Drug Testing and Analysis最新文献

We report the misuse of a novel synthetic glucocorticoid, 9α-fluoro-6α-methylprednisolone (9F6MP) for the first time in camel racing and, to the best of our knowledge, human or other animal sports. During routine post-race drug testing of cameline plasma samples using liquid chromatography-tandem mass spectrometry we encountered an unknown peak with the same selected reaction monitoring traces as a dexamethasone formate adduct but at a different retention time. The product ion mass spectrum of the unknown peak in negative ion mode was identical to dexamethasone. However, significant differences were observed in positive ion mode. Based on mass spectral analysis, we postulated the unknown peak to be a 6-methyl-16-nor isomer of dexamethasone. Following the procurement of a commercial 9F6MP reference material, the unknown peak was successfully identified as this substance. Interestingly, previous research predicted a high potential for glucocorticoid and anti-inflammatory activity for 9F6MP. However, the therapeutic use of 9F6MP in camels has not been approved by any authorities, and any toxicities and side-effects potentially caused by 9F6MP have not been thoroughly evaluated. Therefore, the misuse of 9F6MP should be strictly controlled for the sake of animal welfare and the integrity of camel racing. The information described in this case report will be beneficial for other anti-doping laboratories in both human and animal sports for the purpose of doping control.

In this study, we assess the feasibility of wastewater sampling from manholes as a potential monitoring tool for performance and image-enhancing drugs (PIEDs) at local gyms and general sports centers in Denmark. Wastewater samples from two gyms and two sports centers were analyzed using two LC-MS-based analytical methods (UPLC-MS/MS and UPLC-TOF-MS) to detect the presence of PIEDs. Samples were collected at 3-h intervals during the opening hours on three selected days of the week for each location. The study showed the presence of anabolic agents such as oxandrolone, stanozolol, and ostarine in wastewater sampled from the gyms. All three compounds are class C drugs in the United States, and likewise, illegal to sell and possess in Denmark. Other PIEDs, such as aromatase inhibitors and psychoactive drugs, were detected in several samples from both gyms and sports centers. However, gyms showed a higher occurrence of PIEDs, whereas general sports centers showed a lower occurrence. Although it cannot be used as a traditional prevalence measure, wastewater sampling from manholes is a useful tool for studying the occurrence of PIEDs among recreational athletes at specific locations or events. Wastewater analysis may be used as an easy and cost-effective tool to complement both preventive and control measures, such as information campaigns, surveys, or personal testing. However, not all locations are suitable for manhole sampling, and it is necessary to consider the legal, ethical, and practical aspects before using wastewater analysis to monitor communities or specific populations.

Methyldienolone, a synthetic anabolic androgenic steroid (AAS), has been banned in sports by the World Anti-Doping Agency (WADA) because of its performance-enhancing properties. This study aimed to investigate the main metabolites using in vitro incubation with human liver microsomes (HLM) and to detect them through liquid chromatography-high-resolution mass spectrometry (LC-HRMS) for doping control purposes. A total of six groups of Phase I metabolites, including 17-epimerization, hydroxylation, C3-keto reduction, 18-nor modifications, reduction, and demethylation, as well as five different Phase II metabolites, such as glucuronide conjugates, were characterized, indicating extensive metabolism by HLM. Structural characterization of these metabolites was improved through derivatization with methoxylamine and hydroxylamine, which enabled their detection with higher sensitivity by LC-HRMS. These novel metabolites provide new insights into the metabolism of methyldienolone and may contribute to antidoping analysis. The synthesis of reference materials is necessary to confirm the structure of the proposed metabolites in the future.

Semisynthetic cannabinoids (SSCs) are a novel group of psychoactive substances obtained by chemical modification of phytocannabinoids such as Δ⁹-THC and CBD. Since 2022, their prevalence has rapidly increased on the European illicit drug market, including Poland, where they are mainly detected in cannabis-derived products (plant material, resinous products, e-liquids, and edibles). In this study, 1186 cannabis-type samples seized in Poland (2022-2024) were analyzed at the IFR using GC-MS, UHPLC-PDA, and LC-QTOF/MS. SSCs were found in 113 samples (9.5%), mostly in plant material and resinous products. The predominant compound was hexahydrocannabinol (HHC), consistently detected as a mixture of two epimers, with (9R)-HHC prevailing (typical (9R):(9S) ratio 2.0-2.8:1). In most cases, SSCs were applied to plant material with low Δ⁹-THC content, mainly Chemotypes II and III (approximately 60% of all cases), indicating intentional enrichment of material with limited psychoactive potential. Following legislative changes introduced in Poland in 2023, identifications of controlled SSCs decreased; however, the diversity of noncontrolled compounds increased. Co-occurrence of multiple SSCs, their structural similarity, stereoisomerism, and limited availability or delays in obtaining certified reference standards may complicate routine analysis. Each analytical technique presents specific limitations: GC-MS can cause degradation of acetate forms of SSCs; UHPLC-PDA faces challenges in differentiating compounds with similar UV spectra, whereas LC-QTOF/MS cannot fully distinguish structural or stereoisomeric forms. This study highlights the dynamic nature of the SSC market and the importance of advanced, multimethod analytical approaches for reliable identification.

This study presents LC-HRMS/MS analyses of LGD-4033 post-administration urine samples, following hydrolysis with β-glucuronidase and liquid-liquid extraction, against chemically synthesized molecules that matched previously proposed metabolites, characterized by ¹H and ¹³C NMR. Using this targeted metabolic investigation approach and the direct comparison of retention times and mass spectral data (high-resolution full scan mass accuracy and collision-induced fragmentation patterns), in accordance with WADA's TD2023IDCR provisions, resulted in unambiguous structural elucidation of additional LGD-4033 metabolites, including (a) the epi-long-term dihydroxylated metabolite (M5a); (b) the epi-pyrrolidinone metabolite (M2d); (c) the (R,R)-diastereoisomer of the ring-opened hydroxylated metabolite (M4b); and (d) one of the two detected tris-hydroxylated metabolites (M6a). Additionally, a new, previously undescribed metabolite, which is a hydroxylated derivative of the pyrrolidinone metabolite M2c, was also detected up to 4 days post-administration and coded as M7. Metabolites M5a and M2d are detectable up to 21 days post-administration and can be considered additional long-term markers. These findings expand current knowledge of LGD-4033 metabolism. From a doping control perspective, the proposed synthetic pathways may facilitate the production of reference materials for the detection and identification of a more comprehensive metabolite profile that will increase metabolic certainty in future LGD-4033 adverse analytical findings.

The increasing misuse of opioids in Europe is an alarming trend, leading to severe social and health consequences. Heroin, a highly potent and addictive opioid, remains the main contributor to the health burden associated with opioid use in the region. Illicit drug characterization and profiling offer valuable insights into the complexity of heroin seizures, assisting law enforcement agencies and forensic experts in gathering evidence for legal proceedings. This study provides a comprehensive overview of the composition of heroin seizures and assesses the feasibility of an electrochemical fingerprint approach for the detection of heroin and its associated components. In the initial phase, the primary focus was on developing an electrochemical sensor optimized for heroin detection. The sensor's performance was validated using street samples provided by Sciensano, a Belgian health institute, ensuring its accuracy and reliability in identifying heroin. Once the capabilities of the sensor were demonstrated, the discrimination of alkaloids and cutting agents in seized samples was integrated into a customized software script. Subsequently, an extensive validation process was conducted using a new dataset of heroin seizures from the Belgian National Institute for Criminalistics and Criminology. The follow-up verification confirmed the sensor's effectiveness in detecting heroin, cutting agents, and alkaloids, highlighting its potential as a valuable tool for drug profiling. This portable, user-friendly device with automatic readout could become essential for forensic experts, law enforcement, and harm reduction efforts in addressing the opioid crisis.

Athletes are increasingly using probiotic supplementation to support their overall health, and it can be particularly beneficial for female athletes in managing recurrent urinary tract infections and bacterial vaginosis. One route of probiotic administration for females is vaginal application, which enables direct modulation of the microbiota. While probiotics are widely recognised for their health benefits, their potential impact on urinary steroidal markers monitored in the Steroidal Module of the Athlete Biological Passport remains unexplored. Given the biological overlap between vaginal and urinary microbiomes, bacteria from vaginal probiotics could transfer into urine samples, potentially altering steroid profiles through microbial enzymatic activity. This study investigates whether vaginal probiotic use, specifically Lactobacillus reuteri and plantaraium, could influence urinary steroid markers relevant to the steroidal passport. In vitro and in vivo approaches were employed to evaluate the potential effects of contamination and variability on key steroidal markers. Analyses of in vitro and in vivo experiments suggest that vaginal probiotics do not substantially affect urinary steroid markers monitored in the Athlete Biological Passport. However, some variations were observed that merit further investigation. These findings contribute to a better understanding of how vaginal probiotics might interfere with doping control results, emphasising the need for further research to ensure accurate interpretation of urine steroidal profiles in the female athlete.

For the past couple of years, black market products have appeared and were confirmed to contain genetic products coding for human erythropoietin (EPO). While being prohibited by the World Anti-Doping Agency (WADA), they could be used to produce endogenously more EPO hormone and hence increase performance. In a previous work, we demonstrated the potential of 20-μL dried blood spots (DBS) to detect the presence of EPO transgene in human blood down to 250 copies (12,500 copies/mL), despite lower sensitivity (30-fold) than in 1-mL fresh blood. As the use of DBS as a collection matrix for antidoping is going to expand in the near future, our aim was to develop and validate a new protocol to improve the sensitivity of gene doping detection from DBS. Three DBS devices were evaluated: polymeric Tasso-M20 (TASSO Inc.) and Mitra (Neoteryx), and cellulosic Protein Saver 903 (Whatman). The best results were achieved with polymeric DBS, and a full validation was performed for the detection of the EPO transgene using Tasso M-20 DBS; 1500 copies/mL were detected in 50% of cases and robust detection was obtained at 5000 copies/mL (100 copies transgene in 20-μL DBS) with the four spots of the Tasso device tested over several weeks. The results confirm that polymeric DBS can be used as an alternative to fresh blood for gene doping detection with high sensitivity simplifying also potential reanalysis in the future.

An innovative software tool for the rapid and efficient simulation of the metabolism of new psychoactive substances (NPS) was developed, based on the open-source project mzmine, and applied. NPS are compounds designed to mimic the psychotropic effects of established illicit drugs while circumventing drug legislation. These compounds are developed solely regarding their desired effects, thus possibly leading to harmful side effects including the formation of toxic metabolites. Analytical reference standards, needed to carry out metabolic studies, are not immediately available because emerging NPS are primarily discovered subsequent to drug confiscations. Using these confiscated substances in traditional metabolic in vivo or in vitro studies is often not possible due to the substances being impure or being a part of a mixture of different NPS. Therefore, a software tool was developed to streamline the evaluation of data acquired by the online combination of electrochemistry and mass spectrometry for the simulation of NPS metabolism. Using this tool, it is possible to generate mass voltammograms directly from mass spectrometric raw data. Combining this newly implemented tool with existing filtering algorithms in mzmine, we simulated the metabolism of the synthetic cannabinoid receptor agonist (SCRA) methyl 3,3-dimethyl-2-[1-(pent-4-en-1-yl)-1H-indazole-3-carboxamido] butanoate (MDMB-4en-PINACA) from a mixed solution of different NPS. Fragmentation data indicated that one of the transformation products found for MDMB-4en-PINACA is likely of a quinoid structure. The potential formation of this possibly highly reactive quinoid metabolite could be a first hint for possible causes of adverse side effects frequently reported after the recreational use of MDMB-4en-PINACA and related SCRAs.