The use of pre-precursors to illicitly manufacture 3,4-methylenedioxyphenyl-2-propanone (MDP2P) for the synthesis of 3,4-methylenedioxymethylamphetamine (MDMA) has emerged in clandestine laboratories. Methyl 3-(3,4-methylenedioxyphenyl)-2-methyl glycidate (MMDMG) and ethyl 3-(3,4-methylenedioxyphenyl)-2-methyl glycidate (EMDMG) have recently been seized in clandestine laboratories in New South Wales, Australia. These glycidate esters can be converted to MDP2P via acid or base hydrolysis. This study investigated factors influencing MDP2P yield from MMDMG and EMDMG through both pathways. For acid hydrolysis, hydrochloric, sulphuric, phosphoric and acetic acids were examined at different reaction times and temperatures. Reactions at 100°C produced higher MDP2P yields than those at room temperature, and 18 h was sufficient for complete conversion. Hydrochloric and sulphuric acids gave the highest yields after 18 h at 100°C (79% and 78%, respectively), phosphoric acid gave moderate conversion (~30%), and acetic acid produced poor yields (< 10%). The conversion proceeded via two previously unreported diol-ester intermediates. Base hydrolysis involved heating glycidate esters with sodium hydroxide in alcohol, forming sodium piperonyl methyl ketone (PMK) glycidate, followed by reaction with aqueous acid. Heating MMDMG in methanol or EMDMG in ethanol for 5 h produced sodium PMK glycidate. This intermediate was unstable in acidic aqueous solution and rapidly converted to MDP2P at room temperature. Yields obtained using hydrochloric, sulphuric and phosphoric acids (73%, 71% and 69%, respectively) were comparable to acid hydrolysis, whereas acetic acid produced lower yields (25%). This work may assist law enforcement agencies to predict illicit drug yields from pre-precursors and support investigations into emerging illicit drug synthesis trends.
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.
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.
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-R2 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.
ABSTRACT 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.
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.
ABSTRACT 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.
ABSTRACT With its widespread recreational and increasing medical use, ∆ 9 ‐tetrahydrocannabinol (∆ 9 ‐THC), the main psychoactive compound in cannabis, is regularly subjected to drug testing in clinical and forensic toxicology. ∆ 8 ‐ and ∆ 10 ‐THC have recently entered unregulated drug markets. Their close structural similarity to ∆ 9 ‐THC poses various analytical challenges, with a high risk of misidentification and misinterpretation of bioanalytical data. This study investigated the in vitro metabolic fate of ∆ 8 ‐ and ∆ 10 ‐THC in comparison to ∆ 9 ‐THC using human hepatocytes and high‐performance liquid chromatography coupled to high‐resolution time‐of‐flight analysis (HPLC‐QToF). A comparable metabolism was observed for ∆ 8 ‐THC and ∆ 9 ‐THC, with the formation of the monohydroxy and carboxy metabolites and their glucuronides. In contrast, ∆ 10 ‐THC was found to be extensively glucuronidated (forming ∆ 10 ‐THC‐glucuronide) and monohydroxylated, with only minor formation of a carboxy metabolite. Structural considerations led to the hypothesis that ∆ 10 ‐THC is predominantly hydroxylated at a different site than ∆ 8 ‐THC and ∆ 9 ‐THC. THC isomers should be considered in cannabis drug testing. The differing metabolism of ∆ 10 ‐THC exacerbates the risk of misinterpretation of analytical results.
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.
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.
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.
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.
