Drug Testing and Analysis最新文献

Epristeride, a novel noncompetitive inhibitor of Type II 5α-reductase, has emerged as a potential therapeutic alternative for benign prostatic hyperplasia (BPH). Given that other 5α-reductase inhibitors, such as finasteride and dutasteride, are already monitored for their potential impact on doping control, comprehensive metabolic studies of epristeride are crucial for antidoping. This study investigates the metabolic pathways and metabolites of epristeride using in vitro microsome models, offering preliminary insights into the pharmacokinetics of this drug. Metabolite profiling was performed using liquid chromatography-high resolution mass spectrometry (LC-HRMS), with data acquisition facilitated by Xcalibur 4.2 software and metabolite identification facilitated by Compound Discoverer 3.3. By employing network pharmacology, the potential targets of epristeride are predicted. The binding energy is calculated using AutoDock Vina software to predict its impact on steroid metabolism. The study proposed three primary metabolites of epristeride: two Phase I oxidation products (M1 and M2) and one Phase II glucuronidation product (M3). Pathway analysis revealed that among the five CYP450 isoforms examined, CYP3A4 played a dominant role. The docking results tentatively elucidated five key target proteins (ESR1, CYP19A1, STAT3, AKR1C3, and CYP17A1) with low binding energies, indicating stable interactions. Notably, Phase I metabolites (M1 and M2) showed significant binding potential with these targets, whereas the Phase II metabolite (M3) exhibited lower binding stability. These findings provide a detailed understanding of epristeride's metabolic pathways and its potential biological impacts, offering valuable insights for monitoring its presence as a confounding factor in doping control.

As the tear gas of the highest toxicity, 2-chloroacetophenone (CN) poses a potential threat for exposed individuals. Several fatality cases following exposure to CN are documented, but an unambiguous identification of CN exposure is still missing. Thus, we herein present the identification of in vitro reaction products between CN and endogenous molecules useful as biomarkers. After incubation of human plasma with CN, diverse acetophenone (AcPhen)-adducts were formed with the small molecule thiols homocysteine (HCys), glutathione (GSH), and cystine (Cys-Cys). All adducts were detected by microbore liquid chromatography-electrospray ionization high-resolution tandem mass spectrometry (μLC-ESI MS/HRMS) working in the parallel reaction monitoring (PRM) mode and were characterized as potential biomarkers of CN exposure. Time- and concentration-dependent adduct formations were investigated, and the stability of AcPhen-adducts in plasma during 4 freeze-and-thaw cycles and in the autosampler was tested. The limit of identification (LOI) of identified AcPhen-adducts in vitro was found at about 6 μM (concentration of CN in plasma) but showed quite limited in vitro stability. The AcPhen-adducts herein presented might be beneficial for future studies addressing CN exposure in vivo.

Beta₂-adrenergic agonists are widely used for bronchial relief in respiratory conditions such as asthma and exercise-induced bronchoconstriction. However, this drug class has also been shown to have muscle anabolic properties. Kelch-Like Family Member 41 (KLHL41) is a sarcomeric protein implicated in muscle remodeling and hypertrophy. In this study, we examined the effects of oral clenbuterol, therapeutic inhaled formoterol, and resistance training on KLHL41 protein abundance in human skeletal muscle. KLHL41 levels were measured by immunoblotting in vastus lateralis muscle biopsies from healthy adults following 2 weeks of oral clenbuterol administration, 6 weeks of inhaled formoterol at therapeutic doses, or 8 weeks of resistance training. We also assessed sex differences and the effects of acute versus prolonged interventions. Prolonged oral clenbuterol administration significantly increased KLHL41 abundance compared to placebo (p < 0.001), with a magnitude similar to that observed after resistance training (p < 0.01), whereas therapeutic inhaled formoterol had no effect on KLHL41 levels. Neither acute clenbuterol administration nor a single resistance training session altered KLHL41 abundance, and no sex differences were observed in baseline KLHL41 levels. These findings indicate that beta₂-adrenergic stimulation via oral clenbuterol, but not therapeutic inhalation of formoterol, promotes sarcomeric remodeling through KLHL41-related pathways similar to those activated by resistance training. The distinct effects of these agents on KLHL41 support current anti-doping regulations prohibiting clenbuterol use and highlight KLHL41 as a potential molecular marker of skeletal muscle adaptation to hypertrophic stimuli.

Synthetic products are marketed for specimen substitution and can be difficult to identify because many contain creatinine, urea, and pH buffers designed to meet acceptance criteria of common specimen validity tests. Methods involving DNA analysis and liquid chromatography-mass spectrometry have been successful in detecting synthetic products, but of these require significant sample preparation, lengthy analyses, and/or complex technical analysis. The aims of this study were to characterize the properties of commonly available synthetic urine products in order to determine new methods of detection, or provide information that might help others determine methods to detect synthetic urine products. Physical evaluations, clinical urinalysis, mass spectrometry, and microscopic analysis were used to evaluate 10 synthetic urine products, and results were compared to authentic human specimens. Two of 10 synthetic products exhibited lasting bubbles or foam after shaking. None of the synthetic products contained cells or formed biological elements. In contrast, 99% of 1127 outpatient specimens and 98% of the 250 pain management specimens evaluated in this study contained biological elements. The automated microscopy analyzer used in this study exhibited 100% sensitivity, 98% specificity, required no specimen pretreatment, provided results in less than 2 min, and was interfaced to the laboratory information system for rapid and accurate reporting. Given that normal human urine contains over 1 million cells per liter, microscopic analysis may provide an effective method to differentiate synthetic products from authentic urine. The shake test followed by microscopic analysis should provide an efficient and effective combination to detect synthetic urine products.

This study presents a novel physio-pharmacological framework for assessing the potential performance-enhancing effects of substances and methods included in the 2022 World Anti-Doping Agency (WADA) Prohibited List across 160 Olympic sport disciplines. An expert panel evaluated 31 consolidated pharmacological categories for their capacity to enhance performance across six core physiological athletic demands. Using a calibrated scoring function and bilinear interpolation, we mapped these effects to each sport's dominant physiological profile. Findings confirmed the well-known ergogenic effects of anabolic agents and erythropoietins for power and endurance-based sports, respectively, including the general ergolytic effects of narcotics and cannabinoids. Moreover, the methodology can be used to evaluate any new sport or substance given their respective physiological demands. The study underscores the need for targeted anti-doping strategies, suggesting that risk assessment and test distribution planning should be aligned with sport-specific physiological demands and the relative performance advantages conferred by different doping strategies. This summary of the current physiological knowledge and pharmacological knowledge has the potential to enhance detection efficiency, optimize resource allocation, and refine prohibited substance screening in elite sport.

Flunixin meglumine is widely used to manage pain and inflammation in horses, and its regulation requires robust pharmacokinetic analysis for harmonization. In this study, we conducted a meta-analysis of flunixin disposition using plasma and urine concentration data from 65 horses across four countries to robustly estimate pharmacokinetic parameters in setting screening limits (SLs) for controlling medications in horses. A population (POP) model was developed using nonlinear mixed-effects model analysis. The irrelevant plasma concentration (IPC) and irrelevant urine concentration (IUC) were determined to be 1.9 and 70.2 ng/mL, respectively, with a typical urine-to-plasma ratio (Rss) of 35.9. Using the current International Federation of Horseracing Authorities (IFHA) screening limits (ISLs) (1 ng/mL for plasma; 100 ng/mL for urine), a longer detection time (DT) was observed for plasma than for urine, especially after multiple doses, as plasma ISL corresponds to a slower terminal elimination phase. Increasing the current plasma ISL from 1 to 3 ng/mL-while keeping the current urine ISL at 100 ng/mL-could better align the plasma and urine DTs. As a limitation of this study, both Standardbred and Thoroughbred data were included, and further data collection is needed to fully ascertain potential breed-specific effects. Moreover, this POP model also enabled relatively accurate Bayesian estimation of individual withdrawal times (WTs) from limited data. Clinicians could apply this Bayesian approach to making informed WT recommendations for horses when sufficient data is available. While existing non-POP statistical models remain viable, they may require a more conservative approach to WT estimation than Bayesian methods.

Unintentional doping violations have been reported after secondary skin exposure, including partner contact or contact sports. Methyltestosterone (MT), an anabolic androgenic steroid on the World Anti-Doping Agency Prohibited List, is readily available in Japan as an over-the-counter topical hair-growth preparation and as oral tablets. This study evaluated whether transdermal absorption of MT can be distinguished from oral administration using human urine and dried blood spot (DBS) analyses. Ten men received MT once daily for five consecutive days (five oral, five transdermal). The urinary tetrahydro-metabolites 17α-methyl-5α-androstane-3α,17β-diol (5α-THMT) and 17α-methyl-5β-androstane-3α,17β-diol (5β-THMT) were detectable within hours after administration for both routes. Following transdermal administration, 5α-THMT remained detectable up to 97 h after the final administration, whereas, after oral administration, 5β-THMT persisted longer, up to 265 h. Transdermal application produced a marked increase in 5α-THMT, consistent with high 5α-reductase activity in skin, yielding a significantly higher 5α-THMT/5β-THMT ratio than after oral administration. Nonetheless, individuals with inherently high 5α-reductase activity may exhibit elevated ratios even after oral dosing, warranting careful interpretation. In DBS after oral dosing, parent MT was generally detectable up to 24 h, providing a shorter detection window than urinary tetrahydro-metabolites. Overall, combining urine (tetrahydro-metabolites) and DBS (parent MT) analyses enables effective detection of MT use and supports differentiation of administration routes.

Erythropoietin receptor agonists (ERAs), including continuous erythropoietin receptor activators (CERAs), are potent blood doping substances used to enhance endurance performance by stimulating erythropoiesis. While traditionally detected through direct analysis of urine or serum samples using sarcosyl-polyacrylamide gel electrophoresis (SAR-PAGE) and western blotting, the slow urinary elimination of third-generation ERAs like CERA has shifted anti-doping strategies toward serum-based detection. This study compared the detectability and stability of CERA in urine and serum matrices and evaluated the added value of combining direct detection with hematological profiling. Using samples from a controlled CERA administration study and an authentic case example, we assessed CERA detection in serum, urine, and simulated dried blood spot (DBS) matrices (Tasso-M20). Additionally, we conducted stability experiments by incubating spiked matrices at 37°C for up to 72 h. Our results confirmed the superior stability and consistent detectability of CERA in serum and DBS compared with urine. Moreover, hematological alterations such as increased reticulocytes percentage flagged by the Athlete Biological Passport (ABP) supported targeted serum testing, leading to the successful detection of CERA. These findings highlight the importance of systematic blood collection for both direct and indirect detection strategies. Furthermore, DBS samples showed promising analytical performance and resistance to elevated temperature, suggesting their utility as minimally invasive alternatives in anti-doping programs. Overall, our study reinforces the relevance of blood matrices in the detection of CERA and advocates for the broader integration of blood-based strategies in targeting doping practices with ERAs.

The serum testosterone-to-luteinizing hormone (T/LH) ratio has previously been suggested as a sensitive marker of testosterone use in an anti-doping setting. When measured with an automated immunoassay platform, this ratio represents a quick and cost-effective screening biomarker to further direct isotope ratio mass spectrometry (IRMS) testing efforts in concurrently collected urine samples to confirm testosterone abuse. To evaluate the practicality of implementing the serum T/LH ratio for routine use, testosterone values in 356 serum samples were compared between a "gold-standard" LC-MS/MS method and an automated immunoassay method. Excellent correlation and minimal bias between the two methods were observed, highlighting the validity of the immunoassay method. Next, a testosterone administration study utilizing a transdermal delivery route was conducted to compare the effectiveness of the serum T/LH ratio to the currently used serum testosterone to androstenedione (T/A4) and urinary testosterone to epitestosterone (T/E) ratios. The serum T/LH ratio was more sensitive than the T/A4 ratio and showed similar sensitivity to the urinary T/E ratio with high interindividual variability. Finally, T/LH analysis was conducted on 626 capillary serum samples collected in the field from male Ultimate Fighting Championship athletes. Of the three samples that showed elevated T/LH ratios in this pool, two of the corresponding urine samples were IRMS positive, one of which showed an unremarkable urinary T/E ratio and relatively normal steroid profile. These results indicate serum T/LH ratio monitoring provides a beneficial addition to the anti-doping tool kit and can improve urinary IRMS testing recommendations.