Kratom (Mitragyna speciosa), a plant native to Southeast Asia, has traditionally been used for stimulant and analgesic effects, yet its safety profile remains poorly understood given its complex polypharmacology and rapidly evolving commercial formulations. It contains a wide variety of indole alkaloids, with mitragynine (MTG) as the major constituent and most abundant psychoactive alkaloid. There has been a rapid growth of the kratom market, accompanied by a marked increase in kratom-related exposures, hospitalizations, and deaths in the United States, coinciding with the introduction of kratom-derived semisynthetic products, as reflected in rising poison control center calls reported by the US Centers for Disease Control and Prevention (CDC). In this context, the present study aims to analyze commercially available kratom products collected from multiple locations using a UPLC-MS/MS method, with the objective of characterizing their alkaloid profiles and assessing labeling accuracy and product integrity. Results demonstrated substantial variability in alkaloid composition across tablets, films, and liquid shots, with frequent inconsistencies between labeled and actual contents. Products often contained additional unreported alkaloids or lacked those listed on the label; however, most still included pharmacologically and toxicologically active compounds, such as 7-hydroxymitragynine (7-HMG) and 3-dehydromitragynine (3DMTG), respectively. Overall, these findings indicate inconsistent manufacturing practices, labeling inaccuracies, and potential adulteration, along with the risk of exposure to highly potent mu-opioid receptor (MOR) agonists. Collectively, this highlights the need for improved regulatory oversight and strengthened post-marketing surveillance, including real-time poison control center data, to ensure consumer safety.

Regulatory testing of cannabis and hemp commonly prioritizes As, Cd, Pb, and Hg, yet inductively coupled plasma mass spectrometry (ICP-MS) workflows often require extensive tuning and method setup that can be difficult to implement in resource-limited laboratories. Although general guidance exists, quantitative ranges for critical settings are still sparsely documented for cannabis matrices, and "one-set" conditions may not transfer well across instruments. To address this gap, we developed a resource-efficient, low-run screening strategy intended to reduce trial-and-error, reagent consumption, and instrument time when only a limited number of runs is feasible, using two linked 12-run, resolution-III Plackett-Burman designs generated in R. First, key ICP-MS parameters were screened under QC conditions to define a stable operating region. Second, digestion factors (acid composition/volumes, temperature program, sample mass, and predigestion) were assessed using spiked inflorescence samples. Apparent effects were identified using standard screening diagnostics and supported by two-factor trend visualizations. The screening highlighted three underreported practical contributors-RF power, peristaltic-pump speed, and operation without inrun dilution-as dominant drivers of robustness, together with element-dependent recovery drivers: As was mainly governed by HCl level; Cd and Hg by coupled HCl volume × sample mass trends; and Pb by combined heating-ramp/HNO3 and temperature/predigestion patterns. Because resolution-III designs alias main effects with specific two-factor interactions, the numerical effects should be interpreted as first-pass screening estimates rather than definitive optima and require higher resolution confirmation. Overall, the study provides a quantitative map of factor relevance for cannabis metal digestion and narrows the experimental space for subsequent optimization and validation.

Gene therapy is a powerful tool for treating and correcting severe genetic diseases caused by defective genes. However, the same technology could be exploited to introduce exogenous genes that enhance physical performance and endurance in sports, which not only subverts the principles of fair competition but also poses risks to athletes' health. Exogenous transgenes can be delivered into the human body via viral vectors, and recombinant adeno-associated virus (rAAV) vectors are, to date, considered the safest and most reliable viral vectors in that regard for a prolonged effect. Since 2003, the World Anti-Doping Agency (WADA) has included the term "gene doping" in its Prohibited List of Substances and Methods under Article M3 "Gene and Cell Doping." To date, no confirmed case of gene doping in sport has been officially identified. Over the past two decades, several strategies for long-term detection of rAAV-based transgenes in biological fluids have been proposed. These include direct methods such as polymerase chain reaction (PCR), liquid chromatography-high-resolution mass spectrometry (LC-HRMS), and matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF), and next-generation sequencing (NGS)-based approaches. Indirect methods include detection of recombinant protein products, analysis of immune responses (e.g., neutralizing antibodies, T-cell activation), and "omics" technologies such as proteomics, immunopeptidomics, and metabolomics, as well as combinations thereof. This review aims to provide a general overview of the potential of gene therapy for enhancing athletic performance and methods for detecting rAAV-based gene doping.

Consumption of psychotropic drugs is a major economic and public health challenge in Australia. Wastewater-based epidemiology (WBE) and drug checking have emerged as complementary tools to monitor this market. WBE estimates drug use within communities for target compounds and their metabolites, while drug checking is a free service available in some Australian jurisdictions, which allows individuals to verify drug purity and composition. In this work, wastewater samples were collected from five sites across Queensland (n = 3), New South Wales (n = 1) and Victoria (n = 1) over the 2024-25 New Year holidays, while drug checking was carried out at a site in central Canberra over December-January. Wastewater samples were analysed for alcohol, illicit drugs and new psychoactive substances (NPS) using liquid chromatography-tandem mass spectrometry. Methamphetamine was the most consumed illicit substance across all sites, while cocaine and MDMA showed distinct increases over the New Year period. Among NPS, bromazolam and 2'-fluoro-2-oxo-PCE were most prevalent, while ketamine was consistently detected at regional sites with consumption 2.8-3-fold higher on New Year's days. Drug checking revealed prevalence of MDMA, cocaine and ketamine. NPS were detected in 15%-20% of total drug samples submitted, with N,N-dimethylpentylone and 2'-fluoro-2-oxo-PCE the most frequently found. This study is the first to demonstrate the potential synergy between WBE and drug checking, highlighting how their complementary roles can enhance drug surveillance, early warnings for emerging psychoactive substances and monitoring of changes in drug consumption during high-risk periods.

Monoclonal antibodies (mAbs) targeting nerve growth factor (NGF) represent a novel therapeutic approach for managing acute and chronic pain in humans and animals. However, their potential misuse as pain-masking agents in competitive equine sports raises significant welfare and integrity concerns. To enhance understanding of the elimination profile of anti-NGF mAb if misused in horses, this study describes the first administration of bedinvetmab (a canine anti-NGF mAb) in three geldings, establishing its plasma elimination profile following subcutaneous administration. Bedinvetmab plasma concentrations were determined using a recently developed generic method involving affinity purification with Protein A magnetic beads, followed by tryptic digestion and identification of a proteotypic peptide via capillary flow high performance liquid chromatography coupled with high resolution tandem mass spectrometry (HPLC-HRMS). Significant interindividual variability was observed. Maximum plasma concentrations ranged from approximately 0.97 to 6.0 μg/mL and occurred 1-2 days post-administration. Bedinvetmab was detectable up to the last sampled time point (34 days) in two horses (estimated concentrations: 1.9 and 1.7 μg/mL), while in the third horse, it was detectable only up to 5 days post-administration (0.13 μg/mL). Although the mechanisms underlying this variability were unclear, these results demonstrate that mAbs developed for other species can exert prolonged effects in horses, underscoring the importance of implementing effective doping controls for the misuse of therapeutic mAbs.

This work assessed the correlation between the measurements of A4 and T in serum obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-high resolution mass spectrometry (GC-qTOF). Both procedures using LC-MS/MS and GC-qTOF instruments were validated using the 6PLUS1Multilevel Serum Calibrator Set MassChrom Steroid Panel 2. Positive and negative quality controls and human serum samples were used to check the correlation between these two approaches. Correlation between T and A4 concentrations measured by GC-qTOF and LC-MS/MS was found to be adequate when correlation tests, regression analysis, and Bland-Altman tests were applied. Although the LOQs required in the WADA technical documents for both analytes were not reached under the described assay conditions, the advantages provided by high-resolution mass spectrometry for the detection and quantification of steroids should not be discarded. The technical document for the identification of substances using mass spectrometry instruments does not include acceptance criteria when using HRMS platforms but should be considered due to the recent increase in its use in anti-doping laboratories.

Detection of androgen doping relies on mass spectrometry-based methods to detect natural endogenous and exogenous androgens in urine and serum. To distinguish exogenous administration from natural variation in endogenous serum androgens requires a robust quantitative basis in the variability of serum androgen measurements. The present study therefore aimed to use extensive data from serial antidoping testing of elite male and female athletes to define the reproducibility of serum androgen profiles in male and female athletes. The present study analyzed 5516 samples from 1689 athletes for serum T, DHT, and A4 concentrations and calculated the T/DHT and A4/T ratios from 889 male and 778 female athletes, excluding samples from pregnant, doped, or XY Disorders of Sexual Development (DSD) individuals. As well as the median and interquartile range, the coefficient of variation (CV) and its 95% confidence limits were calculated from three, six, or nine replicate within-person serum samples (309, 146, and 83 for females and 276, 122, and 65 for males, respectively). Variability was greater for females than males for serum T, DHT, and T/DHT ratio (p < 10[-6]) but not significantly different for serum A4 and for A4/T ratio. These data may provide a basis for enhanced detection of T doping and for diagnosis of XY DSD disorders, 5α reductase 2, and 17β hydroxysteroid dehydrogenase 3 deficiencies.

New tadalafil analogues were found to be added illegally to an herbal supplement marketed for the enhancement of sexual function. The compounds were separated using flash column chromatography. The structures of the detected tadalafil analogues were elucidated by HPLC-DAD/MS and nuclear magnetic resonance (NMR) spectroscopy. A protonated molecule at m/z 450.16 was detected by LC-MS, corresponding to a molecular formula of C24H23N3O6 for both of the diastereomeric compounds. These unknown compounds (1 and 2) were identified as tadalafil analogues containing an additional dihydroxyethyl group and were named N-(2,3-dihydroxypropyl)nortadalafil.

Whole-blood samples are widely utilized in the field of doping control. Owing to the limited volume of whole blood collected from athletes and the fact that Tempus Blood RNA Tube and Paxgene Blood RNA Tube are primarily intended for RNA stabilization and require dedicated extraction workflows, this study aimed to develop a more efficient and cost-effective RNA extraction approach using smaller volumes of EDTA-anticoagulated whole blood. RNA was extracted from human whole blood using three different methods: the TRIzol Reagent, the PureLink RNA Mini Kit (Invitrogen), and the QIAamp RNA Blood Mini Kit (Qiagen). Apparent RNA yield and purity were assessed via a NanoDrop spectrophotometer, and RNA suitability for RT-qPCR analysis was evaluated. The results indicate that red blood cell lysis influenced RNA extraction outcomes. Additionally, clear differences in RNA quality and quantity were observed. Among the three methods evaluated, the QIAamp RNA Blood Mini Kit (Qiagen) tended to yield higher apparent RNA concentrations, purity, and integrity. Therefore, under the exploratory conditions tested, the QIAamp RNA Blood Mini Kit (Qiagen) demonstrated comparatively favorable performance for RNA-based biomarker analysis relevant to doping control.

Homologous blood transfusion (HBT) doping, using donor red blood cells (RBCs) to enhance oxygen capacity, remains a persistent challenge for doping control laboratories, especially in specific populations. Current RBC antigen-based flow cytometric detection (double population, DP) has limited sensitivity in genetically homogeneous populations due to common phenotypic overlap. This study aimed to enhance HBT detection by optimizing population-specific RBC antigen panels and incorporating complementary platelet-derived human leukocyte antigen (HLA) markers. Based on antigen-negative frequency, M, N, and Le[b] antigens were evaluated as candidate markers for improved discrimination in the Japanese population. In simulated HBT samples with a 5% mixing ratio, N antigen showed a clearly separated bimodal distribution, indicating strong potential for DP detection. Although Le[b] antigen showed unclear DP, M antigen displayed uniformly high expression, useful for distinguishing expressing populations. In addition, platelet HLA analysis, especially targeting HLA-A24, enabled detection of donor-derived components even with identical RBC antigen phenotypes. Mixed samples containing as low as 1%-5% donor cells produced visually detectable DP signals in the platelet gate. In conclusion, integrating a race-tailored RBC antigen panel with complementary platelet HLA monitoring may provide a potentially improved framework for HBT detection, particularly in ethnically homogeneous populations like Japan. The optimal antigen panel is highly population-specific due to genetic variations, requiring adaptation for other diverse ethnic groups. Future cohort studies are warranted to validate this dual approach and refine detection sensitivity.

A pilot study was conducted to detect meldonium in raw meat samples from young calves slaughtered 15 and 30 days (n = 3 per time-point) after a 15-day course of Emidonol. Meldonium concentration was also assessed in meat samples after cooking before consumption, as well as in urine samples from volunteers after consuming a single 350 g portion of cooked meat. Urine was collected over 48 h. The maximum mean estimated meldonium concentrations in raw meat samples were 5250 ng/g on the 15th day after drug treatment and 3110 ng/g on the 30th day after drug treatment. The mean estimated meldonium concentrations in medium-rare steak cooked for 10 min (15 days after the course) and baked meat were 2960 and 1970 ng/g, respectively; for the "30-day" samples, these values were 1380 and 760 ng/g. The mean maximum estimated urine meldonium concentrations, which peaked at 2-5 h post-consumption, were 386 and 344 ng/mL for the "15-day" meat, and 258 and 219 ng/mL for the "30-day" meat, respectively. Thus, after eating meat samples contaminated with Emidonol, meldonium can be detected in urine 24-28 h post-consumption; during the first 2-14 h, its concentration can be even higher than the WADA MRPL level, which may result in an inadvertent adverse analytical finding for meldonium in sports drug testing.

Pegmolesatide is a novel synthetic erythropoietin-mimetic agent that binds to and activates the erythropoietin receptor (EPOR), thereby stimulating erythropoiesis. Owing to its erythropoiesis-stimulating activity, it has been included in the latest World Anti-Doping Agency (WADA) Prohibited List as an erythropoietin receptor agonist (S2.1.1). The current analytical approach for pegmolesatide in doping analysis mainly relies on bottom-up nano-liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS) in serum. Although accurate and sensitive, this approach involves extensive sample preparation and time-consuming LC analysis, which limits its suitability for high-throughput initial testing procedure (ITP). In this study, an enzyme-linked immunosorbent assay (ELISA), termed the EPOR-anti-PEG ELISA, was developed as a screening method for pegmolesatide in urine and serum. The assay employs EPOR as the capture reagent to enrich pegmolesatide, followed by detection of its polyethylene glycol (PEG) moiety using the anti-PEG monoclonal antibody RM105. Method validation included assessment of selectivity, limit of detection (LOD), dynamic range, reliability, and carryover. Under optimized conditions, the assay showed an LOD of 1 ng/mL in urine, with an adjusted OD450 screening cutoff of 0.029. In serum, with an adjusted OD450 screening cutoff of 0.243, the LOD was 2 ng/mL, which was lower than the limit of identification (LOI) of 7 ng/mL determined by LC-HRMS. Overall, this ELISA provides a streamlined, cost-effective, and high-throughput screening approach for pegmolesatide and may serve as a practical complement to the LC-HRMS method in doping analysis.

Tesofensine (NS-2330) is a pharmacologically active compound with weight-reducing effects in obese patients. Although still under regulatory review, it has been marketed online as a dietary supplement promoted for weight management and metabolic enhancement. Due to its impact on body weight, tesofensine could be relevant in competitive sports, particularly in weight-class disciplines and sports where power-to-weight ratio is decisive. It is classified under "S6 stimulants" on the World Anti-Doping Agency's Prohibited List and is prohibited in-competition only, making detailed knowledge of its metabolism and excretion essential for anti-doping purposes. Although the pharmacological effects and elimination of tesofensine and one dealkylated metabolite were described previously, elimination profiles and structural information on additional metabolites have been limited. In this study, in vitro metabolism experiments were conducted, followed by investigation of urinary metabolism and elimination in six volunteers after ingestion of 483 μg tesofensine as a dietary supplement. Urine was collected for up to 600 h, prepared by solid-phase extraction, and analyzed by LC-HRMS. Four principal metabolites were identified: three dealkylated metabolites (M1-M3) and one hydroxylated and glucuronidated metabolite (M4), supported by MS/MS dissociation patterns. The validated analytical method for human urine showed an LOD of 0.01 ng/mL, 34% recovery, and 8% interday imprecision. Marked interindividual variability was observed, with peak concentrations of 1-4 ng/mL after 4-46 h and detection windows up to 500 h. The findings enhance analytical procedures and suggest that recommended dosing is unlikely to result in concentrations constituting an Adverse Analytical Finding (AAF) under currently applicable stimulant minimum reporting levels.

A package of raw material, deliberately mislabelled as a muscle-building supplement, was obtained online to contain an unknown steroid. In order to accurately identify the composition of the substance, we used a combination of advanced techniques: liquid and gas chromatography coupled with high-resolution mass spectrometry (GC-HRMS and LC-HRMS), alongside nuclear magnetic resonance (NMR) spectroscopy. By combining the strengths of these methods, we successfully identified the compound as 6-Bromo-androst-4-en-3,17-dione (6-BrAED). This substance is a designer steroid of testosterone that is not currently on the World Anti-Doping Agency (WADA) prohibited list. As it is unlisted, there is a serious hidden risk that athletes could take it by accident through contaminated supplements.