Annette Zschiesche

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.

Numerous analogues of the psychedelic and internationally controlled hallucinogen LSD have been described; however, only a limited number have been systematically investigated. This report presents the first case with an analytically confirmed ingestion of ETH‐LAD (6‐ethyl‐6‐nor‐lysergic acid diethylamide), the N ‐ethyl derivative of LSD. An adolescent patient developed moderate to severe symptoms as acute aggression, agitation, unconsciousness, multiple traumatic injuries, metabolic disturbances and persistent psychosis following ETH‐LAD ingestion and required intensive medical therapy with unexpected findings. The concentration of the hallucinogenic new psychoactive substance in serum was determined with 38 ng mL ⁻¹ . After 17 days of hospitalization, the patient was transferred to a psychiatric clinic for further treatment. Physicians must be aware that ETH‐LAD ingestion may, unlike LSD, lead to severe toxic effects, requiring intensive care.

Targeted and untargeted mass spectrometric analyses of urine, a preferred matrix for toxicological screening, require comprehensive spectral libraries of drug metabolites. To detect new synthetic cannabinoids (SCs), biomarkers for newly emerging compounds must be incorporated rapidly. Unfortunately, reference standards for metabolites of new psychoactive substances (NPS) such as SCs are scarce. Since parent SCs are rarely detectable in urine, elucidating their metabolism poses a significant challenge for forensic toxicology laboratories. Untargeted high-resolution mass spectrometry (HRMS) approaches, often complemented by in silico methods, are increasingly vital to forensic toxicology and metabolomics. A pooled human liver microsome (pHLM) assay was used for in vitro generation of phase I metabolites of three prevalent SCs (ADB-BUTINACA, MDMB-BUTINACA, and MDMB-4en-PINACA). Analysis was performed by trapped ion mobility spectrometry-time-of-flight mass spectrometry (timsTOF-MS) using parallel accumulation serial fragmentation (PASEF) acquisition and MetaboScape software with the T-ReX (time-aligned region complete extraction) 4D workflow including in silico metabolite prediction, fragmentation, and collision cross section (CCS) forecasting. In addition, hydrolyzed and nonhydrolyzed SC-positive urine samples were reanalyzed with this workflow. Results showed successful annotation of predicted metabolites for all three SCs, including monohydroxylations and ester hydrolysis. Qualitative pHLM and urine findings were in good agreement. Compound-specific and most abundant in vivo metabolites were incorporated into a targeted UHPLC-QTOF-MS method to analyze 42 urine samples from the casework. Although complex multistep biotransformation reactions (e.g., ADB-BUTINACA dihydrodiol formation) continue to pose a challenge for in silico prediction, this approach is significantly less time-consuming and less labor-intensive than manual evaluation of known metabolic patterns.

Background: The potent synthetic cannabinoid ADB-BUTINACA (also known as ADB-BINACA) was implicated in three fatal intoxications. Post-mortem samples, including femoral and heart blood, urine, gastric content, bile, vitreous humor, cerebrospinal fluid, and various tissues (brain, kidney, liver, lung, muscle), were analyzed. All cases were mixed intoxications with pregabalin, heroin, ketamine, MDPHP, or ethanol. Methods: Post-mortem examinations were performed in all three fatalities. Blood and urine were screened by immunoassay, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for drugs. ADB-BUTINACA was quantified by standard addition in all matrices except vitreous humor and cerebrospinal fluid. For assessing the contribution of ADB-BUTINACA, a Toxicological Significance Score (TSS) was assigned to each case. Metabolite profiles received from analysis of post-mortem matrices were compared with HepG2 cell and human liver microsome data. Additionally, compound stability was assessed over 12 weeks in whole blood. Results: Femoral blood concentrations of ADB-BUTINACA ranged from 4.2–8.2 ng/mL, and in heart blood from 5.7–11 ng/mL. Brain tissue contained 1.0–6.2 ng/g, and vitreous humor 0.33–2.1 ng/mL. TSS for ADB-BUTINACA of 1 – 3 were assigned for all cases. Heart-to-femoral blood ratios (1.3–1.8) indicated relatively low post-mortem redistribution. Metabolite profile assessment indicated that detection of metabolic biomarkers such as the dihydrodiol could be relevant in urine and bile. Matrix storage at −20 °C is highly recommended to avoid stability issues. Conclusion: ADB-BUTINACA was detected in all investigated matrices, with the highest concentrations observed in liver, lung, and kidney tissue. The elevated levels in these organs likely reflect their lipophilicity as well as their involvement in absorption (lungs) and metabolism/excretion processes (liver and kidneys). The results of this study may enhance interpretation of toxicological findings in similar cases.

Urine is one of the preferred matrices for standard toxicological analysis, which makes the inclusion of drug metabolites in targeted and untargeted screening mandatory. Mass spectrometry is key for substance identification, but updating methods for emerging substances like new psychoactive substances (NPS) is challenging due to the limited availability of reference standards for metabolites. This is particularly problematic for drugs that are barely or not detectable at all in urine. Insufficient metabolic knowledge and lack of spectral data carry the risk of false negatives. This study evaluates a non‐targeted workflow using ultrahigh‐performance liquid chromatography‐trapped ion mobility spectrometry time‐of‐flight mass spectrometry (UHPLC‐timsTOF‐MS) and dedicated processing software (MetaboScape), integrating in silico metabolite prediction (BioTransformer), fragmentation (MetFrag), collision cross‐section (CCS) prediction, and library searching. Quetiapine was selected as a model compound. Phase I metabolites were generated via pooled human liver microsomes (pHLMs) and analyzed by UHPLC‐timsTOF‐MS. Features were extracted and annotated with MetaboScape. The workflow successfully annotated 20 phase I metabolites in the pHLM assay, with 13 confirmed by library matching and 18 by BioTransformer. These metabolites were added to a targeted UHPLC‐QTOF‐MS method for analysis of 30 quetiapine‐positive ante‐ and post‐mortem urine samples from forensic casework. This revealed N ‐, O ‐dealkyl and carboxylated metabolites as the most abundant biomarkers in human urine. This integrated approach enables rapid and reliable metabolite detection, supports biomarker discovery, and facilitates routine screening updates, especially for substances without reference standards. Although not intended for exhaustive metabolic characterization, it offers practical applicability in evolving drug landscapes.

Aim: This report presents a series of 16 patients who were admitted to the emergency department following confirmed intake of the potent synthetic cannabinoid ADB‐CHMINACA. Methods: The cases are drawn from a prospective observational study following the recreational use of synthetic cannabinoids. The clinical and analytical data were combined and submitted to a Poison Centre. The severity of poisoning was evaluated on the basis of clinical data, including follow‐up information. The present analysis was conducted exclusively on patients with analytically confirmed intake of ADB‐CHMINACA and treatment in emergency departments for acute adverse effects. Cases with additional relevant serum concentrations of other drugs of abuse that suggested relevant interactions were excluded. Results: Common clinical manifestations included panic attacks, tachycardia, agitation, vomiting and seizures. Most of the patients experienced these symptoms within minutes after inhaling this new psychoactive substance. Serum concentrations of ADB‐CHMINACA varied widely and ranged from 0.2–31 ng/mL, without a clear correlation between severity of the poisoning and drug concentration. Severity of poisoning was moderate to severe, but all patients recovered. Notably, 1 of the patients developed a posterior reversible encephalopathy syndrome, a rare but serious neurological complication characterised by usually transient symptoms like headaches, seizures and altered mental state, often accompanied by brain oedema. In this case, the patient recovered after 4 days. Conclusion: Clinical presentations exhibited by patients with analytically confirmed intake of ADB‐CHMINACA showed similarities to previous reports; neuropsychiatric and cardiovascular symptoms were most prominent. Notable distinctions include the prevalence of panic attacks and trembling.

Forensic entomologists estimate minimum post-mortem intervals (PMI _min ) by measuring the development of larvae on decomposing bodies. Entomotoxicology assumes significance when the deceased had ingested drugs, as these may alter insect development and consequently the accuracy of PMI _min estimations. 5F-ADB, ADB-BUTINACA, and MDMB-4en-PINACA are potent synthetic cannabinoid receptor agonists and drugs of abuse, linked to human intoxications and deaths. We investigated the impact of 5F-ADB on the development of Lucilia sericata (Meigen, 1826) (Diptera: Calliphoridae) larvae and developed a method to detect 5F-ADB, ADB-BUTINACA, MDMB-4en-PINACA, and their metabolites in larvae by liquid chromatography-mass spectrometry. Neonate larvae were exposed to 5F-ADB concentrations that reflect those in human post-mortem specimens. At precise intervals, larvae were killed in near-boiling water, dried, and then their size measured, followed by toxicological analysis. ANOVA demonstrated that only time (= age) significantly affected larval length and mass (through growth). Drug concentrations typically found in post-mortem peripheral blood did not significantly affect larval development, suggesting that 5F-ADB in a corpse would not influence PMI _min estimation with L. sericata . 5F-ADB was detected in trace amounts in larvae that were exposed to post-mortem peripheral blood concentrations. Most larvae from higher concentration treatments contained 5F-ADB levels above the limit of detection of 0.25 µg/kg, but not all samples tested positive. Post-feeding larvae mostly did not contain detectable levels of 5F-ADB, and metabolites were found in none of the larval samples. Actively feeding L. sericata larvae from a corpse may thus be useful to qualitatively identify a prior consumption of 5F-ADB by the deceased.

Purpose: A powder found at a fatality scene, labeled as the synthetic cathinone 3’,4’-methylenedioxy-α-pyrrolidinohexiophenone (MDPHP) and most likely smoked using a crack pipe, was analyzed. The powder was identified as very potent synthetic cannabinoid ADB-BUTINACA/ADB-BINACA with a high purity (> 98%). This case highlights the risks associated with mislabeled novel psychoactive substances (NPS), particularly those purchased online. Methods: The powder was analyzed using liquid chromatography-high resolution mass spectrometry (LC-HRMS), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and nuclear magnetic resonance (NMR) spectroscopy. Comprehensive toxicological screening, including NPS, was performed in urine and blood. ADB-BUTINACA was quantified using a standard addition method on various post-mortem matrices: femoral and heart blood, urine, stomach content, bile fluid and liver tissue. Scalp hair was analyzed via external calibration to assess potential long-term exposure. Results: ADB-BUTINACA concentrations were 34.5 ng/mL in femoral blood, 101 ng/mL in heart blood and 3.1 ng/mL in urine. Traces of MDMB-BUTINACA were found in the powder and hair, but not in other biological matrices. ADB-BUTINACA metabolites were detected in all biological matrices. MDPHP was found at low concentrations (< 2 ng/mL) in blood and urine but not in the powder, indicating prior cathinone use. A toxicological significance score (TSS) of 3 was assigned for this monointoxication with ADB-BUTINACA. Conclusions: This case demonstrates fatal poisoning due to extremely high ADB-BUTINACA concentrations in post-mortem blood samples, emphasizing the severe risks associated with mislabeled substances. It underscores the importance of drug checking services to prevent poisonings and overdoses caused by highly potent NPS.

The rapid emergence of synthetic cannabinoid receptor agonists (SCRAs) poses challenges for drug testing, particularly when analyzing urine samples due to the rapid metabolization of the parent compounds. In early 2023, two novel SCRAs were reported to the European Union Drugs Agency (EUDA): ADMB-3TMS-PrINACA and Cumyl-3TMS-PrINACA, which are both indazole SCRAs featuring a trimethylsilyl propyl moiety connected to the tertiary indazole nitrogen. Peaks corresponding to metabolites of ADMB-BINACA (also known as ADB-BUTINACA) and Cumyl-4CN-BINACA observed with retention time shifts in a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detecting SCRAs were later identified as metabolites of ADMB- and Cumyl-3TMS-PrINACA. Pooled human liver microsome (pHLMs, 25 µmol/L) and pooled human hepatocyte (PHH, 20 µmol/L) assays were performed to generate metabolites. Additionally, human urine samples were analyzed by reversed phase liquid chromatography-quadrupole-time-of-flight-mass spectrometry (LC-QToF-MS), assisted by GLORYx and BioTransformer 3.0 for in silico metabolite prediction. Gas chromatography-mass spectrometry (GC-MS) was used to identify substances in seized materials. In total, 34 metabolites for ADMB-3TMS-PrINACA and 38 for Cumyl-3TMS-PrINACA were tentatively identified. Major biotransformations included side chain monohydroxylation (specific markers) and TMS-group cleavage, likely initiated by oxidative Si-demethylation followed by further hydroxylation resulting in an N -3-OH-propyl metabolite and further oxidation to the respective N -propionic acid. Most of these biomarkers were detected in the blood, urine, and stomach content of a deceased poly-drug user exposed to ADMB-3TMS-PrINACA. Overall, Cumyl-3TMS-PrINACA was more prevalent than ADMB-3TMS-PrINACA in Germany according to routine urine testing. This work provides the first investigation of the metabolic fate and suggests biomarkers for these new SCRAs.