A Novel Impurity-Profiling Workflow with the Combination of Flash-Chromatography, UHPLC-MS, and Multivariate Data Analysis for Highly Pure Drugs: A Study on the Synthetic Cannabinoid MDMB-CHMICA.

In this paper we present a new, versatile workflow for a synthesis impurity profiling concept, using the combination of flash chromatography (F-LC), liquid chromatography coupled to mass spectrometry (LC-MS), and multivariate data analysis. For three highly pure, structurally different synthetic cannabinoids, we demonstrate that via F-LC more than 99% of the main component (API) can be removed from a sample to enrich present impurities and yield combined fractions of targeted synthesis impurities with reproducible chromatographic signatures via LC-MS. The maximum overall relative standard deviation (RSD) of the complete experimental procedure for isolation and measurement of the impurity profiles (FL-C + LC-MS) was found to be 13.8% on average. The impurity signatures of 40 1 kg samples of MDMB-CHMICA (methyl ( S)-2-(1-(cyclohexylmethyl)-1 H-indole-3-carboxamido)-3,3-dimethylbutanoate) from one large seizure by Luxembourg customs were assessed via UHPLC-MS and compared via principle component analysis (PCA) to possibly discriminate between individual synthesis pathways or production batches and to deduce batch sizes. Three of these 40 samples could be identified as outliers, i. a., as a result of a highly abundant impurity with m/ z 498, isolated via F-LC and identified as methyl 2-(2-(1-(cyclohexylmethyl)-1 H-indole-3-carboxamido)-3,3-dimethylbutanamido)-3,3-dimethylbutanoate, most probably manufactured with a varying synthesis pathway. The remaining 37 samples were subdivided via PCA and hierarchical cluster analysis into five clusters between five and ten samples, representing a maximum possible batch size of 10 kg of pure MDMB-CHMICA. Furthermore, the profiling concept was successfully applied to self-produced and seized "spice-products" to extract impurity profiles of MDMB-CHMICA without any ion suppression or chemical interference.

Successful Replication of GWAS Hits for Multiple Sclerosis in 10,000 Germans Using the Exome Array.

Genome-wide association studies (GWAS) successfully identified various chromosomal regions to be associated with multiple sclerosis (MS). The primary aim of this study was to replicate reported associations from GWAS using an exome array in a large German study. German MS cases (n = 4,476) and German controls (n = 5,714) were genotyped using the Illumina HumanExome v1-Chip. Genotype calling was performed with the Illumina Genome Studio(TM) Genotyping Module, followed by zCall. Single-nucleotide polymorphisms (SNPs) in seven regions outside the human leukocyte antigen (HLA) region showed genome-wide significant associations with MS (P values < 5 × 10(-8) ). These associations have been reported previously. In addition, SNPs in three previously reported regions outside the HLA region yielded P values < 10(-5) . The effect of nine SNPs in the HLA region remained (P < 10(-5) ) after adjustment for other significant SNPs in the HLA region. All of these findings have been reported before or are driven by known risk loci. In summary, findings from previous GWAS for MS could be successfully replicated. We conclude that the regions identified in previous GWAS are also associated in the German population. This reassures the need for detailed investigations of the functional mechanisms underlying the replicated associations.

Characterization of the synthetic cannabinoid MDMB-CHMCZCA.

The synthetic cannabinoid MDMB-CHMCZCA was characterized by various spectroscopic techniques including NMR spectroscopy and tandem mass spectrometry. The synthetic sample was found to be of S-configuration by VCD spectroscopy and comparison of the data with DFT calculations, while ECD spectroscopy was found to be inconclusive in this case. The enantiomeric purity of samples from test purchases and police seizures was assessed by a self-developed chiral HPLC method.

Electromigrative separation techniques in forensic science: combining selectivity, sensitivity, and robustness.

In this review we introduce the advantages and limitations of electromigrative separation techniques in forensic toxicology. We thus present a summary of illustrative studies and our own experience in the field together with established methods from the German Federal Criminal Police Office rather than a complete survey. We focus on the analytical aspects of analytes' physicochemical characteristics (e.g. polarity, stereoisomers) and analytical challenges including matrix tolerance, separation from compounds present in large excess, sample volumes, and orthogonality. For these aspects we want to reveal the specific advantages over more traditional methods. Both detailed studies and profiling and screening studies are taken into account. Care was taken to nearly exclusively document well-validated methods outstanding for the analytical challenge discussed. Special attention was paid to aspects exclusive to electromigrative separation techniques, including the use of the mobility axis, the potential for on-site instrumentation, and the capillary format for immunoassays. The review concludes with an introductory guide to method development for different separation modes, presenting typical buffer systems as starting points for different analyte classes. The objective of this review is to provide an orientation for users in separation science considering using capillary electrophoresis in their laboratory in the future.

Distinct genetic programs guide Drosophila circular and longitudinal visceral myoblast fusion.

BACKGROUND: The visceral musculature of Drosophila larvae comprises circular visceral muscles tightly interwoven with longitudinal visceral muscles. During myogenesis, the circular muscles arise by one-to-one fusion of a circular visceral founder cell (FC) with a visceral fusion-competent myoblast (FCM) from the trunk visceral mesoderm, and longitudinal muscles arise from FCs of the caudal visceral mesoderm. Longitudinal FCs migrate anteriorly under guidance of fibroblast growth factors during embryogenesis; it is proposed that they fuse with FCMs from the trunk visceral mesoderm to give rise to syncytia containing up to six nuclei. RESULTS: Using fluorescence in situ hybridization and immunochemical analyses, we investigated whether these fusion events during migration use the same molecular repertoire and cellular components as fusion-restricted myogenic adhesive structure (FuRMAS), the adhesive signaling center that mediates myoblast fusion in the somatic mesoderm. Longitudinal muscles were formed by the fusion of one FC with Sns-positive FCMs, and defects in FCM specification led to defects in longitudinal muscle formation. At the fusion sites, Duf/Kirre and the adaptor protein Rols7 accumulated in longitudinal FCs, and Blow and F-actin accumulated in FCMs. The accumulation of these four proteins at the fusion sites argues for FuRMAS-like adhesion and signaling centers. Longitudinal fusion was disturbed in rols and blow single, and scar wip double mutants. Mutants of wasp or its interaction partner wip had no defects in longitudinal fusion. CONCLUSIONS: Our results indicated that all embryonic fusion events depend on the same cell-adhesion molecules, but that the need for Rols7 and regulators of F-actin distinctly differs. Rols7 was required for longitudinal visceral and somatic myoblast fusion but not for circular visceral fusion. Importantly, longitudinal fusion depended on Kette and SCAR/Wave but was independent of WASp-dependent Arp2/3 activation. Thus, the complexity of the players involved in muscle formation increases from binucleated circular muscles to longitudinal visceral muscles to somatic muscles.

Nephrocystin-4 is required for pronephric duct-dependent cloaca formation in zebrafish.

NPHP4 mutations cause nephronophthisis, an autosomal recessive cystic kidney disease associated with renal fibrosis and kidney failure. The NPHP4 gene product nephrocystin-4 interacts with other nephrocystins, cytoskeletal and ciliary proteins; however, the molecular and cellular functions of nephrocystin-4 have remained elusive. Here we demonstrate that nephrocystin-4 is required for normal cloaca formation during zebrafish embryogenesis. Time-lapse imaging of the developing zebrafish pronephros revealed that tubular epithelial cells at the distal pronephros actively migrate between the yolk sac extension and the blood island towards the ventral fin fold to join the proctodeum and to form the cloaca. Nphp4-deficient pronephric duct cells failed to connect with their ectodermal counterparts, and instead formed a vesicle at the obstructed end of the pronephric duct. Nephrocystin-4 interacts with nephrocystin-1 and Par6. Depletion of zebrafish NPHP1 (nphp1) increased the incidence of cyst formation and randomization of the normal body axis, but did not augment cloaca malformation in nphp4-deficient zebrafish embryos. However, simultaneous depletion of zebrafish Par6 (pard6) aggravated cloaca formation defects in nphp4-depleted embryos, suggesting that nphp4 orchestrates directed cell migration and cloaca formation through interaction with the Par protein complex.

Risk for myasthenia gravis maps to a (151) Pro→Ala change in TNIP1 and to human leukocyte antigen-B*08.

OBJECTIVE: The objective of this study is to comprehensively define the genetic basis of early onset myasthenia gravis (EOMG). METHODS: We have carried out a 2-stage genome-wide association study on a total of 649 North European EOMG patients. Cases were matched 1:4 with controls of European ancestry. We performed imputation and conditional analyses across the major histocompatibility complex, as well as in the top regions of association outside the human leukocyte antigen (HLA) region. RESULTS: We observed the strongest association in the HLA class I region at rs7750641 (p = 1.2 × 10(-92) ; odds ratio [OR], 6.25). By imputation and conditional analyses, HLA-B*08 proves to be the major associated allele (p = 2.87 × 10(-113) ; OR, 6.41). In addition to the expected association with PTPN22 (rs2476601; OR, 1.71; p = 8.2 × 10(-10) ), an imputed coding variant (rs2233290) at position 151 (Pro→Ala) in the TNFAIP3-interacting protein 1, TNIP1, confers even stronger risk than PTPN22 (OR, 1.91; p = 3.2 × 10(-10) ). INTERPRETATION: The association at TNIP1 in EOMG implies disease mechanisms involving ubiquitin-dependent dysregulation of NF-κB signaling. The localization of the major HLA signal to the HLA-B*08 allele suggests that CD8(+) T cells may play a key role in disease initiation or pathogenesis.

Determination of triacetone triperoxide (TATP) traces using passive samplers in combination with GC-MS and GC-PCI-MS/MS methods.

The use of organic peroxides for the preparation of homemade explosives (HMEs) is common among terrorists due to inexpensive precursor chemicals and simple synthetic procedures. Triacetone triperoxide (TATP) is the most notable peroxide explosive, and has been deployed in several terrorist attacks as explosive filling of improvised explosive devices (IEDs). Forensic identification of TATP in pre-blast and post-blast residues, including on-site analysis, poses significant analytical challenges and induces demand for practicable and sensitive detection techniques. This work presents a concept suitable for laboratory and on-site identification of TATP residues in liquid samples (aqueous TATP synthetic waste) and in gas phase. It is based on TATP enrichment from the aqueous or gas phase using different types of passive samplers (polydimethylsiloxane (PDMS) sampling rods and activated carbon sampling tubes (ACST)) and subsequent identification of the explosive by gas chromatography-mass spectrometry (GC-MS) or GC with positive chemical ionization and tandem MS (GC-PCI-MS/MS) analytical techniques. Additionally, investigation of the stability of TATP in aqueous solutions and of the stability of enriched TATP in passive samplers under different storage conditions, as well as development of TATP re-extraction procedures from passive samplers have been performed in this study. The practical use of passive samplers was demonstrated during and after TATP production processes. Moreover, post-blast sampling of TATP under different conditions of controlled blasting events was investigated using the passive sampling concept.

A new synthetic cathinone: 3,4-EtPV or 3,4-Pr-PipVP? An unsuccessful attempt to circumvent the German legislation on new psychoactive substances.

Synthetic cathinones comprise psychostimulants with desired effects like euphoria, increased vigilance, appetite suppression, and-mainly depending on certain structural features-entactogenic properties. 3,4-EtPV (1-(bicyclo[4.2.0]octa-1,3,5-trien-3-yl)-2-(pyrrolidin-1-yl)pentan-1-one) was first mentioned in an online drug forum in September 2021, where its imminent synthesis was announced. The goal was to produce a legal alternative to the phenylethylamines already banned by the German NpSG. In February and June 2022, two samples labeled with the name and molecular structure of 3,4-EtPV were analyzed. The molecular structure of the obviously mislabeled compound was elucidated and comprehensively characterized within the ADEBAR project. The synthetic cathinone identified differed from the declared 3,4-EtPV by a 3,4-propylene bridge instead of a 3,4-ethylene bridge and a piperidine ring instead of a pyrrolidine ring. The short name 3,4-Pr-PipVP (3,4-propylene-2-(1-piperidinyl)valerophenone) was suggested as a semisystematic name in collaboration with the European Monitoring Centre for Drugs and Drug Addiction. Herein, the results of the analyses are discussed and will enable forensic laboratories to update their databases quickly and identify 3,4-Pr-PipVP confidently. 3,4-Pr-PipVP is already scheduled under the German NpSG. This study highlights that there are ongoing efforts to deliberately circumvent generic definitions given, for example, in the German NpSG and that (unintentional?) mislabeling can be an issue. The end user purchasing substances online can never be sure that the material actually supplied will be the one ordered, and he might receive an illicit drug instead of an uncontrolled one. Furthermore, the purity is always unknown, creating health risks due to unexpected effects and potencies.

Pharmacology, prevalence in Germany, and analytical data of cyclobutylmethyl- and norbornylmethyl-type synthetic cannabinoid receptor agonists.

Synthetic cannabinoid receptor agonists (SCRAs) are distributed on the drug market to produce THC-like effects while evading routine drug testing and legislation. The cyclobutylmethyl (CBM) and norbornylmethyl (NBM) side chain specifically circumvented the German legislation and led to the emergence of exploratory SCRAs in 2019-2021. The NBM SCRAs were detected post-amendment of the new psychoactive substances act in 2020, which scheduled all CBM SCRAs. All six SCRAs are full agonists at the cannabinoid receptor 1 compared with Δ9 -tetrahydrocannabinol and CP-55,940. The CBM SCRAs showed binding affinities of Ki : 29.4-0.65 nm and potencies of EC50 : 483-40.1 nm (CBMICA << CBMINACA < CBMeGaClone). The norbornyl derivatives exhibited high affinities (Ki : 1.87-0.25 nm), with indazole being the most affine. Functional activity data confirmed that the indazole derivative tends to be the most potent of all three NBM SCRAs (EC50 : 169-1.78 nm). The sterically demanding NBM side chain increased the affinity and activity of almost all core structures. Future studies should be conducted on similarly voluminous side chain moieties. The 'life cycle' of all SCRAs on the drug market was less than a year. Notably, Cumyl-CBMICA was the most prevalent while also having the weakest cannabimimetic properties. Quantification of Cumyl-CBMICA during peak consumption in late 2019 and early 2020 revealed an increase in the concentration on the herbal material, which, together with forum entries and blog posts, corroborates the low in vitro cannabimimetic properties. Seizure prevalence data indicate that almost all SCRAs continue to be identified in 2022, potentially due to remaining stocks.

PRKCSH/80K-H, the protein mutated in polycystic liver disease, protects polycystin-2/TRPP2 against HERP-mediated degradation.

Autosomal dominant polycystic liver disease (PCLD) is caused by mutations of either PRKCSH or Sec63, two proteins associated with the endoplasmic reticulum (ER). Both proteins are involved in carbohydrate processing, folding and translocation of newly synthesized glycoproteins. It is postulated that defective quality control of proteins initiates endoplasmic reticulum-associated degradation (ERAD), which disrupts hepatic homeostasis in patients with PRKCSH or Sec63 mutations. However, the precise molecular mechanisms are not known. Here, we show that over-expression or depletion of PRKCSH in zebrafish embryos leads to pronephric cysts, abnormal body curvature and situs inversus. Identical phenotypic changes are induced by depletion or over-expression of TRPP2. Increased PRKCSH levels ameliorate developmental abnormalities caused by over-expressed TRPP2, whereas excess TRPP2 can compensate the loss PRKCSH, indicating that the proteins share a common signaling pathway. PRKCSH binds the C-terminal domain of TRPP2, and both proteins co-localize within the ER. Furthermore, PRKCSH interacts with Herp, and inhibits Herp-mediated ubiquitination of TRPP2. Our findings suggest that PRKCSH functions as a chaperone-like molecule, which prevents ERAD of TRPP2. Dysequilibrium between TRPP2 and PRKCSH may lead to cyst formation in PCLD patients with PRKCSH mutations, and thereby account for the overlapping manifestations observed in PCLD and autosomal dominant polycystic kidney disease.

Nonaqueous capillary electrophoresis-mass spectrometry: a versatile, straightforward tool for the analysis of alkaloids from psychoactive plant extracts.

In this study we show that a nonaqueous capillary electrophoresis mass spectrometry (NACE-MS) method carefully optimized by a design of experiment can be applied to a very large number of alkaloids in different plant extracts. It is possible to characterize the pattern of the psychoactive alkaloids in several plant samples and preparations thereof, each presenting different challenges in their analysis. The method is shown to be able to separate structurally closely related substances, diastereomers and further isobaric compounds, to separate members of different alkaloid classes within one run and to tolerate significant matrix load. A comparison with methods presented in the literature reveals that a near-generic NACE-MS method for the fast profiling of alkaloids in forensically relevant plant samples has been developed.

Forensic analysis of mesembrine alkaloids in Sceletium tortuosum by nonaqueous capillary electrophoresis mass spectrometry.

The consumption of legal and illegal drugs follows an organic trend comparable to the current trend in food consumption. The investigation of such drugs is therefore of interest to characterize the active ingredients of plants and drug preparations. A new method of nonaqueous capillary electrophoresis coupled to mass spectrometry (NACE-MS) as a powerful tool for the separation of complex alkaloid mixtures in difficult matrices is presented in this study for the analysis of samples of Sceletium tortuosum and drug products called Kanna made thereof. The method was found to be suitable for the investigation of the alkaloid composition and relative quantification of the ingredients. It proved of value to separate a large number of isobaric compounds, most probably including diastereomers, double-bond isomers, and further structurally closely related compounds. A comparison of plant samples from different vendors, self-fermented samples, and products ready for consumption was made. The high separation power obtained allowed a better description of the chemotypic differences of plant samples as well as Kanna preparations compared to other methods presented in the literature so far. Thus, the use of the NACE-MS enables a new perspective on the alkaloid profile of Sceletium species.

Identification of fentanyl derivatives at trace levels with nonaqueous capillary electrophoresis-electrospray-tandem mass spectrometry (MS(n), n = 2, 3): analytical method and forensic applications.

The identification of fentanyl derivatives at trace levels employing capillary electrophoresis coupled to electrospray ionization tandem mass spectrometry (CE-ESI-MS(n) , n = 2, 3) is presented. The studied synthetic opioid fentanyl and its derivatives have an exceeding analgesic potency which can be up to 8000 times higher that of morphine. Apart from their therapeutical applications, there is an abuse of them in the drug scene as a heroin substitute. The identification of these opioids at trace levels is of further significant forensic interest with respect to recent seizures of clandestine fentanyl laboratories in Germany. In this work, a nonaqueous capillary electrophoresis (NACE)-ESI-MS(n) procedure was developed for the separation and identification of six fentanyl derivatives including fentanyl, cis- and trans-methylfentanyl, sufentanil, alfentanil, and carfentanil. Their fragmentation pattern in MS(n) experiments were investigated as well as the influence of the sheath-liquid mixture and the influence of the inside diameter of the fused silica capillary on the peak shape and the signal to noise ratio. Method validation included determination of the detection limits (about 1-2 nmol/L) and the repeatability of migration time (at most 0.07% relative standard deviation). The NACE-MS procedure was successfully applied for the analysis of real samples from seizures in illegal fentanyl laboratories.

Implementation of a design of experiments to study the influence of the background electrolyte on separation and detection in non-aqueous capillary electrophoresis-mass spectrometry.

Non-aqueous capillary electrophoresis (NACE) background electrolytes are most often composed of a mixture of methanol and acetonitrile (ACN) with soluble ammonium salts added as electrolyte. In this study on NACE-MS, we used a mixture of glacial acetic acid and ACN giving rise to an acidic background electrolyte (BGE) with a very low dielectric constant. Impressive changes in selectivity and resolution were observed for structurally closely related indole alkaloids including diastereomers upon addition of ammonium formate as electrolyte and upon variation of the solvent ratio. In order to obtain best separation and MS detection conditions and to reveal the influence of the parameters of the BGE on separation and detection and vice versa of the MS parameters on separation, an optimization strategy was employed using a design of experiments in a central composite design with response surface methodology. It was proven that at high electroosmotic flow conditions capillary electrophoretic separations and thus optimization can be realized without interference from the coupling to an MS system. Several significantly interacting parameters were revealed, which are not accessible with classical univariate optimization approaches. With this optimization, alkaloid mixtures from a plant extract of Mitragyna speciosa, containing a large number of diastereomeric compounds were successfully separated.

Nontarget screening of production waste samples from Leuckart amphetamine synthesis using liquid chromatography - high-resolution mass spectrometry as a complementary method to GC-MS impurity profiling.

The established approaches of suspect and nontarget screening (NTS) using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) are usually applied in the field of environmental and bioanalytical analysis. Herein, these approaches were employed on a forensic-toxicological application by analyzing different production waste samples from controlled amphetamine synthesis via Leuckart route to evaluate the suitability of this methodology for identification of route-specific organic substances in such waste samples. For analysis, two complementary LC techniques were used to cover a broad polarity spectrum. After data processing and peak picking using the enviMass software and further manual data restriction, 17 features were tentatively identified as suspects, three of which were subsequently identified with reference substances. All suspects had been previously identified in studies, in which gas chromatography-mass spectrometry (GC-MS) was successfully applied for synthesis marker assessment in waste and amphetamine samples. Remaining features with high signal intensity and assigned sum formula were selected for the attempt of structure elucidation. Seven potential synthesis markers were tentatively identified, which were not yet reported, except the sum formula of one compound, and which were partly also detected in real case waste samples afterward. The innovative application of the NTS approach using LC-HRMS for the analysis of aqueous amphetamine synthesis waste samples showed its suitability as extension to GC-MS analysis as it was possible to successfully identify seven new potential marker compounds, which are specific either for the conversion of the pre-precursors α-phenylacetoacetonitrile and α-phenylacetoacetamide to benzyl methyl ketone or for the subsequent Leuckart synthesis route after their conversion.

The ADEBAR project: European and international provision of analytical data from structure elucidation and analytical characterization of NPS.

Novel substances for which none or limited analytical data are available constitute a challenge for police and customs forensic laboratories. The time-consuming process of structural elucidation and acquisition of analytical data has been centralized in the ADEBAR project in Germany, co-funded since 2017 by the EU's Internal Security Fund. The project aims to comprehensively characterize substances relevant for forensic-toxicological casework within the analytical competence network. The analytical datasets are distributed digitally through European and (inter)national channels. Additionally, pharmacological evaluation allows for estimating in vivo potency and potential harm required as scientific evidence for legislative amendments. The ADEBAR project contributes to the availability of analytical data on new substances relevant to the daily work of police and customs laboratories. Since the inception of the ADEBAR project, 549 samples have been registered, and 302 substance reports notified to the EMCDDA, including numerous spectrometric and spectroscopic data. In addition, 3,619 mass spectra have been accumulated in ADEBAR mass spectra databases. A central institution for the structure elucidation and acquisition of valid, high-quality analytical data for police and customs forensic laboratories and forensic medicine institutes is important in the future because there does not seem to be an end to the dynamic of novel NPS appearing on the drug market.

Structure elucidation of the novel synthetic cannabinoid Cumyl-Tosyl-Indazole-3-Carboxamide (Cumyl-TsINACA) found in illicit products in Germany.

New chemical moieties continue to appear in synthetic cannabimimetics (SC), the largest group of new psychoactive substances in the EU. We describe the first comprehensive characterisation of the novel SC Cumyl-Tosyl-Indazole-3-Carboxamide (Cumyl-TsINACA) (N-[2-phenylpropan-2-yl]-1-tosyl-1H-indazole-3-carboxamide) from seized case samples. Structure elucidation was performed within the EU-project ADEBAR plus to facilitate confident identification by other researchers and practitioners worldwide. Characteristic MS fragmentations include the cleavage of the sulfonamide bond (S-N), the aryl sulfone bond (C-S) and the elimination rearrangement of SO2 in the side chain. Cumyl-TsINACA is a full receptor agonist at hCB1 (Emax  = 228%) with very weak binding affinity (Ki  = 292 nm) and low functional activity (EC50  = 31 µm). Thermal degradation of Cumyl-TsINACA was observed under GC conditions. The degree to which the tosyl side chain is cleaved due to pyrolysis primarily depends on solvent, the use of glass wool in the liner and injector temperature. The determination of the constitution by NMR spectroscopy was ambiguous due to the high number of neighbouring, non-proton-bearing atoms. Therefore, other possible structures compatible with the NMR correlations were generated using the WebCocon software. The unambiguous structural evidence was finally obtained by spectra comparison after the synthesis of Cumyl-TsINACA. The low thermal stability, as well as the low affinity and potency, renders this compound unfavourable for the use as a psychoactive substance. Thus, we do not expect widespread adoption of this SC.

Synthetic cannabinoid receptor agonists and their human metabolites in sewage water: Stability assessment and identification of transformation products.

Since their first appearance in 2008, synthetic cannabinoid receptor agonists (SCRAs) remain the most popular new psychoactive substances (NPS) in the EU. Following consumption, these drugs and their metabolites are urinary excreted and enter the sewage system enabling the application of wastewater-based epidemiology (WBE). Knowing the fate of target analytes in sewage water is essential for successful application of WBE. This study investigates the stability of several chemically diverse SCRAs and selected human metabolites under sewage conditions utilizing a combination of liquid chromatography-tandem mass spectrometry and high-resolution mass spectrometry (HRMS). Target analytes included SCRAs with indole (5F-PB-22, PB-22 pentanoic acid), indazole (AMB-FUBINACA, 5F-ADB, 5F-ADB dimethylbutanoic acid), carbazole (MDMB-CHMCZCA, EG-018), and γ-carboline (Cumyl-PeGaClone) chemical core structures representing most of the basic core structures that have occurred up to now. Stability tests were performed using wastewater effluent containing 5% activated sludge as inoculum to monitor degradation processes and formation of transformation products (TPs). The majority of investigated SCRAs, excluding the selected human metabolites, was recalcitrant to microbial degradation in sewage systems over a period of 29 days. Their stability was rather controlled by physico-chemical processes like sorption and hydrolysis. Considering a typical hydraulic in-sewer retention time of 24 h, the concentration of AMB-FUBINACA decreased by 90% thus representing the most unstable SCRA investigated in this study. Among the 10 newly identified TPs, three could be considered as relevant markers and should be included into future WBE studies to gain further insight into use and prevalence of SCRAs on the drug market.

New synthetic cannabinoids carrying a cyclobutyl methyl side chain: Human Phase I metabolism and data on human cannabinoid receptor 1 binding and activation of Cumyl-CBMICA and Cumyl-CBMINACA.

Synthetic cannabinoids (SCs) represent a large group of new psychoactive substances (NPS), sustaining a high prevalence on the drug market since their first detection in 2008. Cumyl-CBMICA and Cumyl-CBMINACA, the first representatives of a new subclass of SCs characterized by a cyclobutyl methyl (CBM) moiety, were identified in July 2019 and February 2020. This work aimed at evaluating basic pharmacological characteristics and human Phase I metabolism of these compounds. Human Phase I metabolites were tentatively identified by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QToF-MS) of urine samples and confirmed by a pooled human liver microsome (pHLM) assay. The basic pharmacological evaluation was performed by applying a competitive ligand binding assay and a functional activation assay (GTPγS) using cell membranes carrying the human cannabinoid receptor 1 (hCB1 ). Investigation of the human Phase I metabolism resulted in the identification of specific urinary markers built by monohydroxylation or dihydroxylation. Although Cumyl-CBMICA was primarily hydroxylated at the indole ring, hydroxylation of Cumyl-CBMINACA mainly occurred at the CBM moiety. Both substances acted as agonists at the hCB1 receptor, although substantial differences could be observed. Cumyl-CBMINACA showed higher binding affinity (Ki = 1.32 vs. 29.3 nM), potency (EC50 = 55.4 vs. 497 nM), and efficacy (Emax = 207% vs. 168%) than its indole counterpart Cumyl-CBMICA. This study confirms that substitution of an indole by an indazole core tends to increase in vitro potency, which is potentially reflected by higher in vivo potency. The emergence and disappearance of SCs distributed via online shops carrying a CBM moiety once more demonstrate the "cat-and-mouse" game between manufacturers and legislation.