Artificially cultured bacterial solutions derived from urine were used to simulate urine degradation. Bacterial activity primarily induced the hydrolysis of glucuronide-conjugated steroids during the early stage, followed by steroid transformation and redistribution among conjugated forms as degradation progressed. By separating and analyzing free and glucuronide-conjugated steroid fractions, the free-to-total ratios of the diols (5αAdiol and 5βAdiol) were demonstrated to be sensitive indicators of degradation progression, particularly in low-testosterone (T)-excreting populations where the Tfree/Ttotal ratio may be difficult to determine. In the early stage of degradation, the concentrations of steroids in the Urinary Markers of the Steroidal Module of the Athlete Biological Passport (ABP-USM) were not significantly affected, even when a small proportion of free steroids was detected. However, as the degradation process advanced, the degradation markers currently used by WADA (the 5αAND/A and 5βAND/Etio ratios) increased rapidly. Once these ratios exceeded 0.1, the concentrations of both free and glucuronide-conjugated steroids underwent notable changes, which may potentially affect the interpretation of the ABP-USM markers.

Esports has undergone rapid expansion over the past two decades, bringing with it challenges analogous to those observed in traditional athletic domains, particularly the misuse of performance-enhancing drugs (PEDs). Doping poses a dual threat to competitive integrity and player health, yet its manifestation in Esports remains insufficiently examined compared with conventional sports. This disparity has contributed to the under-recognition of substance misuse in professional gaming and the emergence of associated health risks among players. A lack of awareness among tournament organisers and regulatory bodies has further impeded the establishment of comprehensive anti-doping frameworks. To address this gap, the Esports Integrity Commission (ESIC) introduced a prohibited substances list tailored to the cognitive and psychological demands of Esports, acknowledging the limitations of the World Anti-Doping Agency (WADA) framework in this context. Substances commonly implicated in Esports doping include nootropics and other cognitive enhancers that improve attention, reaction speed and mental stamina. While stimulants such as medications for attention-deficit/hyperactivity disorder (ADHD) and energy drinks have been widely discussed, the non-medical use of antidepressants remains underexplored and insufficiently regulated despite their inclusion on ESIC monitoring lists. This review provides a critical synthesis of the pharmacology, pharmacokinetics, adverse effects and analytical detection strategies for antidepressants relevant to Esports doping. A co-citation analysis of 3756 references published between 2000 and 2025 was conducted to map the field's intellectual structure. Systematic screening identified 69 key studies that met predefined inclusion criteria and were analysed in detail.

This study investigated the incorporation of estra-4,9-diene-3,17-dione (dienedione) and its metabolites into equine hair following exogenous administration and evaluates the potential application of hair analysis for long-term doping surveillance. Mane hair samples were collected from three Thoroughbred castrated horses before and after oral administration of dienedione and from untreated colts for comparison. Dienedione was detected in post-administration hair from all treated horses but was absent in corresponding pre-administration samples. Segmental distribution patterns indicated contributions from both systemic incorporation via hair follicle and external absorption, such as sweat or sebum, with considerable interindividual variability. Three dienedione metabolites (M3a, M7a and M7b), previously reported to exhibit prolonged persistence in plasma and urine, were also detected in hair from the treated horses and showed similar segmental distribution. The absence of detectable dienedione and its monitored metabolites in hair from untreated colts, despite their endogenous presence in urine, suggests that hair analysis may provide more definitive information for the interpretation of dienedione findings following exogenous administration.

This study describes the detection and temporal distribution of cyclosporine in equine hair following ocular implant administration in a Thoroughbred gelding. Mane hair samples were collected at 172- and 217-day post-implantation and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after alkaline hydrolysis and liquid-liquid extraction. The method achieved an estimated limit of detection of 0.1 pg/mg. Cyclosporine was detected in hair at both sampling time points, with segmental analysis revealing distribution patterns consistent with systemic exposure and incorporation during hair growth. Peak concentrations were observed in distal segments corresponding to the period of implantation, whereas proximal segments in the later sample were below the detection limit, indicating declining exposure over time. To our knowledge, this study provides the first evidence of an exogenous peptide drug being incorporated into equine hair. The prolonged detectability of cyclosporine in hair highlights its potential as a complementary matrix for long-term monitoring of peptide drug exposure in equine sports.

Discrimination between endogenous and exogenous Gamma-hydroxybutyrate (GHB) in hair matrix is a major challenge in the forensic context. According to the UNODC, to verify a possible intake, GHB concentration should be compared with the subject's basal level. Most published studies investigated endogenous GHB on a single hair segment, without considering possible intra-individual variation. The present study aimed to detect endogenous GHB in hair using a newly validated method, with a focus on the relevance of segmental analysis. Two hundred subjects were tested. After the removal of the first 0.5 cm from the proximal end, each hair strand was divided into three segments of 1 cm. A water extraction was performed during the pulverization process, and the extract was injected into a LC-MS/MS system. Data have been statistically analyzed. Overall, endogenous GHB concentration ranged from < LOD (0.1 ng/mg) to 6.6 ng/mg, with 27% of samples having a value < LOD in at least two segments. Within the remaining subjects, neither gender nor age significantly influenced the concentration. On the other hand, results confirmed that hair treatment can modify endogenous GHB concentrations. Intra-individual variability was limited, with the highest percentage of cases showing minimal changes in concentration (0-0.1 ng/mg) between contiguous segments. However, in up to 4% of cases, the variation resulted to be higher than 1 ng/mg between contiguous segments. The present study then confirms the need to perform in forensic cases the segmental analysis, to be sure that any potential increase of GHB depends exclusively on external administration.

Selective androgen receptor modulators (SARMs), such as LGD-4033, are frequently detected in doping control samples and pose significant challenges for result interpretation, particularly at low concentration levels potentially arising from contamination. In this study, a quantitative method for the determination of the carboxylated long-term metabolite (M5-b) of LGD-4033 in human urine was developed and applied to samples obtained from controlled micro-dose administration studies. Urine samples from single- and multiple-dose administration studies (1, 10, and 50 μg) were analyzed following enzymatic hydrolysis and solid-phase extraction using LC-HRMS/MS. Quantification was enabled by the use of a recently synthesized certified reference material, allowing, for the first time, the generation of quantitative excretion data for long-term metabolite under micro-dose conditions. The results demonstrated the rapid formation of long-term metabolite and a clear dose-dependent increase in urinary concentrations. Extended detection windows were observed for all dosing regimens, with long-term metabolite remaining detectable for several days after single-dose administration and for substantially longer periods following consecutive administration (5 days). Accumulation effects during multiple-dose intake resulted in sustained urinary concentrations and prolonged detectability. The quantitative data generated in this study provide an important basis for improved interpretation of adverse analytical findings involving LGD-4033 and support a more evidence-based distinction between different intake scenarios.

Illicit use of messenger ribonucleic acid (mRNA) agents represents an imminent threat to gene doping control in equine sports. The effectiveness and safety of lipid nanoparticle-encapsulated mRNA agents (LNP-mRNA), which have been well-proven during COVID-19 pandemic, have resulted in prominent growth of the interest on LNP-mRNA agents for expressing desired proteins in vivo. The vast number of potential performance-enhancing mRNAs has demanded cost-effective and reliable methods for their detections. This study employed a multimodal analysis approach to detect an LNP-mRNA product ("LNP-epo" hereafter) arising from encapsulation of a black-market product of human erythropoietin (hEPO) mRNA. Lipid nanoparticle (LNP) components were detected by liquid chromatography/high-resolution tandem mass spectrometry after supported liquid extraction; hEPO mRNA was detected by reverse transcription quantitative polymerase chain reaction after RNA extraction; hEPO protein was detected by sandwich enzyme-linked immunosorbent assay. Comparative elimination analysis of LNP-epo in blood samples collected from an administered horse showed the longest detection time achieved by LNP monitoring (at least 34-day postadministration), followed by hEPO mRNA detection (up to 30 days in whole blood; up to 17 days in plasma), and shortest by hEPO protein detection (up to 3 days). Together with its low analysis cost, broad coverage of LNP components, expandable scope and independence from mRNA sequence modifications and the need for specific reagents (e.g., primers, antibodies), the detection of LNP can be a fit-for-purpose screening method for monitoring the misuse of LNP-mRNA in equine sports. The other two approaches could provide insight on the intentional effect elicited by LNP-mRNA.

Nitazenes are highly potent synthetic opioids of increasing concern in clinical and forensic toxicology, thus underscoring the need for sensitive and selective analytical methods for biological specimens. This study describes the development and validation of an LC-MS/MS method for the quantification of nitazene analogs and related metabolites in plasma using micro-QuEChERS sample preparation. The validated concentration range was 0.05-20 ng/mL, with coefficients of determination R[2] > 0.99. The limit of detection (LOD) was 0.025 ng/mL, and the lower limit of quantification (LLOQ) was 0.05 ng/mL. Bias ranged from -2.6% to 15.7%, within-run precision from 3.4% to 12.7%, and between-run precision from 4.4% to 14.9%, as evaluated at three quality control levels. Matrix effects and recovery were analyte-dependent but compatible with satisfactory overall validation performance. No interferences or carryover were observed. Method applicability was demonstrated in an authentic intoxication case, in which N-pyrrolidino protonitazene was quantified at 1.4 ng/mL, representing, to our knowledge, the first clinical case reported in Brazil. The case was clinically characterized by severe opioid toxicity requiring repeated naloxone administration. The proposed method combined low sample volume, simple sample preparation, and suitable analytical performance for targeted analysis of emerging nitazene compounds. These findings support the use of micro-QuEChERS and LC-MS/MS as a practical approach for targeted nitazene quantification in clinical and forensic toxicology.

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.