ABSTRACT Tesamorelin is a growth hormone‐releasing hormone (GHRH) analogue used for reducing excess abdominal fat in HIV‐infected patients suffering from lipodystrophy. Together with other GHRH analogues, it is classified as a doping agent and is prohibited at all times according to the World Anti‐Doping Agency (WADA). To investigate whether current testing methodologies are adequate for detecting tesamorelin abuse, an administration study was performed involving six healthy volunteers and 10 consecutive days of Egrifta (tesamorelin) injections, mimicking a doping protocol. A validated solid‐phase extraction method was used to analyse the samples, reaching limits of detection of 13 pg/mL in urine and 41 pg/mL in serum. To account for the generation of metabolites by proteolytic degradation, tesamorelin was incubated in fresh human serum. Five previously unreported metabolites were found and identified, extending the detectability of tesamorelin abuse. Oxidation of the methionine residue was observed in tesamorelin and metabolites, hence ions to monitor the sulfoxide and sulfone forms were added to the methods. Despite a limit of detection 70 times lower than the WADA minimum required performance level and the inclusion of several metabolites, tesamorelin use could not be detected in post‐administration urine. In serum, tesamorelin metabolites were detected in all six volunteers for up to 2 h after injection, whereas intact tesamorelin was detected in just two volunteers, up to 30 min post‐administration. The outcome of this study shows that tesamorelin has a short detection window and testing methods should focus on metabolites in serum, rather than intact compounds in urine.
Wastewater-based epidemiology (WBE) is a reliable complement to traditional drug-use monitoring approaches such as surveys and law-enforcement statistics, yet evidence from small countries with heterogeneous population settings remains limited. This study applied WBE to estimate community-level consumption and geographical variation of four illicit stimulant drugs (amphetamine, cocaine as benzoylecgonine, methamphetamine and 3,4-methylenedioxymethamphetamine [MDMA]) across the Grand Duchy of Luxembourg, representing urban, periurban and rural contexts. Weekly time-proportional influent wastewater samples were collected during 2024 from five wastewater treatment plants. Target drugs and metabolites were extracted using solid-phase extraction and quantified by liquid chromatography coupled to triple quadrupole mass spectrometry. Consumption estimates were normalised to population size using established correction factors and drug purity estimates from analysis carried out on seizures in 2024. A total of 255 samples were analysed with 100% detection frequency for cocaine, 99.6% for MDMA, 92.5% for amphetamine and 79.2% for methamphetamine. Estimated consumption levels were highest in the treatment plant serving Luxembourg City and lowest in the most rural setting. Significant increases were observed for cocaine and MDMA during weekends. Drug consumption showed linear correlations with population size across all substances. Annual consumption estimates identified cocaine as the most consumed illicit stimulant in Luxembourg. Wastewater-based epidemiology allows a nonbiased estimation of stimulant drug consumption in Luxembourg, revealing distinct spatial patterns with higher consumption in urban than in rural areas, as well as pronounced increases during weekends. These findings demonstrate the utility of this approach in complementing traditional drug surveillance systems and supporting evidence-informed public health responses.
This publication describes the analytical characterization of ZC-B (3-(4-bromo-2,5-dimethoxyphenyl)azetidine, also termed 2C-B-AZET), a novel hallucinogenic substance identified in capsules seized from drug-related cases. The off-white powder recovered from inside the capsules was used without further purification and systematically analyzed using gas chromatography-mass spectrometry (GC-MS), ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS), Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR). Following GC-MS analysis, ZC-B exhibited characteristic fragment ions involving the bromine isotopes at m/z 242/244, m/z 226/228, and m/z 148. The molecular ion could only be observed at very low intensity. Under UHPLC-QTOF-MS/MS conditions, the protonated molecule was detected at m/z 272.0282. The predominant product ions in auto-MS/MS mode were m/z 228.9860 and 198.9752. A search of the mass spectrometry data against the NIST spectral library yielded no matching results. The off-white solid recovered from inside the capsules was directly characterized by NMR spectroscopy for full structural elucidation. Comparison with recent literature unambiguously identified the substance as ZC-B succinate. This established analytical framework was simple and well-suited for the qualitative detection of ZC-B in forensic drug seizures. Furthermore, it serves as a reference for the detection and identification of emerging new psychoactive substances (NPS).
ABSTRACT Doping analysis of erythropoietin receptor agonists (ERAs) is frequently hindered by the “no band” phenomenon, in which endogenous erythropoietin (EPO) becomes undetectable owing to a variety of pre‐analytical factors. The absence of an internal standard (ISTD) poses the risk of an undetected sample preparation failure. Furthermore, the EPO gene variant c.577del presents a substantial analytical challenge. Specifically, the failure to identify variant‐encoded EPO (VAR‐EPO) during reverse immunopurification (RI) may cause false‐positive results for recombinant EPO (rEPO), highlighting the necessity of an ISTD for RI process monitoring. Nevertheless, the degradation of high molecular weight PEG‐modified ISTDs could interfere with ERAs detection. To address these technical limitations, low molecular weight ISTDs represent a superior alternative for methodological development. This study reports the strategic development of two novel low molecular weight ISTDs: a wild‐type EPO‐based designed mutant (INS1) and a mutant EPO encoded by the c.557del variant (INS2). Both mutant proteins were expressed in the H5 insect cell line via the baculovirus expression vector system. The two engineered EPO mutants possess simplified paucimannose‐type N‐glycans, conferring a distinctly lower molecular weight profile than endogenous EPO and mammalian cell‐expressed rEPO. Functionally, INS1 is designed to monitor the integrity of the initial testing procedure (ITP), whereas INS2 is engineered for process monitoring of RI to enable accurate rEPO confirmation. The results demonstrate that the developed low molecular weight ISTDs can effectively monitor procedural integrity and improve the detection performance of ERAs analytical assays.
ABSTRACT Hair analysis is widely used to retrospectively monitor long‐term drug exposure in forensic toxicology, yet the possibility of external contamination can complicate result interpretation. This challenge applies not only to common drugs of abuse but also to synthetic cannabinoids (SCs), one of the largest groups of new psychoactive substances (NPS) on the European drug market. During abstinence control, a suspect's scalp hair sample taken in August 2023 was found positive for MDMB‐4en‐PINACA (15 pg/mg) along with several other prevalent SCs, for example, ADB‐BUTINACA, EDMB‐PINACA, and JWH‐210, despite a claimed 2‐year abstinence period. A multi‐matrix, multi‐method analytical approach was applied to the scalp hair, washing solutions, and a cutout of the baseball cap worn during and after the alleged period of SC use, comprising gas chromatography–mass spectrometry (GC‐MS) and liquid chromatography‐quadrupole time‐of‐flight mass spectrometry (LC‐QToF‐MS) screening and liquid chromatography–tandem mass spectrometry (LC‐MS/MS) quantification. The cap extract showed an identical SC profile to the hair sample, with MDMB‐4en‐PINACA at approximately 22 ng/cm 2 . No SCs introduced to the European market after the claimed beginning of abstinence (end of July 2021) were detected. Nicotine, caffeine, and their metabolites verified cap wearing. Hair analyses in our lab since mid‐2021 identified MDMB‐4en‐PINACA as the most prevalent SC (34.8% of positive samples). Thus, contamination of hair by wearing the cap is assumed to be the cause for the presence of SCs in hair. Laboratories engaged in hair analysis for abstinence testing should be aware that drug detection in hair could also result from external contamination by clothing.
