reduction in HbA1c concentration was not associated with increased ZMP or AICAR concentrations.Conclusions: MTX therapy probably does not produce a chronic increase in erythrocyte ZMP or urinary AICAR concentrations. Collectively, our data do not support the hypothesis that MTX improves glucose homeostasis through chronic accumulation of ZMP. [ABSTRACT FROM AUTHOR]

Prenylamine is a vasodilator of phenylalkylamine structure and was used for the treatment of angina pectoris, until reports of undesirable effects including ventricular tachycardia led to a decreasing use of the drug in the 1980s. Metabolic N-dealkylation of orally ingested prenylamine can liberate amphetamine in humans and cause positive findings for amphetamine in doping and forensic analysis. In 2010, the World Anti-Doping Agency (WADA) classified prenylamine as a non-specified stimulant according to the 2010 Prohibited List, thus banning its use in sports in-competition. Supporting the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based detection method, a post-administration urine sample following a single oral prenylamine ingestion (Segontin® 60 mg) was analyzed for urinary metabolites. The LC-separated analytes were ionized in positive electrospray ionization (ESI) mode and detected as protonated ions using an AB Sciex TripleTOF 5600 quadrupole-time-of-flight hybrid mass spectrometer. Over 40 phase I metabolites were detected, including previously unknown mono- bis-, tris- and tetra-hydroxylated prenylamine, several hydroxylated and methoxylated prenylamine metabolites and (hydroxylated) diphenylpropylamine. Investigation of the collision-induced dissociation behaviours of the metabolites by high resolution/high accuracy mass spectrometry allowed for the assignment of the nature and the site of observed metabolic transformations. The most abundant phase I metabolite was confirmed as p-hydroxy-prenlyamine by chemical synthesis and stable isotope labelling of reference material. An existing routine screening assay based on direct injection and LC-MS/MS analysis of urine was modified and validated according to common guidelines, in order to allow for the detection of p-hydroxy-prenylamine in sports drug testing. The assay demonstrated the ability to detect the target metabolite at 0.1 ng/ml at intra- and inter-day imprecisions below 10%. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The misuse of the sympathomimetic and anabolic agent clenbuterol has been frequently reported in professional sport and in the livestock industry. In 2010, a team of athletes returned from competition in China and regular doping control samples were taken within the next two days. All urine samples contained low amounts (pg/ml) of clenbuterol, drawing the attention to a well-known problem: the possibility of an unintended clenbuterol intake with food. A warning that Chinese meat is possibly contaminated with prohibited substances according to international anti-doping regulations was also given by Chinese officials just before the Bejing Olympic Games in 2008. To investigate if clenbuterol can be found in human urine, a study was initiated comprising 28 volunteers collecting urine samples after their return from China. For the quantification of clenbuterol at a low pg/ml level, a very sensitive and specific isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed using liquid/liquid re-extraction for clean-up with a limit of detection and quantification of 1 and 3 pg/ml, respectively. The method was validated demonstrating good precision (intra-day: 2.9-5.5 %; inter-day: 5.1-8.8%), accuracy (89.5-102.5%) and mean recovery (81.4%). Clenbuterol was detectable in 22 (79%) of the analyzed samples, indicating a general food contamination problem despite an official clenbuterol prohibition in China for livestock. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

To indicate homologous or autologous blood transfusion in sports drug testing, quantification of increased urinary concentrations of di(2-ethylhexyl) phthalate (DEHP) metabolites presents a promising approach; however, the possible intra-individual variation of the metabolite concentrations over time has not been well characterized. The aim of this study was to explore the intra-individual variability of urinary DEHP metabolites among seven volunteers without special occupational exposure to DEHP during one week (n = 253) in order to investigate the possibility of increased urinary concentrations of the metabolites caused by, for example, residential, dietary, or environmental exposure. Quantification of three DEHP metabolites - mono(2-ethylhexyl) phthalate, mono(2-ethyl-5-oxohexyl) phthalate, and mono(2-ethyl-5-hydroxyhexyl) phthalate - was accomplished after enzymatic hydrolysis of urinary glucuronide conjugates and direct injection using isotope-dilution liquid chromatography-tandem mass spectrometry. Although urinary concentrations of DEHP metabolites showed considerable intra-individual variation, no increased values were observed comparable to the concentrations measured in urine specimens collected after blood transfusion. Copyright © 2011 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

A new multi-target approach based on liquid chromatography - electrospray ionization tandem mass spectrometry (LC-(ESI)-MS/MS) is presented to screen for various classes of prohibited substances using direct injection of urine specimens. With a highly sensitive new generation hybrid mass spectrometer classic groups of drugs - for example, diuretics, beta2-agonists - stimulants and narcotics are detectable at concentration levels far below the required limits. Additionally, more challenging and various new target compounds could be implemented. Model compounds of stimulant conjugates were studied to investigate a possible screening without complex sample preparation. As a main achievement, the integration of the plasma volume expanders dextran and hydroxyethyl starch (HES), commonly analyzed in time-consuming, stand-alone procedures, is accomplished. To screen for relatively new prohibited compounds, a common metabolite of the selective androgen receptor modulator (SARMs) andarine, a metabolite of growth hormone releasing peptide (GHRP-2), and 5-amino-4-imidazolecarboxyamide ribonucleoside (AICAR) are analyzed. Following a completely new approach, conjugates of di(2-ethylhexyl) phthalate (DEHP) metabolites are monitored to detect abnormally high levels of plasticizers indicating for illicit blood transfusion. The assay was fully validated for qualitative purposes considering the parameters specificity, intra- (3.2-16.6%) and inter-day precision (0.4-19.9%) at low, medium and high concentration, robustness, limit of detection (1-70 ng/ml, dextran: 30 µg/ml, HES: 10 µg/ml) and ion suppression/enhancement effects. The analyses of post-administration and routine doping control samples demonstrates the applicability of the method for sports drug testing. This straightforward and reliable approach accomplishes the combination of different screening procedures resulting in a high-throughput method that increases the efficiency of the labs daily work. Copyright © 2011 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The article focuses on a study related to the analysis of incorporation of meldonium drug in erythrocytes. Topics discussed include a monitoring program of the World Anti-Doping Agency, application of drug for prevention of diabetes, neurodegenerative disorders & bronchopulmonary diseases and a study protocol approved by local ethics committee of the National Institute for Sports Research.

RATIONALE: The issue of illicit performance enhancement spans human and animal sport in presumably equal measure, with prohibited substances and methods of doping conveying both ways. Due to the proven capability of unbound ionic cobalt (Co2+) to stimulate erythropoiesis in humans, both human and equine anti-doping regulations have listed cobalt as a banned substance, and in particular in horse drug testing, thresholds for cobalt concentrations applying to plasma and urine have been suggested or established. Recent reports about the finding of substantial amounts of undeclared nickel in arguably licit performance- and recovery-supporting products raised the question whether the ionic species of this transition metal (Ni2+), which exhibits similar prolyl hydroxylase inhibiting properties to Co2+, has been considered as a substitute for cobalt in doping regimens. METHODS: Therefore, a pilot study with 200 routine post-competition doping control horse urine samples collected from animals participating in equestrian, gallop, and trotting in Europe was conducted to provide a first dataset on equine urinary Ni2+ concentrations. All specimens were analyzed by conventional inductively coupled plasma mass spectrometry (ICP-MS) to yield quantitative data for soluble nickel. RESULTS: Concentrations ranging from below the assay's limit of quantification (LOQ, 0.5 ng/mL) up to 33.4 ng/mL with a mean value (± standard deviation) of 6.1 (±5.1) ng/mL were determined for the total nickel content. CONCLUSIONS: In horses, nickel is considered a micronutrient and feed supplements containing nickel are available; hence, follow-up studies are deemed warranted to consolidate potential future threshold levels concerning urine and blood nickel concentrations in horses using larger sets of samples for both matrices and to provide in-depth insights by conducting elimination studies with soluble Ni2+-salt species. [ABSTRACT FROM AUTHOR]

Terbutaline is a fast-acting β2-adrenergic agonist used in the treatment of obstructive pulmonary diseases. Doping control for β2-agonists, which are forbidden in sports by the World Anti-doping Agency (WADA), is performed in screening by liquid chromatography/mass spectrometry after hydrolysis of phase-II metabolites. In this study, the mono-sulfoconjugated phase-II metabolite of terbutaline was synthesized and the chemical structure was characterized by 1H-nuclear magnetic resonance spectrometry and high resolution/high accuracy Orbitrap mass spectrometry. The metabolite was designated as the phenolic esterified compound, which has been mentioned in most literature reports but has not been verified so far. The benzylic esterified compound was also synthesized and characterized by high-resolution/high accuracy Orbitrap mass spectrometry but was not detectable in urine samples from an excretion study performed after a single application of one terbutaline capsule (7.5 mg terbutaline sulfate salt). The phenolic sulfate of terbutaline was detected for two to four days after administration, whereas the unchanged terbutaline was detected for four to five days. A glucuronidated, disulfated or trisulfated phase-II metabolite of terbutaline was not found. The measurement of phase-II metabolites is planned to be incorporated into existing screening procedures to allow a faster sample preparation. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The use of prescribed antidepressants by athletes has not been restricted in human sports since 2003, after the antidepressants bupropion and amineptine were removed from the list of prohibited substances. Recent awareness of antidepressants has been stimulated by reports from the media concerning possible misuse of antidepressants among healthy athletes. The prevalence of antidepressants has been monitored over the past ten years with screening procedures routinely used by WADA-accredited laboratories. The growth in antidepressant use among athletes peaked in 2007 and 2008 after a modest increase over the first eight years of this survey. Pharmacy prescriptions for antidepressants in Germany did not show a correlated growth during this period. The increasing variety of antidepressant medications has led to a continued increase in the diversity of antidepressant substances used by athletes and the 'normal' population. The number of different sports affected by the presence of antidepressants has increased in the past decade, especially in endurance sports. The predominance of female antidepressant users in the normal population was reflected in the athletes' group. We concluded from our results that the development of antidepressant prevalence in elite sports did not correlate with that among the general public in Germany. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The anti-doping rules of national and international sport federations ban any use of tetrahydrogestrinone (THG) in human as well as in horse sports. Initiated by the THG doping scandals in human sports a method for the detection of 3-keto-4,9,11-triene steroids in horse blood and urine was developed. The method comprises the isolation of the analytes by a combination of solid phase and liquid–liquid extraction after hydrolysis and solvolysis of the steroid conjugates. The concentrations of THG in blood and urine samples were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A THG excretion study on horses was conducted to verify the method capability for the analysis of postadministration urine samples. In addition, blood samples were collected to allow for determination of the pharmacokinetics of THG in horses. Following the administration of a single oral dose of 25 μg THG per kg bodyweight to 10 horses, samples were collected at appropriate intervals. The plasma levels of THG reached maximal concentrations of 1.5–4.8 ng/mL. Twenty-four hours after the administration plasma levels returned to baseline. In urine, THG was detectable for 36 h. Urinary peak concentrations of total THG ranged from 16 to 206 ng/mL. For the 10 horses tested, the mean plasma clearance of THG was 2250 mL/h/kg and the plasma elimination half-life was 1.9 h. [ABSTRACT FROM AUTHOR]

Erythropoietin (EPO) promotes the production of red blood cells, the key factor in the regulation of the oxygen transport, and has been abused by athletes for performance enhancement in endurance sports. Current methods to detect EPO misuse are based on isoelectric focussing (IEF), double blotting, and chemiluminescence detection. A new approach utilizing SDS-PAGE mobilities of target analytes is presented. Employing two internal standards (novel erythropoiesis stimulating protein and recombinant rat EPO), the assay provides a tool which allows the calculation of relative mobility values for endogenous urinary EPO and recombinant epoetins (e.g., Dynepo™) and, thus, the distinction of these analytes in doping control samples. A reference group of 53 healthy volunteers and samples originating from a Dynepo™ (epoetin delta) excretion study conducted with a single person were analyzed and led to a significant discrimination of endogenous urinary and recombinant EPO. A clear differentiation was accomplished over a period of four days post-administration of a single injection of 50 IU/kg body weight. Hence, the method may be useful as a screening procedure in doping control or as complementary confirmation tool to the established IEF assay. [ABSTRACT FROM AUTHOR]

The aim of the present study was to improve and validate a flow cytometric method for the detection of homologous blood transfusion in doping control analysis. A panel of eight different primary antibodies and two different phycoerythrin-conjugated secondary antibodies was used for the detection of different blood populations. The flow cytometer used in this study was the BD FACSArray® instrument. Mixed red blood cell populations were prepared from phenotype known donors. Linearity, specificity, recovery, precision, robustness and interday-precision were tested for every primary antibody used in the presented assay. The technique of signal amplification was utilized for an improved separation of antigens with weak or heterozygous expression to improve the interpretation of histograms. The resulting method allowed to clearly identify mixed red blood cell populations in homologous blood transfusion samples containing 0.3–2.0% of donor blood. [ABSTRACT FROM AUTHOR]

Urine manipulation in sports drug testing has become a serious problem for doping control laboratories, and recent scandals in elite endurance sports have revealed the problem of urine manipulation presumably using proteases, which will impede the detection of drugs such as erythropoietin (EPO) or other peptide hormones. Using commonly accepted analytical strategies, a protocol was developed enabling the determination elevated protease activities in doping control specimens followed by the visualization of protein degradation and identification of proteases such as chymotrypsin, trypsin and papain. Therefore, protease detection kits based on fluorescein isothiocyanate-labeled casein were employed, and protease concentrations greater than 15 μg/ mL of urine entailed subsequent 1-dimensional gel electrophoretic visualization of urinary proteins. The presence of 20 μg of proteases per mL of urine caused a complete degradation of proteins usually observed in urinary matrices (‘trace of burning’), while respective proteases were still detected in spiked urine samples after 10 days of storage at + 4 and -20 °C. Identification of target proteases at respective molecular weights was accomplished using bottom-up sequencing approaches based on in-gel digestion of separated enzymes followed by capillary liquid chromatography — Orbitrap tandem mass spectrometry. [ABSTRACT FROM AUTHOR]

The Federation Equestre Internationale has permitted the use of altrenogest in mares for the control of oestrus. However, altrenogest is also suspicious to misuse in competition horses for its potential anabolic effects and suppression of typical male behaviour, and thus is a controlled drug. To investigate the pharmacokinetics of altrenogest in horses we conducted an elimination study. Five oral doses of 44 μg/kg altrenogest were administered to 10 horses at a dose interval of 24 h. Following administration blood and urine samples were collected at appropriate intervals. Altrenogest concentrations were measured by liquid chromatography-tandem mass spectrometry. The plasma levels of altrenogest reached maximal concentrations of 23–75 ng/mL. Baseline values were achieved within 3 days after the final administration. Urine peak concentrations of total altrenogest ranged from 823 to 3895 ng/mL. Twelve days after the final administration concentrations were below the limit of detection (ca 2 ng/mL). [ABSTRACT FROM AUTHOR]

The article informs that erythropoiesis-stimulating products contain undeclared cobalt and nickel species. Topics discussed include presence of anabolic agents in products used by athletes, observing personal environment of elite athletes, and administration of products enriched with bioavailable cobalt species to athletes.