Drug Testing and Analysis最新文献

As of 2025, the distribution of products claiming to contain so-called semisynthetic cannabinoids has continued. In most cases, the names of tetrahydrocannabinol (THC) analogs contained in these products are labeled; however, the actual components are often unknown. In this study, we identified the compounds in products that claim to contain THC analogs. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-photodiode array-mass spectrometry (LC-PDA-MS) were used to analyze six products. Seven compounds were isolated from products via silica gel column chromatography, and their structures were determined by ¹H, ¹³C NMR, and various two-dimensional NMR techniques, including H-H correlation spectroscopy, heteronuclear multiple quantum coherence, heteronuclear multiple-bond correlation, and nuclear Overhauser effect spectroscopy. The compounds detected in products A and B were identified as Δ⁸-THCM and Δ⁹-THCM. From product C, 9(R)-hexahydrocannabiphorol methyl ether (9(R)-HHCPM) was identified. The compounds isolated from product D were identified as 2-allyl-Δ⁸-THC. Δ⁹-THC methyl carbonate and Δ⁸-iso-THC methyl carbonate were isolated and identified from product E. The compounds isolated from product F were identified as 10(S)-Hydroxy-9(R)-HHC. This study is the first report on THC analogs having methylcarbonated hydroxyl groups at the C1 position of THC in commercial products. The newly detected THC analogs are potential health hazards if used in general; there are no toxicity data for any of these compounds. In addition, with their unregulated synthesis and the by-product/residues that might have concerns exist. Thus, there are concerns regarding the distribution of products containing new THC analogs.

3,4-Methylenedioxymethamphetamine (MDMA) remains unapproved for therapeutic use despite the promising results of MDMA-assisted psychotherapy. There is a need to better understand the safety, pharmacokinetics, and toxicology of possible MDMA-based prodrugs. Like lisdexamfetamine, amino acid prodrugs of MDMA may enable more controlled systemic exposure, but their metabolic activation pathways and metabolites are not known yet. This study investigated the bioactivation and metabolism of the MDMA prodrugs, MDMA-tryptophan (MDMA-Trp), MDMA-lysine (MDMA-Lys), and MDMA-glycine (MDMA-Gly), in zebrafish embryos (ZE), pooled human liver S9 fraction (pHLS9), pooled fresh human whole blood (pFHWB), and human urine after microdosing (HMD). It elucidated mechanistic activation routes and identified screening targets relevant for drug testing and safety assessment. In ZE, MDMA-Trp underwent hydroxylation and N-dealkylation prior to amide cleavage, indicating a stepwise bioactivation pathway that differs from direct conversion observed for the other prodrugs. All three prodrugs were cleaved to MDMA in ZE, pHLS9, and HMD, with known MDMA metabolites additionally formed in ZE and pHLS9, whereas no metabolites were detected in pFHWB, suggesting that amide cleavage is not mediated in blood under the tested conditions. Unique urine screening targets were identified only for MDMA-Trp, while biomarkers for MDMA-Lys and MDMA-Gly consisted of MDMA and known MDMA metabolites. This study demonstrated conversion of amino acid prodrugs to MDMA in pHLS9- and ZE-based systems and in humans after microdosing, but not in blood. There is a need for further studies such as their pharmacokinetic profiles in humans.

Tetrahydro sulfate metabolites are well-established long-term biomarkers of methyltestosterone use that can be detected intact by LC-MS/MS. However, their identification using product ion spectra under collision-induced dissociation conditions in negative ion mode is an analytical challenge under the provisions of the WADA TD2023IDCR. In this study, six out of eight potential tetrahydro-methyltestosterone sulfate metabolites were microscale-synthesized to facilitate both the structural elucidation of the detected metabolites and the development of direct identification methods. Their identification was based on their GC-MS/(MS) analysis after TMS derivatization or LC-HRMS/(MS) analysis following derivatization of the free 17β-hydroxy group with carbonyldiimidazole (CDI), producing 17β-OH-imidazole carbamate derivatives. The resulting derivatives were detectable in both negative and positive ion modes, enabling their identification through characteristic product ion spectra. Urine samples from two 17α-methyltestosterone excretion studies were analyzed using these methods, and detection/identification time windows of intact sulfate metabolites were estimated under TD2023IDCR and compared with those obtained from GC-MS/(MS) analysis of the glucuronide fraction after hydrolysis. Overall, the inclusion of the tetrahydro-methyltestosterone sulfate metabolites significantly extends the detection time window for methyltestosterone abuse. Still, the established identification time window was similar to, or shorter than, that derived from the glucuronide fraction analysis.

Prohibited gene editing in horses (either in embryos or via cell culture and cloning) can result in both desired and undesired outcomes. If left undetected, changes can proliferate within the population in subsequent generations, posing a major threat to welfare and breed integrity.

Despite the recent success in introducing supramolecular solvents (SUPRAS)-based extraction to drug analysis, its application and robustness in day-to-day regular urine testing have yet to be demonstrated. Moreover, the applicability of SUPRAS in equine doping control testing remains unexplored. In this work, we have successfully developed for the first time a simple, rapid, inexpensive, and environmentally friendly SUPRAS extraction method for analyzing 76 prohibited substances of different classes (selective androgen receptor modulators, hypoxia-inducible factor prolyl hydroxylase inhibitors, angiotensin II receptor antagonists, benzodiazepines, etc.) in hydrolyzed horse urine with liquid chromatography-mass spectrometry (LC/MS) for detection. The developed 1,2-hexanediol-based SUPRAS-LC/MS method has been fully validated, and its applicability and robustness in day-to-day testing of horse urine have also been demonstrated. This work marks a significant milestone in the advancement of green and sustainable drug testing methodology in equine sports, offering a novel approach to address one of the complexities inherent in equine doping control.

In clandestine laboratories, amphetamine is predominantly synthesized via the Leuckart route. In recent years, a trend is observed: Capacities of illicit production facilities for synthetic drugs in Europe increased and fewer small-scale laboratories are encountered by police and customs authorities. One of the designer pre-precursors currently applied is methyl α-phenylacetoacetate (MAPA), which can be converted into the key synthesis educt benzyl methyl ketone by acidic hydrolysis. Besides replacements of former designer pre-precursors due to scheduling (e.g., α-phenylacetoacetonitrile [APAAN]), another trend for synthesis is observed since 2019, namely, the alkaline hydrolysis of the reaction intermediate N-formylamphetamine into the free amphetamine base during the second step of the Leuckart route instead of the previously predominantly applied acidic hydrolysis using concentrated hydrochloric acid. In this study, 28 samples of products and production waste seized from a dismantled large-scale amphetamine laboratory in Germany that used MAPA as designer pre-precursor and the modified alkaline Leuckart step two were analyzed by a nontargeted liquid chromatography-high-resolution mass spectrometry approach for tentative identification of specific markers for the use of MAPA and the alkaline hydrolysis of N-formylamphetamine. After peak picking, 23 features were identified as suspects and further seven new features were tentatively identified. These seven potential marker compounds appeared to be indicative for the pre-precursor conversion step and were found in production waste and in amphetamine base product samples. Additionally, there were hints for the formation of high molecular weight compounds during the modified Leuckart step two.

The steroidal module of the Athlete Biological Passport (ABP) longitudinally monitors five ratios between urinary concentrations of endogenous anabolic and androgenic steroids. Even though it has improved detection of testosterone doping, the interpretation of data from multiple discrete biomarkers is complex. This study sought to create a single score to identify doping rather than relying on the interpretation of each parameter alone. A Bayesian model was used to define an ABP sequence probability for each biomarker to assess the extremity of a measurement relative to the expected levels from ABP. This was used to discriminate between doped and presumed clean individuals based upon pattern classification of biomarkers using classification algorithms. Data were obtained from laboratory-controlled experimental studies as well as routine doping control tests. A laboratory model (where classifier is trained using the laboratory-controlled data only) and a mixed model (where classifier is trained on combined laboratory-controlled and doping control data) were developed and tested on the doping control data. Logistical regression was seen to have the best classification performance across the methods used, with the Abnormal Steroid Profile Score (ASPS) representing the estimated probability from the logistical regression model. Classifier performance produced an AUC of 0.67 and 0.75 when trained on the laboratory model and the mixed model, respectively, with T/E and 5α-Diol/5β-Diol representing the main biomarkers driving the ASPS. These findings demonstrate that the ASPS can discriminate between the doping status of individuals, even if a mixture of steroids, administration methods and doses are used.

Recombinant human erythropoietin (rHuEPO) is often misused in endurance sports due to its potent erythropoietic effects. While transcriptomic biomarkers hold promise for detecting rHuEPO use beyond conventional testing windows, many proposed gene markers may also respond to physiological stimuli such as exercise or altitude. This study compared 153 previously reported rHuEPO-responsive genes in whole blood with transcripts identified during exercise (GEPREP database) and high-altitude exposure (four independent studies). For the exercise dataset, gene-level statistical outputs were obtained directly from the GEPREP database, while biological relevance was calculated using Cohen's d. Analyses of altitude and rHuEPO datasets followed the original statistical procedures described in each study. Among the 153 rHuEPO-responsive genes, 94 overlapped with altitude and 34 with exercise. However, 50 genes remained unaffected by either exercise or altitude stimuli. Enriched in post-translational regulation and intracellular transport pathways, these genes represent promising candidate transcriptomic markers of rHuEPO administration. This work provides a refined gene panel that reduces the likelihood of false positives and requires further experimental validation before integration into RNA-based detection tests.