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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.

Contains fulltext : 252186.pdf (Publisher’s version ) (Open Access), BACKGROUND AND AIMS: Detection of autoantibodies is a mainstay of diagnosing autoimmune hepatitis (AIH). However, conventional autoantibodies for the workup of AIH lack either sensitivity or specificity, leading to substantial diagnostic uncertainty. We aimed to identify more accurate serological markers of AIH with a protein macroarray. APPROACH AND RESULTS: During the search for more-precise autoantibodies to distinguish AIH from non-AIH liver diseases (non-AIH-LD), IgG antibodies with binding capacities to many human and foreign proteins were identified with a protein macroarray and confirmed with solid-phase ELISAs in AIH patients. Subsequently, polyreactive IgG (pIgG) was exemplarily quantified by reactivity against human huntingtin-interacting protein 1-related protein in bovine serum albumin blocked ELISA (HIP1R/BSA). The diagnostic fidelity of HIP1R/BSA binding pIgG to diagnose AIH was assessed in a retrospective training, a retrospective multicenter validation, and a prospective validation cohort in cryoconserved samples from 1,568 adults from 10 centers from eight countries. Reactivity against HIP1R/BSA had a 25% and 14% higher specificity to diagnose AIH than conventional antinuclear and antismooth muscle antibodies, a significantly higher sensitivity than liver kidney microsomal antibodies and antisoluble liver antigen/liver pancreas antigen, and a 12%-20% higher accuracy than conventional autoantibodies. Importantly, HIP1R/BSA reactivity was present in up to 88% of patients with seronegative AIH and in up to 71% of AIH patients with normal IgG levels. Under therapy, pIgG returns to background levels of non-AIH-LD. CONCLUSIONS: pIgG could be used as a promising marker to improve the diagnostic workup of liver diseases with a higher specificity for AIH compared to conventional autoantibodies and a utility in autoantibody-negative AIH. Likewise, pIgG could be a major source of assay interference in untreated AIH.

Background Insulin is essential for the regulation of glucose metabolism and exhibits anabolic/anticatabolic characteristics. Oversupply and undersupply pose acute health risks for the human organism. Objective Recombinantly produced insulins are classified as doping agents in sports and prohibited for individuals who do not suffer from insulin-dependent diabetes mellitus. In addition, the administration of insulins has been suspected and proven in a series of (attempted) suicides and homicides. The required analytical approaches necessitate particular sensitivity and specificity, applied to a variety of different available biological matrices. Methods By means of scientific articles published over the past 10 years on the development of analytical approaches for insulins in doping control samples and in forensic contexts as well as case reports, the progress in insulin detection concerning instrumentation, sample type and preparation is presented. Results and conclusion Similar approaches concerning the analysis of insulins have been established in doping controls and forensics; however, the available test matrices can be significantly different. While sports drug testing almost exclusively relies on blood and urine samples, matrices collected postmortem are considerably more variable and can include, e.g. vitreous humor and skin tissue. Depending on different case-related factors, such as prevailing medical conditions or postmortem intervals, specific matrices and sample collection strategies are advantageous. Especially the immediate preservation of dried blood spots as well as the collection of (suspected) injection sites appear warranted in order to facilitate subsequent insulin analyses.

Corticotropin-releasing hormone (CRH), a peptide hormone whose secretion leads to adrenal cortisol release, is classified as prohibited substance by the World Anti-Doping Agency (WADA). In order to comprehensively enforce anti-doping regulations, a detection method for CRH in blood (serum and plasma) is required. In this study, two different immunoaffinity purification strategies were optimized and employed for sample preparation, followed by nano-ultra high performance liquid chromatography (UHPLC) coupled to high resolution/high accuracy tandem mass spectrometry (HRMS/MS). For that purpose, a CRH primary polyclonal antibody was immobilized to either IgG-covered paramagnetic particles or a monolithic protein A/G surface. The first approach using magnetic beads was fully validated in both human plasma and serum, while the Mass Spectrometric Immunoassay (MSIA (TM)) procedure was cross-validated for selected parameters. The resulting assays' LLODs were estimated at 200 pg mL(-1) (magnetic beads) and 500 pg mL(-1) (MSIA (TM)). In addition to human CRH, also animal analogs such as ovine and bovine CRH were found to be traceable using the established approach. For all target analytes comprising of 41 amino acids (approximately 4.7 kDa), high-resolution/high-accuracy product ion mass spectra were generated, and diagnostic y-and b-ions were identified. Proof-of-concept data were obtained by the analysis of plasma samples collected in the course of CRH stimulation blood tests. Specimens were collected from three patients 15 and 30 min after intravenous application of a 100 mu g single dose of human CRH, yielding plasma concentrations between 7.6 and 18.9 ng mL(-1).

RationaleThe 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. MethodsTherefore, 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. ResultsConcentrations 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. ConclusionsIn 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.

Nutritional supplements are one of the main sources of doping substances. Counterfeit preparations often contain stimulants, anabolic-androgenic steroids, beta 2 agonists, or peptide hormones. These substances are mostly not named on the list of ingredients, but the products are promoted by aggressive advertising, with reference to their effects - especially fat loss, boosting effects for training, and muscle build-up. One of the reasons for the expansion of the market for counterfeit dietary supplements is the easy availability of doping substances. The health risks associated with consuming such preparations are unpredictable. Fundamentally, athletes should take dietary supplements only if their consumption is indicated from a nutritional perspective, and the recommendation is to avoid preparations promoted by extreme advertising and from unsafe sources. The Cologne List is a database that includes nutritional supplements with a low risk of doping, which have been checked for anabolic steroids and stimulants and have tested negative.

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-beta-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-naive, 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-beta-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) x fasting insulin ( U/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 HbAlc 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.