Multidimensional chromatography coupled to tandem mass spectrometry (MS/MS), including simple sample preparation with protein precipitation, anion conversion with ammonium hydroxide, and solid-phase extraction using mixed-mode anion exchange in a 96-well plate format, has been validated for rapid simultaneous analysis of human insulin and its six analogs (lispro, glulisine, glargine, degludec, detemir, and aspart) in human plasma. This method is critical for clinical diagnostics, forensic investigations, and anti-doping efforts due to the widespread use of these substances. In the present study, improved chromatographic resolution was achieved using a first-dimension trap-and-elute configuration with an XBridge C18 (2.1 × 20 mm, 3.5 μm) trap column combined with second dimension separation on a Cortecs Ultra-High-Performance Liquid Chromatography (UHPLC) C18+ (2.1 × 100 mm, 1.6 μm) analytical column implemented within a two-dimensional-LC-MS/MS system. The total chromatographic run time was 11 min. This setup increases both the resolution and sensitivity of the method. A mobile phase consisting of 0.8% formic acid (FA) in water and 0.7% FA in acetonitrile was used for gradient elution. Bovine insulin was used as the internal standard. MS detection was performed in positive electrospray ionization mode, and the ion suppression due to matrix effects was evaluated. Validation criteria included linearity, precision, accuracy, recovery, lower limit of quantitation, matrix effect, and stability tests with and without protease inhibitor cocktail under different conditions (short-term stability, long-term stability, and freeze-thaw stability). The concentration range for all insulins was 50-15 000 pg/mL, with limits of quantification below the therapeutic reference range for all analytes. Intra-run precision ranged from 1.1% to 5.7%, inter-run precision from 0.7% to 5.9%, and overall recovery from 96.9% to 114.3%. The validated method has been implemented successfully by the Department of Forensic Medicine at our hospital for the investigation of unexplained deaths.
This review systematically compiles sports-related drugs, substances, and methodologies based on the most frequently detected findings from prohibited lists published annually by the World Anti-Doping Agency (WADA) between 2003 and 2021. Aligned with structure of the 2023 prohibited list, it covers all proscribed items and details the pharmacokinetics and pharmacodynamics of five representatives from each section. Notably, it explores significant metabolites and metabolic pathways associated with these substances. Adverse analytical findings are summarized in tables for clarity, and the prevalence is visually represented through charts. The review includes a concise historical overview of doping and WADA's role, examining modifications in the prohibited list for an understanding of evolving anti-doping measures.
- MeSH
- Doping in Sports * MeSH
- Performance-Enhancing Substances pharmacokinetics MeSH
- Humans MeSH
- Substance Abuse Detection methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Background: In recent years, there has been intensive discussion about the positive effect of nicotine usage on enhancing sports performance. It is frequently applied through a non-burned tobacco form before physical activity. Nicotine is under the World Anti-Doping Agency (WADA) 2021 monitoring program. Therefore, study results that reveal either positive or negative effects are expected. This is the pilot study that reports the effect of 8 mg dose of nicotine on performance and perceived pain. Material and Methods: This research aimed to explore the oral intake effect of a high-nicotine dose (8 mg) on the maximum anaerobic performance and other selected physical performance parameters in healthy, well-trained adult athletes (n = 15, age 30.7 ± 3.6, BMI 25.3 ± 1.7). The cross-sectional study protocol included the oral administration of either sublingual nicotine or placebo tablets before the anaerobic load assessed by a standardized 30 s Wingate test of the lower limbs. Afterward, the Borg subjective perception of pain (CR 10) and Borg rating of perceived exertion (RPE) were evaluated. Wilcoxon signed-rank test was used for the analysis of data with a 0.05 level of significance. Results: The results revealed that oral administration of an 8 mg nicotine dose does not significantly improve any of the physical performance parameters monitored. We only reported the statistically significant positive effect in RPE (p = 0.03). Conclusion: Lower perception of pain intensity that we reported after nicotine application might be an important factor that affects performance. However, we did not report any improvement in physical performance parameters.
- MeSH
- Anaerobiosis MeSH
- Pain * drug therapy MeSH
- Adult MeSH
- Humans MeSH
- Nicotine * pharmacology MeSH
- Pain Perception MeSH
- Pilot Projects MeSH
- Cross-Sectional Studies MeSH
- Athletes MeSH
- Physical Exertion MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Publication type
- Journal Article MeSH
New screen-printed sensor with a boron-doped diamond working electrode (SP/BDDE) was fabricated using a large-area linear antenna microwave chemical deposition vapor system (LA-MWCVD) with a novel precursor composition. It combines the advantages of disposable printed sensors, such as tailored design, low cost, and easy mass production, with excellent electrochemical properties of BDDE, including a wide available potential window, low background currents, chemical resistance, and resistance to passivation. The newly prepared SP/BDDEs were characterized by scanning electron microscopy (SEM) and Raman spectroscopy. Their electrochemical properties were investigated by cyclic voltammetry and electrochemical impedance spectroscopy using inner sphere ([Fe(CN)6]4-/3-) and outer sphere ([Ru(NH3)6]2+/3+) redox probes. Moreover, the applicability of these new sensors was verified by analysis of the anti-inflammatory drug lornoxicam in model and pharmaceutical samples. Using optimized differential pulse voltammetry in Britton-Robinson buffer of pH 3, detection limits for lornoxicam were 9 × 10-8 mol L-1. The oxidation mechanism of lornoxicam was investigated using bulk electrolysis and online electrochemical cell with mass spectrometry; nine distinct reaction steps and corresponding products and intermediates were identified.
- MeSH
- Boron * chemistry MeSH
- Electrodes MeSH
- Electrolysis * MeSH
- Oxidation-Reduction MeSH
- Spectrum Analysis, Raman MeSH
- Publication type
- Journal Article MeSH
In the second part of this study, a systematic comparison was made between two ion fragmentation acquisition modes, namely data-independent acquisition (DIA) and DIA with ion mobility spectrometry (IMS) technology. These two approaches were applied to the analysis of 192 doping agents in urine. Group I included 102 compounds such as stimulants, diuretics, narcotics, and β2-agonists, while Group II contained 90 compounds included steroids, glucocorticoids, and hormone and metabolic modulators. Important method parameters were examined and compared, including the fragmentation, sensitivity, and assignment capability with the minimum occurrence of false positive hits. The results differed between Group I and II in number of detected fragments when exploring the MS/MS spectra. In Group I only 13%, while in the Group II 64% of the substances had a higher number of fragments in DIA-IMS mode vs. DIA. In terms of sensitivity, the performance of the two modes with and without activated IMS dimension was identical for about 50% of the doping agents. The sensitivity was higher without IMS, i.e. in simple DIA mode, for 20-40% of remaining doping agents. Despite this sensitivity reduction with IMS, 82% of compounds from both Groups met the minimum required performance level (MRPL) criteria of the World Anti-Doping Agency (WADA) when the DIA-IMS mode was applied. Automated data processing is important in routine doping analysis. Therefore, processing methods were optimized and evaluated for the prevalence of false peak assignments by analysing the target substances at different concentrations in urine samples. Overall, a significantly higher number of misidentified compounds was observed in Group II, with an almost 2-fold higher number of misidentifications in DIA compared to DIA-IMS. This result highlights the benefit of the IMS dimension to reduce the rate of false positive in screening analysis. The optimized UHPLC-IM-HRMS method was finally applied to the analysis of urine samples from administration studies including nine doping agents from both Groups. However, to limit the number of interferences from the biological matrix, an emphasis is needed on the adequate settings of the data processing method.
Oxycodone is a widely prescribed, full agonist opioid analgesic. As such, it is used clinically to treat different kinds of painful conditions, with a relatively high potential for doping practices in athletes. In this paper, different classic and innovative miniaturised matrices from blood and urine have been studied and compared, to evaluate their relative merits and drawbacks within therapeutic drug monitoring (TDM) and to implement new protocols for anti-doping analysis. Plasma, dried blood spots (DBS) and dried plasma spots (DPS) have been studied for TDM purposes, while urine, dried urine spots (DUS) and volumetric absorptive microsamples (VAMS) from urine for anti-doping. These sampling techniques were coupled to an original bioanalytical method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the evaluation and monitoring of the levels of oxycodone and its major metabolites (noroxycodone and oxymorphone) in patients under pain management and in athletes. The method was validated according to international guidelines, with good results in terms of precision, extraction yield and accuracy for all considered micromatrices. Thus, the proposed sampling, pre-treatment and analysis are attractive strategies for oxycodone determination in human blood and urine, with advanced options for application to derived micromatrices. Microsampling procedures have significant advantages over classic biological matrices like simplified sampling, storage and processing, but also in terms of precision (<9.0% for DBS, <7.7% for DPS, <7.1% for DUS, <5.3% for VAMS) and accuracy (>73% for DBS, >78% for DPS, >74% for DUS, >78% for VAMS). As regards extraction yield, traditional and miniaturised sampling approaches are comparable (>67% for DBS, >74% for DPS, >75% for DUS, >75% for VAMS). All dried matrices have very low volumes, leading to a significant advantage in terms of analysis feasibility. On the other hand, this also leads to a corresponding decrease in the overall sensitivity.
- MeSH
- Chromatography, Liquid methods MeSH
- Doping in Sports methods MeSH
- Plasma chemistry MeSH
- Humans MeSH
- Miniaturization methods MeSH
- Urine chemistry MeSH
- Drug Monitoring methods MeSH
- Morphinans blood urine MeSH
- Specimen Handling methods MeSH
- Blood Specimen Collection MeSH
- Oxycodone blood urine MeSH
- Oxymorphone blood urine MeSH
- Tandem Mass Spectrometry methods MeSH
- Body Fluids chemistry MeSH
- Dried Blood Spot Testing methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Mezinárodní výzkumy dokládají, že látky zvyšující výkon představují závažný problém jak ve výkonnostním, tak rekreačním sportu a ovlivňují též adolescentní sportovce. V tomto článku prezentujeme postoje českých adolescentů (sportujících na rekreační a výkonnostní úrovni, N = 2 526) vůči dopingu a jejich záměry doping užít. Výsledky byly získány v rámci výzkumného projektu „Doping českých adolescentů: Rozšíření, vztahy a zkušenosti”, který byl podpořen Světovou antidopingovou agenturou (WADA), realizován v letech 2014–2016 a zahrnoval 2 851 adolescentů ze všech krajů České republiky. Byl užit dotazník hodnotící postoje vůči dopingu a podvádění ve sportu, záměry užít doping a chování ve vztahu k dopingu. Bylo zjištěno, že 19 % českých sportujících adolescentům byl doping nabídnut. Dále byl zjištěn relativně malý záměr české populace užít doping; respondenti byli více znepokojeni možnými zdravotními důsledky dopingu než důsledky morálními. Z psychosociálního hlediska podléhají adolescenti zejména sociálním tlakům a očekáváním ohledně výsledků ve sportovních soutěžích a fyzického vzhledu a mají tendenci k rizikovému chování s možnými dlouhodobými negativními důsledky. Výsledky ukázaly nezbytnost antidopingové prevence, kde důležitou roli hraje užší sociální prostředí adolescentů.
International research shows that performance enhancing drugs represent a serious problem both in competitive and leisure sports, affecting adolescent athletes as well. In this paper we present attitudes of Czech adolescents (involved in sport at recreational and performance level, N = 2 526) towards doping and their doping intentions. The results were gained in frame of the research project ”Doping in Czech adolescents: Prevalence, correlates and experiences” supported by the World Anti-Doping Agency. The study was realized in 2014–2016 and included 2 851 adolescents from all regions of the Czech Republic. Data gained via the questionnaire assessing attitudes toward doping and cheating in sports, doping intentions and doping behavior, we found that 19 % of Czech sporting adolescents were offered doping. We observed relatively low doping intentions within our population; the respondents appeared to be more discouraged by possible health consequences, rather than by moral aspects of doping. From a psychological perspective, adolescents are especially susceptible to social pressures and expectations regarding results in sport competitions and physical appearance and tend to participate in risky behavior with possible harmful long-term effects. Results showed the necessity of antidoping prevention where the important role is played by the closer adolescents’ social environment.
- MeSH
- Doping in Sports * prevention & control statistics & numerical data trends MeSH
- Risk Assessment MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Surveys and Questionnaires MeSH
- Psychomotor Performance drug effects MeSH
- Social Environment MeSH
- Socioeconomic Factors MeSH
- Youth Sports MeSH
- Athletic Performance MeSH
- Health Risk Behaviors MeSH
- Check Tag
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Male MeSH
- Female MeSH
This work describes the development of two methods involving supported liquid extraction (SLE) sample treatment followed by ultra-high performance liquid chromatography or ultra-high performance supercritical fluid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS and UHPSFC-MS/MS) for the screening of 43 anabolic agents in human urine. After evaluating different stationary phases, a polar-embedded C18 and a diol columns were selected for UHPLC-MS/MS and UHPSFC-MS/MS, respectively. Sample preparation, mobile phases and MS conditions were also finely tuned to achieve highest selectivity, chromatographic resolution and sensitivity. Then, the performance of these two methods was compared to the reference routine procedure for steroid analyses in anti-doping laboratories, which combines liquid-liquid extraction (LLE) followed by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). For this purpose, urine samples spiked with the compounds of interest at five different concentrations were analyzed using the three analytical platforms. The retention and selectivity of the three techniques were very different, ensuring a good complementarity. However, the two new methods displayed numerous advantages. The overall procedure was much faster thanks to high throughput SLE sample treatment using 48-well plates and faster chromatographic analysis. Moreover, the highest sensitivity was attained using UHPLC-MS/MS with 98% of the doping agents detected at the lowest concentration level (0.1ng/mL), against 76% for UHPSFC-MS/MS and only 14% for GC-MS/MS. Finally, the weakest matrix effects were obtained with UHPSFC-MS/MS with 76% of the analytes displaying relative matrix effect between -20 and 20%, while the GC-MS/MS reference method displayed very strong matrix effects (over 100%) for all of the anabolic agents.
- MeSH
- Anabolic Agents urine MeSH
- Chromatography, Gas MeSH
- Doping in Sports prevention & control MeSH
- Liquid-Liquid Extraction MeSH
- Humans MeSH
- Substance Abuse Detection methods MeSH
- Steroids urine MeSH
- Chromatography, Supercritical Fluid methods MeSH
- Tandem Mass Spectrometry methods MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Trimetazidin je antianginózní lék, který je řazen do skupiny tzv. metabolických modulátorů. Mechanismem jeho antianginózního a antiischemického účinku je inhibice β-oxidace mastných kyselin a přesun výroby energie v podobě molekul ATP na oxidaci glukózy, což je z hlediska spotřeby kyslíku efektivnější způsob výroby energie v ischemickém myokardu. Trimetazidin je lék poměrně starý, registrován je od roku 1978. Jeho indikací je profylaktická léčba stabilní anginy pectoris. Evropská léková agentura (EMA) doporučuje trimetazidin v léčbě anginy pectoris jako lék druhé volby, především do kombinace s konvenčními antianginózními léky, zejména β-blokátory. To je zcela v souladu s doporučeními Evropské kardiologické společnosti i České kardiologické společnosti a také se současným indikačním omezením SÚKL. V současnosti probíhá mortalitně/morbiditní studie ATPCI, která má zhodnotit účinnost a bezpečnost trimetazidinu v léčbě chronických forem ICHS, konkrétně u nemocných po prodělané perkutánní koronární intervenci. Trimetazidin se přechodně dostal i na seznam látek zneužívaných ve sportu jako doping.
Trimetazidine is an anti-anginal drug included in the group of so-called metabolic modulators. The mechanism of its anti-anginal and anti-ischaemic effects is inhibition of β -oxidation of fatty acids and energy transfer in the form of ATP molecules to glucose oxidation which is a more effective method of energy production in ischaemic myocardium in term of oxygen consumption. Trimetazidine is a relatively old drug – it has been approved since 1978. Its indication is prophylactic treatment of stable angina pectoris. European medicines agency (EMA) recommends trimetazidine for the treatment of angina pectoris as a second-choice drug, particularly in combination with conventional anti-anginal drugs, especially β -blockers. This is quite consistent with the guidelines of both European Cardiology Society and Czech Cardiology Society, and also current indication restriction by SÚKL. A mortality/morbidity study ATPCI is currently underway that should evaluate the efficacy and safety of trimetazidine in the treatment of chronic CAD, specifically in patients after percutaneous coronary intervention. Trimetazidine has been temporarily included even in the list of substances used in sports as doping.
- MeSH
- Angina Pectoris * drug therapy MeSH
- Doping in Sports MeSH
- Coronary Circulation drug effects MeSH
- Coronary Disease * drug therapy MeSH
- Humans MeSH
- Meta-Analysis as Topic MeSH
- Cannabinoid Receptor Modulators physiology MeSH
- Drug-Related Side Effects and Adverse Reactions MeSH
- Trimetazidine * administration & dosage pharmacokinetics adverse effects therapeutic use MeSH
- Check Tag
- Humans MeSH
In the last decade, several immunoassays have been published as the alternative/complementary rapid methods for steroid analysis in food supplements. The present review shows a significant amount of food supplements containing banned anabolic androgenic steroids that are not declared as ingredients thus presenting risk for consumers and may lead to positive results in anti-doping controls. Traditional methods for analysis of steroids such as LC/MS and GC/MS were used for monitoring suspect food supplements.
- MeSH
- Anabolic Agents * administration & dosage adverse effects toxicity MeSH
- Chromatography, Affinity methods utilization MeSH
- Chromatography, Gas methods utilization MeSH
- Doping in Sports * MeSH
- Enzyme-Linked Immunosorbent Assay methods utilization MeSH
- Hypertension chemically induced MeSH
- Infertility chemically induced MeSH
- Blood Coagulation Disorders chemically induced MeSH
- Chemical and Drug Induced Liver Injury MeSH
- Humans MeSH
- Tendon Injuries chemically induced MeSH
- Lipid Metabolism Disorders chemically induced MeSH
- Dietary Supplements * adverse effects MeSH
- Steroids * analysis MeSH
- Testosterone * analogs & derivatives adverse effects MeSH
- Legislation, Drug MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
