Acute psychotropic, autonomic, and endocrine effects of 5,6-methylenedioxy-2-aminoindane (MDAI) compared with 3,4-methylenedioxymethamphetamine (MDMA) in human volunteers: A self-administration study.

The acute psychoactive, autonomic, and endocrine effects of the new psychoactive substance (NPS) 5,6-methylenedioxy-2-aminoindane (MDAI; 3.0 mg/kg, range 180-228 mg) were investigated in six healthy volunteers (four males, two females) in a non-blinded fashion without placebo. Subjective, cardiovascular, and endocrine responses were compared with two different doses of 3,4-methylenedioxymethamphetamine (MDMA) (75 mg and 125 mg) described in previously published placebo-controlled studies, which used identical outcome measures including Visual Analogue Scales (VAS), the Adjective Mood Rating Scale (AMRS), and the 5 Dimensions of Altered States of Consciousness (5D-ASC) scale. MDAI was well tolerated and produced subjective effects comparable with those of 125 mg MDMA. MDAI increased blood pressure similar to 125 mg MDMA but did not increase heart rate or body temperature. MDAI increased cortisol and prolactin levels and could be detected in serum about 20 min post ingestion and remained detectable at least for 4 days. In urine, MDAI was detectable over a period of at least 6 days. Further clinical investigations are warranted to assess whether MDAI could serve as drug with medicinal properties.

Allosteric activation of vinculin by talin.

The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single-molecule magnetic tweezers experiments, Molecular Dynamics simulations, actin-bundling assays, and adhesion assembly experiments in live cells, we here describe a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding, which reinforces the binding to talin at a rate of 0.03 s-1. This allosteric transition can compete with force-induced dissociation of vinculin from talin only at forces up to 10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The 'allosteric vinculin mutant' is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere.

Intrinsically disordered region of talin's FERM domain functions as an initial PIP2 recognition site.

Focal adhesions (FAs) mediate the interaction of the cytoskeleton with the extracellular matrix in a highly dynamic fashion. Talin is a central regulator, adaptor protein, and mechano-sensor of FA complexes. For recruitment and firm attachment at FAs, talin's N-terminal FERM domain binds to phosphatidylinositol 4,5-bisphosphate (PIP2)-enriched membranes. A newly published autoinhibitory structure of talin-1, where the known PIP2 interaction sites are covered up, lead us to hypothesize that a hitherto less examined loop insertion of the FERM domain acts as an additional and initial site of contact. We evaluated direct interactions of talin-1 with a PIP2 membrane by means of atomistic molecular dynamics simulations. We show that this unstructured, 33-residue-long loop strongly interacts with PIP2 and can facilitate further membrane contacts, including the canonical PIP2 interactions, by serving as a flexible membrane anchor. Under force as present at FAs, the extensible FERM loop ensures talin maintains membrane contacts when pulled away from the membrane by up to 7 nm. We identify key basic residues of the anchor mediating the highly dynamic talin-membrane interaction. Our results put forward an intrinsically disordered loop as a key and highly adaptable PIP2 recognition site of talin and potentially other PIP2-binding mechano-proteins.

Longitudinal strand ordering leads to shear thinning in Nafion.

Proton-exchange membrane fuel cells (PEMFC) offer a promising energy generation alternative for a wide range of technologies thanks to their ecological friendliness and unparalleled efficiency. At the heart of these electrochemical cells lies the membrane electrode assembly with its most important energy conversion components, the Proton Exchange Membrane. This component is created through the use of printing techniques and Nafion inks. The physicochemical properties of the ink, such as its viscosity under shear, are critical for the finished product. In this work we present non-equilibrium Molecular Dynamics simulations using a MARTINI based coarse-grained model for Nafion to understand the mechanism governing the shear viscosity of Nafion solutions. By simulating a Couette flow and calculating density maps of the Nafion chains in these simulations we shed light on the process that leads to the experimentally observed shear thinning effects of Nafion solutions under flow. We observe rod-shaped Nafion microstructures, 3 nm in size on average, when shear flow is absent or low. Higher shear rates instead break these structures and align Nafion strands along the direction of the flow, resulting in lower shear viscosities. Our work paves the way for a deeper understanding of the dynamic and mechanical properties of Nafion including studies of more complex CL and PEM inks.

Extraordinary long detection window of a synthetic cannabinoid metabolite in human urine - Potential impact on therapeutic decisions.

Synthetic cannabinoids (SCs) have become established drugs of abuse. They play an increasing role in drug therapy, where abstinence control testing is required. Differentiation between recent drug uptake and uptake in the distant past is important for drug therapy. This study aimed to evaluate the detection window of a metabolite commonly used as a consumption marker for AB-FUBINACA and AMB-FUBINACA (synonym: FUB-AMB) in urine analysis. The acidic hydrolysis metabolite was quantified in urine samples of a drug user by applying a validated analytical method. The concentration profile of the metabolite was correlated with usage data of the subject. Pharmacokinetic properties of AB-FUBINACA were collected by analysis of serum and urine samples from a controlled administration study (single oral ingestion of AB-FUBINACA). Thirteen urine samples were taken without advance notice over 2 years. The metabolite was detected in the first urine sample at 0.77 ng/mg creatinine and subsequently in concentrations ranging from 0.06 to 0.29 ng/mg creatinine. Usage data showed credible abstinence from SCs during this period. The pharmacokinetic properties observed within the controlled self-administration study supported the hypothesis of distribution into deeper compartments and long-lasting elimination (serum concentration-time curve showing biphasic kinetics). An elimination phase of over 1 year after the last drug uptake seems plausible in cases of extensive consumption. To avoid misinterpretation of positive findings, we recommend testing patients with known SC use at the beginning of the abstinence program and to re-test continuously at short time intervals. These data enable the correct interpretation of analytical findings.

Advances in molecular simulations of protein mechanical properties and function.

Single-molecule force spectroscopy and classical molecular dynamics are natural allies. Recent advances in both experiments and simulations have increasingly facilitated a direct comparison of SMFS and MD data, most importantly by closing the gap between time scales, which has been traditionally at least 5 orders of magnitudes wide. In this review, we will explore these advances chiefly on the computational side. We focus on protein dynamics under force and highlight recent studies that showcase how lower loading rates and more statistics help to better interpret previous experiments and to also motivate new ones. At the same time, steadily increasing system sizes are used to mimic more closely the mechanical environment in the biological context. We showcase some of these advances on atomistic and coarse-grained scale, from asymmetric membrane tension to larger (multidomain/multimeric) protein assemblies under force.

Detection and phase I metabolism of the 7-azaindole-derived synthetic cannabinoid 5F-AB-P7AICA including a preliminary pharmacokinetic evaluation.

In June 2018, a 'research chemica'l labeled 'AB-FUB7AICA' was purchased online and analytically identified as 5F-AB-P7AICA, the 7-azaindole analog of 5F-AB-PINACA. Here we present data on structural characterization, suitable urinary consumption markers, and preliminary pharmacokinetic data. Structure characterization was performed by nuclear magnetic resonance spectroscopy, gas chromatography-mass spectrometry, infrared and Raman spectroscopy. Phase I metabolites were generated by applying a pooled human liver microsome assay (pHLM) to confirm the analysis results of authentic urine samples collected after oral self-administration of 2.5 mg 5F-AB-P7AICA. Analyses of pHLM and urine samples were performed by liquid chromatography-time-of-flight mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS). An LC-MS/MS method for the quantification of 5F-AB-P7AICA in serum was validated. Ten phase I metabolites were detected in human urine samples and confirmed in vitro. The main metabolites were formed by hydroxylation, amide hydrolysis, and hydrolytic defluorination, though - in contrast with most other synthetic cannabinoids - the parent compound showed the highest signals in most urine samples. The compound detection window was more than 45 hours in serum. The concentration-time profile was best explained by a two-phase pharmacokinetic model. 5F-AB-P7AICA was detected in urine samples until 65 hours post ingestion. Monitoring of metabolite M07, hydroxylated at the alkyl chain, next to parent 5F-AB-P7AICA, is recommended to confirm the uptake of 5F-AB-P7AICA in urinalysis. It seems plausible that the shift of the nitrogen atom from position 2 to 7 (e.g. 5F-AB-PINACA to 5F-AB-P7AICA) leads to a lower metabolic reactivity, which might be of general interest in medicinal chemistry.

Human phase I metabolism of the novel synthetic cannabinoid 5F-CUMYL-PEGACLONE.

PURPOSE: 5F-CUMYL-PEGACLONE is a recently emerged γ-carbolinone derived synthetic cannabinoid. The present study aimed to identify phase I metabolites to reliably prove consumption of the substance by urine analysis and to differentiate from the uptake of the non-fluorinated analog CUMYL-PEGACLONE. METHODS: For metabolite characterization, phase I metabolites were analyzed by liquid chromatography-high resolution mass spectrometry after incubation with pooled human liver microsomes. Reliability of the biomarkers was evaluated by analysis of human urine samples (n = 20) by liquid chromatography-triple quadrupole tandem mass spectrometry. Sample preparation included ß-glucuronidase treatment followed by liquid-liquid extraction. RESULTS: In total, 15 metabolites were detected in vivo and characterized. Metabolic reactions were primarily observed at the γ-carbolinone core and the 5-fluoropentyl chain, and included N-dealkylation, hydroxylation, hydrolytic defluorination, formation of a dihydrodiol, oxidation to the pentanoic acid metabolite and formation of the propionic acid metabolite. Six of these metabolites were identical with phase I metabolites of CUMYL-PEGACLONE, which must be considered for interpretation of analytical findings in urine samples. CONCLUSIONS: 5F-CUMYL-PEGACLONE was subject to extensive metabolism in humans. The propionic acid metabolite was the most abundant metabolite in all urine samples and should be targeted when maximum sensitivity is needed (e.g., drug abstinence control). However, this metabolite also occurs in the biotransformation of the non-fluorinated analog and is, therefore, not a compound-specific marker. For differentiation, a metabolite hydroxylated at the γ-carbolinone core showed to be the most reliable marker and should be used as an additional target analyte.

5F-Cumyl-PINACA in 'e-liquids' for electronic cigarettes: comprehensive characterization of a new type of synthetic cannabinoid in a trendy product including investigations on the in vitro and in vivo phase I metabolism of 5F-Cumyl-PINACA and its non-fluorinated analog Cumyl-PINACA.

PURPOSE: In recent years e-liquids used in electronic cigarettes have become an attractive alternative to smoking tobacco. A new trend is the use of e-liquids containing synthetic cannabinoids (SCs) instead of smoking cannabis or herbal mixtures laced with SCs. In the frame of a systematic monitoring of the online market of 'legal high' products, e-liquids from online retailers who also sell herbal blends were bought. METHODS: The products were analyzed by gas chromatography-mass spectrometry. In some of the e-liquids an unknown compound was detected which was identified as the SC 5F-Cumyl-PINACA (1-(5-fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indazole-3-carboxamide) by nuclear magnetic resonance analysis. To investigate the phase I metabolism of this new class of compounds, 5F-Cumyl-PINACA and its non-fluorinated analog Cumyl-PINACA were incubated with pooled human liver microsomes (pHLM). Cumyl-PINACA was additionally ingested orally (0.6 mg) by a volunteer in a controlled self-experiment. To assess the relative potency of Cumyl-PINACA a set of SCs were characterized using a cAMP assay. RESULTS: Metabolism of 5F-Cumyl-PINACA and Cumyl-PINACA showed similarities with AM-2201 and JWH-018. The main metabolites were formed by hydroxylation at the N-pentyl side chain. The main metabolites detected in the volunteer's urine sample were the same as in the pHLM assay. All SCs tested with the cAMP assay were full agonists at the CB1 receptor. Cumyl-PINACA was the most potent SC among the tested compounds and showed an EC50 value of 0.06 nM. CONCLUSIONS: The increasing popularity of e-liquids particularly among young people, and the extreme potency of the added SCs, pose a serious threat to public health. To our knowledge, this is the first report describing the tentative identification of human in vivo metabolites of Cumyl-PINACA and 5F-Cumyl-PINACA.

Phase I metabolic profiling of the synthetic cannabinoids THJ-018 and THJ-2201 in human urine in comparison to human liver microsome and cytochrome P450 isoenzyme incubation.

Despite the increasing relevance of synthetic cannabinoids as one of the most important classes within "New Psychoactive Substances", there is still a lack of knowledge concerning their metabolism in humans. Due to the extensive metabolism of synthetic cannabinoids, metabolites are necessarily the best target analytes in urine, posing additional challenges to forensic analysis. The aims of this study were to identify appropriate urinary targets indicating intake of THJ-018 or THJ-2201 as well as to elucidate the most important cytochrome P450 isoenzymes within the metabolism of THJ-018 and THJ-2201 in vitro. For this purpose, the in vitro metabolism of THJ-018 and THJ-2201 was initially established using pooled human liver microsomes. The results obtained were compared to previously published in vitro results as well as to the results of the metabolic profiles from selected recombinant cytochrome P450 isoenzymes and from 23 urine samples from forensic cases. LC-HRMS was used to conduct product ion scans and to examine the metabolite spectra. For THJ-018, 17 different metabolite groups containing 33 different metabolites and isomers were detected after microsomal incubation, with the major metabolic pathways being monohydroxylation at the pentyl chain and of the naphthyl moiety as well as dihydroxylation of both residues. For THJ-2201, 19 different metabolite groups and 46 different metabolites and isomers were observed. The major metabolic pathways were monohydroxylation at the naphthyl moiety and oxidative defluorination. Significant contribution to the in vitro metabolism of THJ-018 and THJ-2201 originated from CYP2B6, CYP2C19, CYP3A4, and CYP3A5. As several cytochrome P450 isoenzymes are involved in the metabolism of these synthetic cannabinoids, a co-consumption with other drugs is unlikely to have an impact on their metabolism.

Synthetic cannabinoids in hair - Pragmatic approach for method updates, compound prevalences and concentration ranges in authentic hair samples.

Since the first detection of synthetic cannabinoids (SCs) in so-called 'legal high' products (e.g. 'Spice') sold as legal alternatives to marihuana, the rapid development of this class of designer drugs poses a great challenge for analytical laboratories. The aim of this study was the comprehensive validation of an up-to-date LC-MS/MS method for detection of SCs in human hair for the purpose of drug abstinence testing and evaluation of a pragmatic re-validation approach for frequent method adaption. The validation demonstrated low quantification limits (0.5-5.0 pg mg-1) and acceptable selectivity, linearity, accuracy, and precision for 72 SCs. High matrix effects have been taken into consideration as a major limitation of the method. The partial re-validation approach proved to be an appropriate compromise between reduced validation effort and sufficient control of the method performance enabling analysts to keep pace with the dynamics of the drug market. The analysis of 294 authentic samples resulted in 163 positive samples and showed a broad concentration range (<1.0-5,700 pg mg-1) for 52 SCs in hair with up to 17 different compounds detected in a single hair sample. Periods of detection between one and 58 months were observed for single compounds in hair. Regarding the interpretation of analytical findings semi-quantitative concentrations were considered sufficient for a rough classification of the intensity of drug exposure in (i) passive exposure or exposure in the distant past (lower pg mg-1 range), (ii) more intense exposure (elevated concentration range, >20 pg mg-1 (upper 25th-percentile)), and (iii) heavy/recent exposure (>150 pg mg-1).

Stability of Biological Membranes upon Mechanical Indentation.

Mechanical perturbations are ubiquitous in living cells, and many biological functions are dependent on the mechanical response of lipid membranes. Recent force-spectroscopy studies have captured the stepwise fracture of stacks of bilayers, avoiding substrate effects. However, the effect of stacking bilayers, as well as the exact molecular mechanism of the fracture process, is unknown. Here, we use atomistic and coarse-grained force-clamp molecular dynamics simulation to assess the effects of mechanical indentation on stacked and single bilayers. Our simulations show that the rupture process obeys the laws of force-activated barrier crossing, and stacking multiple membranes stabilizes them. The rupture times follow a log-normal distribution which allows the interpretation of membrane rupture as a pore-growth process. Indenter hydrophobicity determines the type of pore formation, the preferred dwelling region, and the resistance of the bilayer against rupture. Our results provide a better understanding of the nanomechanics underlying the plastic rupture of lipid membranes.

Phase I metabolism of the carbazole-derived synthetic cannabinoids EG-018, EG-2201, and MDMB-CHMCZCA and detection in human urine samples.

Synthetic cannabinoids (SCs) are a structurally diverse class of new psychoactive substances. Most SCs used for recreational purposes are based on indole or indazole core structures. EG-018 (naphthalen-1-yl(9-pentyl-9H-carbazol-3-yl)methanone), EG-2201 ((9-(5-fluoropentyl)-9H-carbazol-3-yl)(naphthalen-1-yl)methanone), and MDMB-CHMCZCA (methyl 2-(9-(cyclohexylmethyl)-9H-carbazole-3-carboxamido)-3,3-dimethylbutanoate) are 3 representatives of a structural subclass of SCs, characterized by a carbazole core system. In vitro and in vivo phase I metabolism studies were conducted to identify the most suitable metabolites for the detection of these substances in urine screening. Detection and characterization of metabolites were performed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry (LC-ESI-QToF-MS). Eleven in vivo metabolites were detected in urine samples positive for metabolites of EG-018 (n = 8). A hydroxypentyl metabolite, most probably the 4-hydroxypentyl isomer, and an N-dealkylated metabolite mono-hydroxylated at the carbazole core system were most abundant. In vitro studies of EG-018 and EG-2201 indicated that oxidative defluorination of the 5-fluoropentyl side chain of EG-2201 as well as dealkylation led to common metabolites with EG-018. This has to be taken into account for interpretation of analytical findings. A differentiation between EG-018 and EG-2201 (n = 1) uptake is possible by the detection of compound-specific in vivo phase I metabolites evaluated in this study. Out of 30 metabolites detected in urine samples of MDMB-CHMCZCA users (n = 20), a metabolite mono-hydroxylated at the cyclohexyl methyl tail is considered the most suitable compound-specific consumption marker while a biotransformation product of mono-hydroxylation in combination with hydrolysis of the terminal methyl ester function provides best sensitivity due to its high abundance.

Metabolism of Nine Synthetic Cannabinoid Receptor Agonists Encountered in Clinical Casework: Major in vivo Phase I Metabolites of AM-694, AM-2201, JWH-007, JWH-019, JWH-203, JWH-307, MAM-2201, UR-144 and XLR-11 in Human Urine Using LC-MS/MS.

BACKGROUND: `Herbal mixtures` containing synthetic cannabinoid receptor agonists (SCRAs) are promoted as legal alternative to marihuana and are easily available via the Internet. Keeping analytical methods for the detection of these SCRAs up-to-date is a continuous challenge for clinicians and toxicologists due to the high diversity of the chemical structures and the frequent emergence of new compounds. Since many SCRAs are extensively metabolized, analytical methods used for urine testing require previous identification of the major metabolites of each compound. OBJECTIVE: The aim of this study was to identify the in vivo major metabolites of nine SCRAs (AM- 694, AM-2201, JWH-007, JWH-019, JWH-203, JWH-307, MAM-2201, UR-144, XLR-11) for unambiguous detection of a drug uptake by analysis of urine samples. METHOD: Positive urine samples from patients of hospitals, detoxification and therapy centers as well as forensic-psychiatric clinics were analyzed by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole time-of-flight mass spectrometry (LCqToF- MS) for investigation of the major in vivo metabolites. RESULTS: For all investigated SCRAs, monohydroxylation, dihydroxylation and/or formation of the Nhexanoic/ pentanoic acid metabolites were among the most abundant metabolites detected in human urine samples. Substitution of the fluorine atom was observed to be an important metabolic reaction for compounds carrying an N-(5-fluoropentyl) side chain. N-Dealkylated metabolites were not detected in vivo. CONCLUSION: The investigated metabolites facilitate the reliable detection of drug uptake by analysis of urine samples. For distinction between uptake of the fluorinated and the non-fluorinated analogs, the N-(4-hydroxypentyl) metabolite of the non-fluorinated analog was identified as a useful analytical target and consumption marker.

Phase I metabolism of the recently emerged synthetic cannabinoid CUMYL-PEGACLONE and detection in human urine samples.

Indole-, indazole-, or azaindole-based synthetic cannabinoids (SCs), bearing a cumyl substituent are a widespread, recreationally used subgroup of new psychoactive substances (NPS). The latest cumyl-derivative, CUMYL-PEGACLONE, emerged in December 2016 on the German drug market. The substance features a novel γ-carboline core structure, which is most likely synthesized to bypass generic legislative approaches to control SCs by prohibiting distinct core structures. Using liquid chromatography-tandem mass spectrometry and liquid chromatography-high resolution mass spectrometry techniques, the main in vivo phase I metabolites of this new substance were detected. A pooled human liver microsome assay was applied to generate in vitro reference spectra of CUMYL-PEGACLONE phase I metabolites. Additionally, 30 urine samples were investigated leading to 22 in vivo metabolites. A metabolite mono-hydroxylated at the γ-carbolinone core system and a metabolite with an additional carbonyl group at the pentyl side chain were evaluated as highly specific and sensitive markers to proof CUMYL-PEGACLONE uptake. Moreover, 3 immunochemical assays commonly used for SC screening in urine were tested for their capability of detecting the new drug but failed due to insufficient cross-reactivity.

Acute side effects after consumption of the new synthetic cannabinoids AB-CHMINACA and MDMB-CHMICA.

INTRODUCTION: In 2014, the "European Monitoring Centre for Drugs and Drug Addiction" (EMCDDA) reported on 30 novel synthetic cannabinoids (SCs). Among these were indole- and indazole-based valine derivatives with a cyclohexylmethyl side chain (e.g., AB-CHMINACA and MDMB-CHMICA), which represent a new class of SCs. METHODS: A prospective observational study of patients treated in emergency departments (EDs) after the intake of SCs was conducted. Clinical and laboratory data were combined and reported to a poison control centre. Serum and/or urine samples of ED patients were analyzed using LC-MS/MS. RESULTS: Forty four patients (39 male, five female, 12-48 years) were included. AB-CHMINACA (MDMB-CHMICA) was identified in 20 (19) serum samples, and in 21 (25) urine samples, respectively. In 19 of the cases, more than one SC was present. Other psychoactive substances (mainly amfetamines) were identified in seven cases, but in five out of these in urine samples only. Based on the Poison Severity Score, severity of poisoning was minor (4), moderate (31) or severe (9). Most frequently reported neuropsychiatric symptoms were CNS-depression (n = 21, 61%), disorientation (n = 20, 45%), generalized seizures (n = 12, 27%), combativeness (n = 8, 18%) and extreme agitation (n = 7, 16%). Duration of symptoms lasting 24 hours or longer occurred in 15 cases (34%). DISCUSSION: The prevalence of certain neuropsychiatric symptoms was higher in our study than in former reports after the intake of SCs of the aminoalkylindole-type (first generation) SCs. In addition, severe poisoning and duration of symptoms were also higher. CONCLUSIONS: In this study, the valine derivative AB-CHMINACA and the tert-leucine derivative MDMB-CHMICA ("third generation of SCs") seem to be associated with more severe clinical toxicity than was previously reported in patients exposed to earlier generation SCs such as JWH-018. However, this observation needs to be confirmed with a larger cohort of patients with analytically confirmed abuse of third generation SCs. The rapid turnover of SCs on the drug market together with the occurrence of SCs such as AB-CHMINACA and MDMB-CHMICA is alarming, especially because of the unexpectedly high frequency of neuropsychiatric symptoms.

Detection of the recently emerged synthetic cannabinoid 5F-MDMB-PICA in 'legal high' products and human urine samples.

Indole or indazole-based synthetic cannabinoids (SCs) bearing substituents derived from valine or tert-leucine are frequently abused new psychoactive substances (NPS). The emergence of 5F-MDMB-PICA (methyl N-{[1-(5-fluoropentyl)-1H-indol-3-yl]carbonyl}-3-methylvalinate) on the German drug market is a further example of a substance synthesized in the context of scientific research being misused by clandestine laboratories by adding it to 'legal high' products. In this work, we present the detection of 5F-MDMB-PICA in several legal high products by gas chromatography-mass spectrometry (GC-MS) analysis. To detect characteristic metabolites suitable for a proof of 5F-MDMB-PICA consumption by urine analysis, pooled human liver microsome (pHLM) assays were performed and evaluated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) techniques to generate reference spectra of the in vitro phase I metabolites. The in vivo phase I metabolism was investigated by the analysis of more than 20 authentic human urine specimens and compared to the data received from the pHLM assay. Biotransformation of the 5-fluoropentyl side chain and hydrolysis of the terminal methyl ester bond are main phase I biotransformation steps. Two of the identified main metabolites formed by methyl ester hydrolysis or mono-hydroxylation at the indole ring system were evaluated as suitable urinary biomarkers and discussed regarding the interpretation of analytical findings. Exemplary analysis of one urine sample for 5F-MDMB-PICA phase II metabolites showed that two of the main phase I metabolites are subject to extensive glucuronidation prior to renal excretion. Therefore, conjugate cleavage is reasonable for enhancing sensitivity. Commercially available immunochemical pre-tests for urine proved to be unsuitable for the detection of 5F-MDMB-PICA consumption. Copyright © 2017 John Wiley & Sons, Ltd.

Activity-Based Detection of Consumption of Synthetic Cannabinoids in Authentic Urine Samples Using a Stable Cannabinoid Reporter System.

Synthetic cannabinoids (SCs) continue to be the largest group of new psychoactive substances (NPS) monitored by the European Monitoring Center of Drugs and Drugs of Abuse (EMCDDA). The identification and subsequent prohibition of single SCs has driven clandestine chemists to produce analogues of increasing structural diversity, intended to evade legislation. That structural diversity, combined with the mostly unknown metabolic profiles of these new SCs, poses a big challenge for the conventional targeted analytical assays, as it is difficult to screen for "unknown" compounds. Therefore, an alternative screening method, not directly based on the structure but on the activity of the SC, may offer a solution for this problem. We generated stable CB1 and CB2 receptor activation assays based on functional complementation of a split NanoLuc luciferase and used these to test an expanded set of recent SCs (UR-144, XLR-11, and their thermal degradation products; AB-CHMINACA and ADB-CHMINACA) and their major phase I metabolites. By doing so, we demonstrate that several major metabolites of these SCs retain their activity at the cannabinoid receptors. These active metabolites may prolong the parent compound's psychotropic and physiological effects and may contribute to the toxicity profile. Utility of the generated stable cell systems as a first-line screening tool for SCs in urine was also demonstrated using a relatively large set of authentic urine samples. Our data indicate that the stable CB reporter assays detect CB receptor activation by extracts of urine in which SCs (or their metabolites) are present at low- or subnanomolar (ng/mL) level. Hence, the developed assays do not only allow activity profiling of SCs and their metabolites, it may also serve as a screening tool, complementing targeted and untargeted analytical assays and preceding analytical (mass spectrometry based) confirmation.

Immunoassay screening in urine for synthetic cannabinoids - an evaluation of the diagnostic efficiency.

BACKGROUND: The abuse of synthetic cannabinoids (SCs) as presumed legal alternative to cannabis poses a great risk to public health. For economic reasons many laboratories use immunoassays (IAs) to screen for these substances in urine. However, the structural diversity and high potency of these designer drugs places high demands on IAs regarding cross-reactivity of the antibodies used and detection limits. METHODS: Two retrospective studies were carried out in order to evaluate the capability of two homogenous enzyme IAs for the detection of currently prevalent SCs in authentic urine samples. Urine samples were analyzed utilizing a 'JWH-018' kit and a 'UR-144' kit. The IA results were confirmed by an up-to-date liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) screening method covering metabolites of 45 SCs. RESULTS: The first study (n=549) showed an 8% prevalence of SCs use (LC-MS/MS analysis) among inpatients of forensic-psychiatric clinics, whereas all samples were tested negative by the IAs. In a second study (n=200) the combined application of both IAs led to a sensitivity of 2% and a diagnostic accuracy of 51% when applying the recommended IA cut-offs. Overall, 10 different currently prevalent SCs were detected in this population. The results can be explained by an insufficient cross-reactivity of the antibodies towards current SCs in combination with relatively high detection limits of the IAs. CONCLUSIONS: In light of the presented study data it is strongly recommended not to rely on the evaluated IA tests for SCs in clinical or forensic settings. For IA kits of other providers similar results can be expected.

In vitro metabolism of the synthetic cannabinoid 3,5-AB-CHMFUPPYCA and its 5,3-regioisomer and investigation of their thermal stability.

Recently, the pyrazole-containing synthetic cannabinoid N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(cyclohexylmethyl)-3-(4-fluorophenyl)-1H-pyrazole-5-carboxamide (3,5-AB-CHMFUPPYCA) has been identified as a 'research chemical' both in powdered form and as an adulterant present in herbal preparations. Urine is the most common matrix used for abstinence control and the extensive metabolism of synthetic cannabinoids requires implementation of targeted analysis. The present study describes the investigation of the in vitro phase I metabolism of 3,5-AB-CHMFUPPYCA and its regioisomer 5,3-AB-CHMFUPPYCA using pooled human liver microsomes. Metabolic patterns of both AB-CHMFUPPYCA isomers were qualitatively similar and dominated by oxidation of the cyclohexylmethyl side chain. Biotransformation to monohydroxylated metabolites of high abundance confirmed that these species might serve as suitable targets for urine analysis. Furthermore, since synthetic cannabinoids are commonly administered by smoking and because some metabolites can also be formed as thermolytic artefacts, the stability of both isomers was assessed under smoking conditions. Under these conditions, pyrolytic cleavage of the amide bond occurred that led to approximately 3 % conversion to heat-induced degradation products that were also detected during metabolism. These artefactual 'metabolites' could potentially bias in vivo metabolic profiles after smoking and might have to be considered for interpretation of metabolite findings during hair analysis. This might be relevant to the analysis of hair samples where detection of metabolites is generally accepted as a strong indication of drug use rather than a potential external contamination. Copyright © 2016 John Wiley & Sons, Ltd.