A multivariate Bayesian learning approach for improved detection of doping in athletes using urinary steroid profiles

Biomarker analysis of athletes' urinary steroid profiles is crucial for the success of anti-doping efforts. Current statistical analysis methods generate personalised limits for each athlete based on univariate modelling of longitudinal biomarker values from the urinary steroid profile. However, simultaneous modelling of multiple biomarkers has the potential to further enhance abnormality detection. In this study, we propose a multivariate Bayesian adaptive model for longitudinal data analysis, which extends the established single-biomarker model in forensic toxicology. The proposed approach employs Markov chain Monte Carlo sampling methods and addresses the scarcity of confirmed abnormal values through a one-class classification algorithm. By adapting decision boundaries as new measurements are obtained, the model provides robust and personalised detection thresholds for each athlete. We tested the proposed approach on a database of 229 athletes which includes longitudinal steroid profiles classified as normal, atypical, or confirmed abnormal. Our results demonstrate improved detection performance, highlighting the potential value of a multivariate approach in doping detection.Comment: 25 pages, main manuscript pgs. 1-19, appendix A pgs. 19-22, appendix B pgs. 23-2

Combination of carbon isotope ratio with hydrogen isotope ratio determinations in sports drug testing

Carbon isotope ratio (CIR) analysis has been routinely and successfully applied to doping control analysis for many years to uncover the misuse of endogenous steroids such as testosterone. Over the years, several challenges and limitations of this approach became apparent, e.g., the influence of inadequate chromatographic separation on CIR values or the emergence of steroid preparations comprising identical CIRs as endogenous steroids. While the latter has been addressed recently by the implementation of hydrogen isotope ratios (HIR), an improved sample preparation for CIR avoiding co-eluting compounds is presented herein together with newly established reference values of those endogenous steroids being relevant for doping controls. From the fraction of glucuronidated steroids 5β-pregnane-3α,20α-diol, 5α-androst-16-en-3α-ol, 3α-Hydroxy-5β-androstane-11,17-dione, 3α-hydroxy-5α-androstan-17-one (ANDRO), 3α-hydroxy-5β-androstan-17-one (ETIO), 3β-hydroxy-androst-5-en-17-one (DHEA), 5α- and 5β-androstane-3α,17β-diol (5aDIOL and 5bDIOL), 17β-hydroxy-androst-4-en-3-one and 17α-hydroxy-androst-4-en-3-one were included. In addition, sulfate conjugates of ANDRO, ETIO, DHEA, 3β-hydroxy-5α-androstan-17-one plus 17α- and androst-5-ene-3β,17β-diol were considered and analyzed after acidic solvolysis. The results obtained for the reference population encompassing n = 67 males and females confirmed earlier findings regarding factors influencing endogenous CIR. Variations in sample preparation influenced CIR measurements especially for 5aDIOL and 5bDIOL, the most valuable steroidal analytes for the detection of testosterone misuse. Earlier investigations on the HIR of the same reference population enabled the evaluation of combined measurements of CIR and HIR and its usefulness regarding both steroid metabolism studies and doping control analysis. The combination of both stable isotopes would allow for lower reference limits providing the same statistical power and certainty to distinguish between the endo- or exogenous origin of a urinary steroi

Simplified quantification of insulin, its synthetic analogs and C-peptide in human plasma by means of LC-HRMS.

The quantification of peptide hormones by means of liquid chromatography (LC) coupled to mass spectrometry (MS) or other techniques (e.g. immunoassays) has been a challenging task in modern analytical chemistry. Especially for insulin, its synthetic analogs, and C-peptide, reliable determinations are urgently needed due to their diagnostic value in the management of diabetes and insulin resistance and because of the illicit use of insulin as a performance-enhancing agent in professional sports or as an effective toxin in forensic toxicology. The concomitant measurement of C-peptide and insulin offers an established tool for the diagnostic workup of hypoglycemia (endogenous vs. exogenous hyperinsulinemia), characterizing hepatic insulin clearance, and the assessment of beta-cell function (insulin secretion). Thus, the present approach offers the possibility to determine human insulin and its synthetic analogs (lispro, glulisine, aspart, glargine metabolite, degludec, detemir, porcine, and bovine) and C-peptide simultaneously after sample preparation utilizing protein precipitation and a mixed-mode cation-exchange solid-phase extraction, and subsequent detection by LC-high resolution MS. The method was fully validated regarding the following parameters: specificity, limit of detection (0.2 ng/mL), limit of quantification (0.6 ng/mL), recovery (40-90%), accuracy (78-128%), linearity, precision (< 21%), carry over, robustness, and matrix effects. The proof-of-concept was shown by analyzing authentic plasma samples from adults with class II obesity and prediabetes collected in the course of an oral glucose tolerance test. All sample preparation steps were controlled by two stable isotope-labeled internal standards, namely [[2 H10 ] Leu B6, B11, B15, B17 ]-insulin, and [[13 C6 ] Leu 26, 30 ] C-peptide

Effect of changes in the deuterium content of drinking water on the hydrogen isotope ratio of urinary steroids in the context of sports drug testing

The hydrogen isotope ratio (HIR) of body water and, therefore, of all endogenously synthesized compounds in humans, is mainly affected by the HIR of ingested drinking water. As a consequence, the entire organism and all of its synthesized substrates will reflect alterations in the isotope ratio of drinking water, which depends on the duration of exposure. To investigate the effect of this change on endogenous urinary steroids relevant to doping-control analysis the hydrogen isotope composition of potable water was suddenly enriched from -50 to 200 ‰ and maintained at this level for two weeks for two individuals. The steroids under investigation were 5β-pregnane-3α,20α-diol, 5α-androst-16-en-3α-ol, 3α-hydroxy-5α-androstan-17-one (ANDRO), 3α-hydroxy-5β-androstan-17-one (ETIO), 5α-androstane-3α,17β-diol, and 5β-androstane-3α,17β-diol (excreted as glucuronides) and ETIO, ANDRO and 3β-hydroxyandrost-5-en-17-one (excreted as sulfates). The HIR of body water was estimated by determination of the HIR of total native urine, to trace the induced changes. The hydrogen in steroids is partly derived from the total amount of body water and cholesterol-enrichment could be calculated by use of these data. Although the sum of changes in the isotopic composition of body water was 150 ‰, shifts of approximately 30 ‰ were observed for urinary steroids. Parallel enrichment in their HIR was observed for most of the steroids, and none of the differences between the HIR of individual steroids was elevated beyond recently established thresholds. This finding is important to sports drug testing because it supports the intended use of this novel and complementary methodology even in cases where athletes have drunk water of different HIR, a plausible and, presumably, inevitable scenario while travelin

Do dried blood spots have the potential to support result management processes in routine sports drug testing?—Part 3: LC–MS/MS‐based peptide analysis for dried blood spot sampling time point estimation

Along with the recent acknowledgement of the World Anti-Doping Agency to use dried blood spot (DBS) samples for routine doping control purposes, there have been propositions to use DBS as a matrix that allows regular proactive remotely supervised self-sampling, providing potential longitudinal monitoring of an athlete's exposure to doping agents. However, several organizational aspects have to be considered before implementation, such as the verification of the sample collections time point. Based on a previous untargeted proteomics workflow utilizing liquid chromatography–high-resolution mass spectrometry (LC–HRMS) to identify protein/peptide markers to define the time since deposition of a bloodstain, the aim of the current study was to develop a targeted LC–HRMS/MS analytical method for promising peptidic target analytes. A long-term DBS storage experiment was carried out over a 3-month period (sample collection time points: 0, 2, 4, 7, 14, 21, 28, 42, 56, 70, 84 and 91 days) with DBS samples of 10 volunteers for longitudinal investigation of signal abundance changes of targeted peptide sequences at different storage temperatures (room temperature [RT], 4°C and −20°C). Prior to experimental analysis, LC–HRMS/MS method characteristics were successfully assessed, including intraday precision, carryover and sample extract stability. For estimation of DBS sample collection time points, ratios of two peptides that originate from the same protein prior to tryptic digestion were created. Two targeted peptide area ratios were found to significantly increase after being stored at RT for 28 days, representing potential markers for future use in routine doping controls that contribute to advancing complementary avenues in anti-doping

Pharmacokinetic modelling of orally administered cannabidiol and implications for medication control in horses

Cannabidiol (CBD) products gain increasing popularity amongst animal owners and veterinarians as an alternative remedy for treatment of stress, inflammation or pain in horses. Whilst the use of cannabinoids is banned in equine sports, there is limited information available concerning CBD detection times in blood or urine. The aim of this study was to determine the pharmacokinetic properties of CBD following oral administration in the horse to assist doping control laboratories with interpreting CBD analytical results. Part 1: dose escalation study: Single oral administration of three escalating doses of CBD paste (0.2 mg/kg, n = 3 horses; 1 mg/kg, n = 3; 3 mg/kg, n = 5) with >7 days wash-out periods in between. Part 2: multiple dose study: oral administration of CBD paste (3 mg/kg, n = 6) twice daily for 15 days. Multiple blood and urine samples were collected daily throughout both studies. Following study part 2, blood and urine samples were collected for 2 weeks to observe the elimination phase. Concentrations of CBD, its metabolites and further cannabinoids were evaluated using gas-chromatography/tandem-mass-spectrometry. Pharmacokinetic parameters were assessed via two approaches: population pharmacokinetic analysis using a nonlinear mixed-effects model and non-compartmental analysis. AUC0–12 h and Cmax were tested for dose proportionality. During the elimination phase, the CBD steady-state urine to serum concentration ratio (Rss) was calculated. Oral CBD medication was well-tolerated in horses. Based on population pharmacokinetics, a three-compartment model with zero-order absorption most accurately described the pharmacokinetic properties of CBD. High volumes of distribution into peripheral compartments and high concentrations of 7-carboxy-CBD were observed in serum. Non-compartmental analysis identified a Cmax of 12.17 ± 2.08 ng/mL after single administration of CBD (dose: 3 mg/kg). AUC0–12 h showed dose proportionality, increase for Cmax leveled off at higher doses. Following multiple doses, the CBD terminal half-life was 161.29 ± 43.65 h in serum. Rss was 4.45 ± 1.04. CBD is extensively metabolized and shows high volumes of tissue distribution with a resulting extended elimination phase. Further investigation of the potential calming and anti-inflammatory effects of CBD are required to determine cut-off values for medication control using the calculated Rss

Historical Study on the Irrigation Reservoirs in Sasayama District

(1)江戸時代において極めて米作率の高かつた篠山藩の灌漑水源は, その7割余が溜池に依存し, その造営は江戸初頭から中期にかけて過半が完成された。斯る短期間に幕藩制のもとで造営が急がれたのは, 庄園制以来隷属下におかれていた耕作農民に独立の機会を与え, 本百姓化して直接的に彼らから領主が生産物地代を確保するに外ならなかつた。そしてその当初の目的は達せられ, 近世村落の成立によつて農業生産上の諸計劃が, その成員の自律性によつて或る程度維持されていた。(2)池の位置は造営の容易性から山腹の谷に多く散在し, 且池元たる山村の耕地に導水の優先権があつたゝめに, 水田の多くを有し水を特に要する盆地内が常に旱損に見舞れ, 江戸時代を通じておくれた技術によつて農業が支えられていた。(3)盆地内は水に窮乏し乍らも, 溜池が農業の発展に果した役割は大であつた。(溜池のない湧水灌漑による地域に比し)そして江戸中期以後藩経済の窮乏に乗じ, 小作料取得の土地所有者の中から藩に代り池を造営する者があらわれ, 或は村方所有の池を集中して, この地方での大地主に成長した

ArylPropionamide-Derived Selective Androgen Receptor Modulators: Liquid Chromatography-Tandem Mass Spectrometry Characterization of the in Vitro Synthesized Metabolites for Doping Control Purposes. Drug Metab. Dispos

ABSTRACT: Selective androgen receptor modulators (SARM) are a prominent group of compounds for being misused in sports owing to their advantageous anabolic properties and reduced side effects. To target the preventive doping control analysis in relevant compounds, the challenge is to predict the metabolic fate of a new compound. For aryl-propionamide-derived SARM, an in vitro assay employing microsomal and S9 human liver enzymes was developed to simulate phase-I and phase-II metabolic reactions. In vitro metabolic profiles and the structure-metabolic relationship were compared between four structurally modified substrates. Accurate mass measurements were used to characterize the synthesized metabolites, and also collision-induced dissociation was examined to suggest the methodological approach to monitor the prohibited use of aryl-propionamide-derived drug candidates. Subsequent phase-I and phase-II metabolic reactions were successfully combined in one in vitro assay. The main routes of phase-I modifications involved the hydrolysis of ether linkage, monohydroxylation, and hydrolytic cleavage of the amide bond. Nitro-reduction and deacetylation were reactions observed for substrates possessing the corresponding functionality. SARM metabolites were analyzed in negative ion electrospray ionization and detected as deprotonated species [M-H] ؊ . The main metabolic modifications were observed to occur in the B-ring side, and collision-induced dissociation resulted in the product ions originating from the A-ring side of the compound. These structure-specific ions may be monitored as target ions in the routine doping control. For decades, steroidal androgens have been clinically used in the treatment of diseases related to androgen deficiency, including muscle-wasting, osteoporosis, and benign prostate hyperplasia, but recently their suitability for hormone replacement therapy of aging men and regulation of male fertility has been under investigation (NegroVilar, 1999; In the development of new aryl-propionamide-derived SARM with higher pharmacological activity, better selectivity, and more advantageous pharmacokinetic properties, several structure-activity and in vitro and in vivo metabolic studies have been carried out We thank the Manfred Donike Institute for Doping Analysis, Cologne, and the Federal Office of Sports, Switzerland, for supporting the presented work. Article, publication date, and citation information can be found a

Preliminary data on the potential for unintentional antidoping rule violations by permitted cannabidiol (CBD) use.

According to the World Anti-Doping Agency (WADA) regulations, cannabinoids use is prohibited in competition except for cannabidiol (CBD) use. For an adverse analytical finding (AAF) in doping control, cannabinoid misuse is based on identification of the pharmacologically inactive metabolite 11-nor-delta-9-carboxy-tetrahydrocannabinol-9-carboxylic acid (carboxy-THC) in urine at a concentration greater than 180 ng/ml. All other (minor) cannabinoids are reported as AAF when identified, except for CBD that has been explicitly excluded from the class of cannabinoids on WADA\u27s Prohibited List since 2018. However, due to the fact that CBD isolated from cannabis plants may contain additional minor cannabinoids, the permissible use of CBD can lead to unintentional violations of antidoping regulations. An assay for the detection of 16 cannabinoids in human urine was established. The sample preparation consisted of enzymatic hydrolysis of glucuronide conjugates, liquid-liquid extraction, trimethylsilylation, and analysis by gas chromatography/tandem mass spectrometry (GC-MS/MS). Spot urine samples from CBD users, as well as specimens obtained from CBD administration studies conducted with 15 commercially available CBD products, were analyzed, and assay characteristics such as selectivity, reproducibility of detection at the minimum required performance level, limit of detection, and limit of identification were determined. An ethical committee approved controlled single dose commercially available CBD products administration study was conducted to identify 16 cannabinoids in urine samples collected after ingestion or application of the CBD products as well as their presence in spot urine samples of habitual CBD users. Variable patterns of cannabinoids or their metabolites were observed in the urine samples, especially when full spectrum CBD products were consumed. The presence of minor cannabinoids or their metabolites in an athlete\u27s in-competition urine sample represents a substantial risk of an antidoping rule violation

Behavioral observations, heart rate and cortisol monitoring in horses following multiple oral administrations of a cannabidiol containing paste (part 2/2)

As a remedy against stress and anxiety, cannabidiol (CBD) products are of increasing interest in veterinary medicine. Limited data is available describing the actual effectiveness of CBD in horses. The aim of this study (part 2 of 2) was to analyze stress parameters via behavioral observation, heart rate monitoring and assessment of blood and saliva cortisol levels in healthy horses treated repeatedly with a CBD containing paste. Twelve horses were randomly assigned to a treatment or a control group. Two pastes were orally administered in a double-blinded study design, one paste containing CBD and one paste without active ingredient. Both pastes were administered twice daily over 15 days (dose: 3 mg CBD/kg). Behavioral observations were conducted daily using a sedation score and a rating of facial expressions, based on the previously described facial sedation scale for horses (FaceSed) and the Horse Grimace Scale. Blood and saliva samples were obtained regularly to determine cortisol levels throughout the study. Cortisol levels were analyzed by means of liquid chromatography/tandem mass spectrometry (LC/MS/MS). Behavioral observations and cortisol levels were compared between groups. Prior to paste administration, a novel object test was performed and the horses’ reaction to loading on a trailer was recorded. Both tests were repeated after 13 days of paste application. Movement patterns such as different gaits during the novel object test were evaluated and an ethogram was designed to assess exhibited behavioral traits. Cardiac beat-to-beat (R-R) intervals were recorded throughout and evaluated using heart rate (HR) and heart rate variability (HRV) parameters. Blood and saliva samples for cortisol analysis were taken before and after the tests. Daily behavioral observations and cortisol levels did not differ between the treatment and the control group. Similarly, analysis of movement patterns, HR, HRV and cortisol levels during the novel object test and trailer test did not identify significant differences between the groups. Regularly administered oral CBD (3 mg/kg BID over 15 days) had no statistically significant effect on behavioral observations, cortisol levels, HR and HRV in horses. Further research is required to establish adequate doses and indications for the use of CBD in horses