Betamethasone sodium phosphate (BETP) is a glucocorticosteroid used in humans. The primary objective was to estimate irrelevant plasma (IPC) and urine (IUC) concentrations using a pharmacokinetic/pharmacodynamic approach based on systemic clearance estimated from intravenous (IV) pharmacokinetic data. As a secondary objective, detection times (DTs) following IV, intramuscular (IM), and intranasal jet (INJ) administration were estimated for medication control. Plasma and urine concentrations were obtained from a total of 20 horses in Japan and Germany, with some horses contributing to more than one route after single IV (0.06 mg/kg, n = 7; 0.04 mg/kg, n = 8), single IM (20 mg/horse, n = 7), and multiple INJ (4 mg/horse for 5 days, n = 6) administration. The data were analyzed using a nonlinear mixed-effects model. The plasma clearance was 276 mL/kg/h, and the steady-state urine-to-plasma ratio was 25.8. For IV administration at 0.06 and 0.04 mg/kg every 24 h, the IPCs were 0.018 and 0.012 ng/mL, and the corresponding IUCs were 0.47 and 0.31 ng/mL, with the latter IUC close to the current International Federation of Horseracing Authorities international screening limit (ISL) (0.2 ng/mL). All horses fell below the ISL within 72 h after single IV administration at 0.06 and 0.04 mg/kg and after single IM administration, whereas all horses fell below the ISL within 48 h after INJ administration. The applicability of the estimated DTs should be interpreted according to the regulatory framework governing the use of human BETP formulations and their routes of administration in the relevant jurisdiction.

Lysergic acid diethylamide (LSD) analogues have emerged as new psychoactive substances (NPS) since the mid-2010s, and novel derivatives continue to appear on the recreational drug market. Since late 2022, products marketed as "1D-LSD" have been advertised on X (formerly Twitter) and other websites. Despite various online references portraying 1D-LSD as 1-(1,2-dimethylcyclobutane-1-carbonyl)-LSD, our chemical analysis of a product marketed as "1D-LSD" revealed that it actually contained 1-(thiophene-2-carbonyl)-LSD (1T-LSD), which has the same nominal mass. Another group subsequently reported similar findings in a blotter paper printed "1D-LSD.". In this study, we synthesized 1D-LSD as a mixture of four diastereomers, reflecting the stereoisomers of the commercially available 1,2-dimethylcyclobutane-1-carboxylic acid. The mixture was separated into two groups, each consisting of two diastereomers, through preparative liquid chromatography. The correspondence between the fractions and diastereomers was established using nuclear magnetic resonance spectroscopy aided by quantum chemical calculations. The synthesized 1D-LSD was further characterized using liquid chromatography/mass spectrometry, gas chromatography/mass spectrometry, and Fourier-transform infrared spectroscopy. Additionally, the incorporated carboxylic acid was recovered through alkaline hydrolysis, and analysis of the recovered acid confirmed the presence of all four diastereomers. The analytical properties of 1D-LSD were compared with those of 1T-LSD. Our comparative analysis revealed that the two compounds exhibited markedly different analytical properties, indicating that confusion between them is unlikely when appropriate analytical methods are applied.

Benzodiazepines are widely prescribed psychoactive drugs frequently encountered in forensic and clinical toxicology. Hair analysis is a useful tool for retrospective drug monitoring, but its effectiveness depends on highly sensitive and reliable analytical methods due to the complexity of the matrix and the typically low concentrations of analytes, especially after single-dose exposure. This study aimed to develop, validate, and compare two LC-MS/MS methods for the simultaneous identification and quantification of 38 benzodiazepines and their metabolites in 25 mg of hair. Both methods used identical procedures for sampling, washing, and instrumental analysis, differing only in the extraction step. In the first method, 500 μL of M3 solution was added to cut hair and incubated at 100°C for 60 min. In the second, methanol was added to pulverized hair and incubated overnight at 50°C. Extracts were evaporated, reconstituted in 500 μL of M3, and 3 μL injected into the LC-MS/MS system. Validation with spiked hair and quality control samples showed limits of quantification ranging from 2 to 46 pg/mg, with linear calibration curves up to 200 pg/mg. Imprecision was ≤ 20% (n = 9), and most analytes showed process efficiencies above 70%. Application to 72 real samples demonstrated that the M3 extraction provided lower detection limits than the methanol method, making it more suitable for identifying single-dose exposure. Both methods are suitable for benzodiazepine screening in forensic toxicology, particularly when sample amounts are limited.

This study presents findings from the European Syringe Collection and Analysis Project Enterprise (ESCAPE) conducted in Greece between 2022 and 2024. Used syringes were collected through harm reduction services and needle distribution and collection programs coordinated by EOPAE in Athens, Thessaloniki, and Patras, and analyzed using standardized ESCAPE extraction procedures followed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). A total of 2122 syringes were included in the study. Substantial geographical variability in injected drug-use patterns was identified across the participating cities. Athens demonstrated persistent opioid predominance accompanied by high cocaine co-detection, extensive adulteration, and frequent multiple drug co-detections. Methamphetamine was detected exclusively in Athens, demonstrating a transient increase during 2023. Thessaloniki exhibited a progressively cocaine-dominant profile, with cocaine detections increasing markedly during the study period, while opioids remained consistently prevalent. In contrast, Patras demonstrated a distinct buprenorphine-centered profile associated with comparatively lower adulteration prevalence and fewer multiple drug category co-detections. Increasing detections of benzodiazepines, methadone, and pregabalin were observed across all cities during 2024. Fentanyl was detected in all participating cities during the final study year, although prevalence remained low. Adulterants, including caffeine and paracetamol, were highly prevalent in Athens and Thessaloniki but substantially less frequent in Patras. The present findings demonstrate substantial regional heterogeneity in injected drug-use patterns within Greece and support syringe residue analysis as a rapid and highly informative surveillance approach capable of identifying temporal drug-market changes, emerging synthetic opioids, adulteration patterns, and evolving patterns of injected drug use.

The new synthetic opioid (NSO) market has been reshaped by the emergence of an opioid class known as "nitazenes," 2-benzylbenzimidazole opioids spanning a wide potency range, with several analogues surpassing the potency of fentanyl. The recently identified compound 5-cyano isotodesnitazene, also referred to as isotocyanazene or isotocyanozene, represents a nitazene analogue with a previously unreported 5-cyano substitution. This study determined the in vitro μ-opioid receptor (MOR) activation potential of 5-cyano isotodesnitazene and related "cyanazene" analogues, in comparison with their desnitazene and 5-nitro-substituted counterparts. Using a cell-based β-arrestin 2 recruitment assay, 5-cyano isotodesnitazene (EC50 = 71.5, Emax = 117% vs. fentanyl) demonstrated lower potency than its 5-nitro analogue isotonitazene (EC50 = 2.55, Emax = 130%) and fentanyl (EC50 = 15.1, Emax = 99.9%) while exhibiting comparable activity to isotodesnitazene (EC50 = 49.2, Emax = 113%). Similar in vitro trends were observed for the other three cyanazene analogues examined (5-cyano protodesnitazene, 5-cyano etodesnitazene, and 5-cyano metodesnitazene), indicating that a 5-cyano substitution does not compensate for the potency loss generally associated with removal of the 5-nitro group in the nitazene scaffold. Additionally, MOR activation was confirmed in an authentic 5-cyano isotodesnitazene sample. All cyanazenes and isotodesnitazene were detectable using BTNX nitazene test strips (Version 2.0). The emergence of 5-cyano isotodesnitazene highlights the continued structural diversification of the nitazene scaffold, underscoring the importance of timely pharmacological characterization, targeted harm reduction strategies, and close monitoring of newly emerging NSO.

The emergence of nitazene compounds in the illicit drug supply is a significant and increasing concern due to their high potency and rapid evolution in several parts of the world. Nitazenes create challenges for harm reduction drug checking services, where there is a need to provide relevant information about the composition of drug samples to people who use drugs. While targeted paper spray mass spectrometry has demonstrated abilities for drug checking, it is difficult to keep analysis methods up to date for the rapidly evolving drug supply. We have developed a precursor ion scan method that monitors four common nitazene product ions paired with tooling to streamline data interpretation. Twenty-five control samples received at a community-level drug checking service were used in method development and evaluation to ensure direct applicability to illicit drug samples. Using the developed method, three nitazene compounds, N -pyrrolidino protonitazene, N -desethyl etonitazene, and N -pyrrolidino ethylene isotonitazene, were detected across 18 samples where a nitazene was not previously detected upon preliminary targeted analysis. This approach demonstrates rapid, nontargeted screening for the detection of emerging nitazenes, ultimately allowing more complete information about the composition of drug samples to be provided to people who use drugs, as well as public health monitoring and epidemiology. Data collected through drug checking service delivery found that nitazene compounds were most commonly detected in place of expected pharmaceutical opioids, combined with benzodiazepines, and in complex opioid mixtures alongside fentanyl. These findings highlight the wide-ranging presence of nitazene compounds in the illicit drug supply.

Ethyl glucuronide in urine (uEtG) is a biomarker that objectively detects alcohol consumption. However, what uEtG values represent regarding the last drinking event remains unclear. This study investigated whether the recency or amount consumed at the last event are associated with multiple uEtG cutoffs indicative of light to heavy drinking. Adults (n = 349) recruited for an alcohol use disorder clinical trial submitted urine samples and self-reported alcohol measures at baseline and twice weekly during a 4-week observation period prior to treatment. The ThermoFisher DRI immunoassay was used to quantify uEtG levels (0-2000 ng/mL). Self-report measures included the number of hours since last drink (Hours), number of standard drinks at that event (Amount), and ratios of these measures (Hours:Amount and Amount:Hours). Three uEtG cutoffs were assessed (100, 500, and 2000 ng/mL) and generalized linear mixed modeling applied to compare self-report measures below versus above each uEtG cutoff. A total of 1676 uEtG samples with self-reported data were included. Comparisons across cutoffs were significant for Hours (p < 0.001), while Amount was only significant at the 2000 ng/mL cutoff (p = 0.001). Hours also had the strongest association with uEtG results across cutoffs (pseudo-R[2] range = 0.474-0.530). Higher uEtG results indicated more recent drinking, rather than higher levels of alcohol consumption, and the period of detection of uEtG was shorter than previously reported. Even at the low threshold of 100 ng/mL, uEtG was best conceptualized as a measure of alcohol consumption in the prior 24 h, with higher uEtG levels being associated with fewer hours since last drink.

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.