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Presents a letter to the editor about the performance of activated protein C-resistance tests.

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., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

The production of beef carries significant environmental repercussions on a worldwide level. Considering that the production of beef in Alpine mountainous regions, such as South Tyrol (Italy), constitutes a modest yet progressively growing segment within the local agricultural sector focus must be put on minimizing the environmental impact of producing one kilogram of meat, while also accounting for the carbon sequestered by Alpine pastures in such marginal areas. To this end 20 beef farms distributed in the South Tyrolean region (Italy) were divided based on the age at slaughter of the beef cattle: 10 farms with a slaughter age of 12 months (SA12) and 10 farms with a slaughter age of 24 months (SA24). Live cycle assessment (LCA) approach was used, and the impact was estimated using two functional units (FU): 1 kg of live weight (LW) and 1 kg of carcass weight (CW). Global warming potential (GWP 100 , kg CO 2 -eq), acidification potential (AP, g SO 2 -eq), and eutrophication potential (EP, g PO 4 -eq) were investigated. Furthermore, within the account, the carbon sequestered by pastures and permanent grassland has been included for estimated the overall carbon footprint. In terms of GWP 100 , the SA12 system proved to be significantly lower for both two functional units under studies, with reductions of 8.5 % and 7.4 % in terms of LW and CW, respectively, compared to the SA24 system, specifically, the SA12 system showed an environmental impact in terms of GWP 100 of 19.5 ± 1.1 kg CO 2 -eq/kg LW, which was significantly lower than the SA24 system that exhibited a value of 22.9 ± 1.1 kg CO 2 -eq/kg LW (P < 0.05). When accounting for the carbon sequestered within the system, the observed values in terms of GWP 100 are significantly lower for SA12 compared to SA24, 17.6 ± 1.5 vs. 20.9 ± 1.5 kg CO 2 -eq/Kg LW (P < 0.05), and 29.2 ± 2.5 vs. 38.7 ± 2.5 kg CO 2 -eq/Kg CW (P < 0.01). These differences are due to less purchase of concentrated feed and greater use of natural resources such as pastures and permanent grasslands. The research indicated that the production of beef in the Alpine region of South Tyrol predominantly occurs within extensive parameters, leading to a satisfactory environmental profile, also including the C sequestration., Competing Interests: Declaration of competing interest The authors affirm that they possess no identifiable conflicting financial involvements or personal affiliations that might have seemed to impact the research presented in this manuscript., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Synthetic cannabinoid receptor agonists (SCRAs, "Spice") are a diverse group of recreational drugs, with their structural and pharmacological variability still evolving. Forensic toxicologists often rely on previous reports to assess their role in intoxication cases. This work provides detailed information on the "Spice"-related fatalities around Munich, Germany, from 2014 to 2020. All cases underwent an autopsy. Pharmaceutical and illicit drugs were detected and quantified in post-mortem peripheral blood or liver by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Based on circumstantial evidence, only those cases for which a prior consumption was suspected underwent additional analyses for SCRAs and other new psychoactive substances in post-mortem blood, liver or antemortem specimens. Drug concentrations, pathological findings at autopsy and case histories were considered to assess and rank the SCRAs' involvement in each death. Concentration ranges for the individual substances in blood were defined and their distribution patterns over the investigated period were determined and correlated with their legal status and local police seizures. We identified 41 different SCRAs among 98 fatalities. 91.8% were male, at a median age of 36 years. SCRAs played a causative role in 51%, contributory role in 26%, and an insignificant role in 23% of cases. In correlation with local police seizures and legal status, 5F-ADB was the most prevalent in our cases, followed by 5F-MDMB-PICA and AB-CHMINACA. Cumyl-CBMICA and 5F-MDMB-P7AICA were among the least frequently detected SCRAs. "Spice"-related fatalities and SCRAs' causative role have significantly decreased among our cases since the German New Psychoactive Substances Act., (© 2023. The Author(s).)

Context: New psychoactive substances (NPS) have become an ongoing threat to public health. To prevent the emergence and spread of NPS, a new German law, the 'NpSG' took effect in November 2016. This study presents an overview of analytically confirmed synthetic cannabinoid (SC) intoxications from January 2015 to December 2018. In order to demonstrate effects of the NpSG, the results of 23 month before and 25 month after the introduction of the law were compared., Methods: Within the scope of a prospective observational study blood and urine samples were collected from emergency patients with suspected NPS intoxication. Comprehensive drug analyses were performed by LC-MS/MS analysis., Results: In the period considered, 138 patients were included. Within these, SC intake was verified in 65 patients (73%) in the period before the law change, and in 30 patients (61%) after. The median age increased significantly from 19.5 to 26 years. Seizures and admission to the ICU were reported significantly less frequently (seizures 29% versus 6.7%, p  = 0.0283; ICU admission 42% versus 13%, p  = 0.0089). 34 different SCs were detected, including four SCs (Cumyl-PEGACLONE, 5 F-MDMB-P7AICA, EG-018, 5 F-Cumyl-P7AICA) not covered by the NpSG at the time of detection. In the first period the most prevalent SC was MDMB-CHMICA ( n  = 24). 5 F-ADB was the most prevalent SC overall, detected in 7 patients (11%) in the first, and in 24 patients (80%) in the second period., Conclusion: The number of SC intoxications decreased overall after the implementation of the NpSG. The shift in the detected SCs can be considered a direct effect of the NpSG but unfortunately the market supply does not appear to have been reduced. Although changes in the age distribution and in the severity of intoxications may be seen as secondary effects of the law, the main objectives of the new law to prevent the emergence and spread of further chemical variations of known scheduled drugs, have apparently not been achieved from the perspective of this study.

Agricultural livestock production ranks among the most environmental impactful industry sectors at the global level, and within the livestock sector, beef production accounts for a large proportion of environmental damage. Beef production in Alpine mountain regions, such as in South Tyrol (Italy), is a small, but increasing agricultural sector. Thus, the aim of this study was to examine the environmental impact of different organic and conventional beef production systems in South Tyrol and to compare their environmental impact and effect on biodiversity under Alpine production conditions. Live cycle assessment (LCA) approach was used and 1 kg of live weight (LW) was chosen as functional unit (FU). Global warming potential (GWP, kg CO 2 -eq), acidification potential (AP, g SO 2 -eq), eutrophication potential (EP, g PO 4 -eq), non-renewable energy use (NRE, MJ-eq), land occupation (LO, m 2 organic land/year) and biodiversity damage potential (BDP) expressed in potential disappeared fraction (PDF) were investigated. The study involved 18 beef cattle farms in the South Tyrolean region: Conventional calf-fattening farms (CCF = 6), organic suckler cow farms (SCF = 6), and conventional heifer/ox fattening farms (HOF = 6). The CCF system showed a higher environmental impact compared to SCF and HOF systems for all impact categories (P < 0.05). Between the organic and the conventional system (SCF and HOF), no significant differences (P > 0.05) were found for most of the considered impact categories (means ± SEM per FU): GWP: 19.8 vs 17.1 ± 4.2 kg CO 2- eq, AP: 11.4 vs 9.3 ± 4.7 g SO 2 -eq, EP: 4.1 vs 2.8 ± 1.2, NRE: 21.9 vs 13.8 ± 7 MJ-eq, SCF and HOF respectively. Only for LO (70.8 vs 44.1 ± 17.7 m 2 organic/y, P < 0.01, SCF and HOF respectively) and the effect on BDP (-1.93 vs -0.85 ± 0.35, PDF, P < 0.01, SCF and HOF respectively) differences between organic and conventional production methods could be revealed. The study showed that beef cattle husbandry in the Alpine area has a satisfactory environmental performance. In particular, the systems studied showed a positive impact in terms of biodiversity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Pharmaceutical research not only provides the basis for the development of new medicinal products but also for the synthesis of new drugs of abuse. 3-Fluorophenmetrazine (3-FPM), a fluorinated derivative of the anorectic phenmetrazine, was first patented in 2011 and appeared on the drug market in 2014. Though invented for potential medical purposes, pharmacokinetic data on this compound, crucial for interpreting forensic as well as clinical cases, are not available. Therefore, a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the detection of 3-FPM in serum, urine, and oral fluid was developed, validated for urine and serum, and used to quantify 3-FPM in samples obtained during a controlled self-experiment. The method proved to be linear, selective and sufficiently sensitive. The limits of detection (LODs) were 0.1 ng/mL, 0.2 ng/mL, and 0.05 ng/mL in serum, urine, and oral fluid. Inter-day precision and intra-day precision (RSD) in serum samples were below 6.3% and below 8.5%, respectively. The highest serum concentration (c max ) of 210 ng/mL was reached 2.5 hours (t max ) after ingestion. The elimination half-life and the volume of distribution were calculated to be approx. 8.8 hours and 400 L (5.3 L/kg). 3-FPM could be detected in serum and urine up to 82 hours and 116 hours, respectively. It was still detected in the last oral fluid sample taken 55 hours after ingestion. 3-FPM was mainly excreted unchanged. Main metabolic reactions were aryl-hydroxylation and N-hydroxylation. Interestingly, the product of oxidative ring opening (2-amino-1-(3-fluorophenyl)propan-1-ol) showed the largest window of detection in the self-experiment., (© 2019 John Wiley & Sons, Ltd.)

The number of newly appearing benzodiazepine derivatives on the new psychoactive substances (NPS) drug market has increased over the last couple of years totaling 23 'designer benzodiazepines' monitored at the end of 2017 by the European Monitoring Centre for Drugs and Drug Addiction. In the present study, three benzodiazepines [flunitrazolam, norflurazepam, and 4'-chlorodiazepam (Ro5-4864)] offered as 'research chemicals' on the Internet were characterized and their main in vitro phase I metabolites tentatively identified after incubation with pooled human liver microsomes. For all compounds, the structural formula declared by the vendor was confirmed by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC MS/MS), liquid chromatography-quadrupole time of flight-mass spectrometry (LC-QTOF-MS) analysis and nuclear magnetic resonance (NMR) spectroscopy. The metabolic steps of flunitrazolam were monohydroxylation, dihydroxylation, and reduction of the nitro function. The detected in vitro phase I metabolites of norflurazepam were hydroxynorflurazepam and dihydroxynorflurazepam. 4'-Chlorodiazepam biotransformation consisted of N-dealkylation and hydroxylation. It has to be noted that 4'-chlorodiazepam and its metabolites show almost identical LC-MS/MS fragmentation patterns to diclazepam and its metabolites (delorazepam, lormetazepam, and lorazepam), making a sufficient chromatographic separation inevitable. Sale of norflurazepam, the metabolite of the prescribed benzodiazepines flurazepam and fludiazepam, presents the risk of incorrect interpretation of analytical findings., (© 2018 John Wiley & Sons, Ltd.)

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)-1 H -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 CB 1 receptor. Cumyl-PINACA was the most potent SC among the tested compounds and showed an EC 50 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., Competing Interests: The authors declare that they have no conflict of interest.This article does not contain any studies with animals performed by any of the authors. Although this study included a self-administration experiment, such scientific human experiments do not necessitate approval by the university ethics committee in Germany.

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., (Copyright © 2018 John Wiley & Sons, Ltd.)

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.

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 )., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The number of new psychoactive substances (NPS) that have emerged on the European market has been rapidly growing in recent years, with a particularly high number of new compounds from the group of synthetic cannabinoid receptor agonists. There have been various political efforts to control the trade and the use of NPS worldwide. In Germany, the Act to control the distribution of new psychoactive substances (NpSG) came into force in November 2016. In this new act, two groups of substances were defined, the group "cannabimimetics/synthetic cannabinoids" covering indole, indazole, and benzimidazole core structures, and a second group named "compounds derived from 2-phenethylamine." Shortly after, the first retailers of "herbal blends" promoted new products allegedly not violating the German NpSG. We describe the identification and structural elucidation of one of the first synthetic cannabinoids not being covered by the NpSG, 5-pentyl-2-(2-phenylpropan-2-yl)-2,5-dihydro-1H-pyrido[4,3-b]indol-1-one. For isolation of the substance a flash chromatography separation was applied. The structure elucidation was performed using gas chromatography-mass spectrometry (GC-MS), gas chromatography-solid state infrared spectroscopy (GC-sIR), liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry (LC-ESI-qToF-MS) and nuclear magnetic resonance (NMR) analysis. Additionally, binding affinity towards the cannabinoid receptors CB 1 and CB 2 and efficacy in a cAMP accumulation assay were measured, showing full agonistic activity and high potency at both receptors. The new compound bears a γ-carboline core structure circumventing the German NpSG and the generic definitions in other national laws. As a semi-systematic name for 2-cumyl-5-pentyl-gamma-carbolin-1-one CUMYL-PEGACLONE is suggested., (Copyright © 2017 John Wiley & Sons, Ltd.)

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., (Copyright © 2017 John Wiley & Sons, Ltd.)

Objective: The potent hallucinogenic drug 25I-NBOMe has recently emerged on the drug market. We present a case with analytically confirmed 25I-NBOMe intoxication from the prospective study "SPICE II Plus"., Case Report: Because of a severe headache a 42-year-old man took one sip of a pediatric analgesic syrup, which had been refilled with a self-made solution of 25I-NBOMe in ethanol. Thirty minutes later restlessness occurred. On arrival in the emergency department mydriasis, strong sweating, disorientation, and agitation were noticed. Within short time the patient developed severe agitation, coenesthesia, and complex hallucinations. In blood serum samples obtained at admission revealed the presence of 25I-NBOMe (34 ng/mL), 2C-I (12 ng/mL) and 25I-NBOH (<1 ng/mL) (LC-ESI-MS/MS). The presumed analgesic syrup contained 25I-NBOMe (2800 μg/mL), and besides ethanol no other compounds were detected. After six hours, the symptoms resolved without further complications., Conclusions: This is a unique case of an analytically confirmed, accidental ingestion of 25I-NBOMe in a drug naïve adult. The finding of 2C-I in the serum sample 50 minutes after intake indicates a fast metabolic breakdown of 25I-NBOMe due to first-pass metabolism.

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.

Cyclic-di-GMP is an ubiquitous second messenger in bacteria. Several c-di-GMP receptor proteins have been identified to date, and downstream signalling pathways are often mediated through protein-protein interactions. The photoreceptor Cph2 from the cyanobacterium Synechocystis sp. PCC 6803 comprises three domains related to c-di-GMP metabolism: two GGDEF and one EAL domain. It has been shown that the C-terminal GGDEF domain acts as blue-light triggered c-di-GMP producer thereby inhibiting motility of the cells in blue light. The specific function of the other two c-di-GMP related domains remained unclear. In this study, we test knockout mutants of potential interaction partners of Cph2 for altered phototactic behaviour. Whereas wild-type cells are non-motile under high-intensity red light of 640 nm, the mutant Δslr1143 displays positive phototaxis. This phenotype can be complemented by overexpression of full-length Slr1143, which also results in an increased cellular c-di-GMP concentration. However, the non-motile phenotype of wild-type cells under high-intensity red light appears not to be due to an elevated cellular c-di-GMP content. Using co-precipitation and yeast two-hybrid assays, we demonstrate that the GGDEF domain of Slr1143 interacts with the EAL and the GGDEF domains of Cph2. However, under the test conditions, the interaction of the two proteins is not light-dependent. We conclude that Slr1143 is a new Cph2-interacting regulatory factor which modulates motility under red light and accordingly we propose Cip1 (Cph2-interacting protein 1) as a new designation for this gene product.

Among the recently emerged synthetic cannabinoids, MDMB-CHMICA (methyl N-{[1-(cyclohexylmethyl)-1H-indol-3-yl]carbonyl}-3-methylvalinate) shows an extraordinarily high prevalence in intoxication cases, necessitating analytical methods capable of detecting drug uptake. In this study, the in vivo phase I metabolism of MDMB-CHMICA was investigated using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry (LC-ESI-Q ToF-MS) techniques. The main metabolites are formed by hydrolysis of the methyl ester and oxidation of the cyclohexyl methyl side chain. One monohydroxylated metabolite, the ester hydrolysis product and two further hydroxylated metabolites of the ester hydrolysis product are suggested as suitable targets for a selective and sensitive detection in urine. All detected in vivo metabolites could be verified in vitro using a human liver microsome assay. Two of the postulated main metabolites were successfully included in a comprehensive LC-ESI-MS/MS screening method for synthetic cannabinoid metabolites. The screening of 5717 authentic urine samples resulted in 818 cases of confirmed MDMB-CHMICA consumption (14%). Since the most common route of administration is smoking, smoke condensates were analyzed to identify relevant thermal degradation products. Pyrolytic cleavage of the methyl ester and amide bond led to degradation products which were also formed metabolically. This is particularly important in hair analysis, where detection of metabolites is commonly considered a proof of consumption. In addition, intrinsic activity of MDMB-CHMICA at the CB 1 receptor was determined applying a cAMP accumulation assay and showed that the compound is a potent full agonist. Based on the collected data, an enhanced interpretation of analytical findings in urine and hair is facilitated. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

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., (Copyright © 2016 John Wiley & Sons, Ltd.)

Diguanylate cyclases are enzymes that use two GTP molecules to produce one molecule cyclic dimeric guanosine monophosphate (c-di-GMP). This cyclic dinucleotide is an ubiquitous prokaryotic second messenger that controls a variety of cell functions. Several proteins have been described which contain a photoreceptor domain fused to a diguanylate cyclase. The cyanobacterial light sensor Cph2 is responsible for the blue-light induced synthesis of c-di-GMP in Synechocystis sp. PCC 6803. Here, we provide a detailed protocol for an in vitro enzymatic assay with a purified photoreceptor protein using light as the crucial reaction parameter for c-di-GMP synthesis. The assay is accomplished under continuous illumination with light of different quality with inactivation of the enzyme by heat denaturation. Analytics are performed using HPLC-UV.

Aim of this study was the investigation of the genotoxic properties of XLR-11 [1-(5-fluoropentyl)-1H-indol-3-yl](2,2,3,3-tetramethylcyclopropyl)methanone, a widely consumed synthetic cannabinoid (SC), and of the benzoyl indole RCS-4 (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone). We characterized the DNA-damaging properties of these drugs in different experimental systems. No evidence for induction of gene mutations was detected in bacterial (Salmonella/microsome) tests, but clear dose-dependent effects were found in in vitro single cell gel electrophoresis (SCGE) assays with human lymphocytes and with buccal- and lung-derived human cell lines (TR-146 and A-549). These experiments are based on the determination of DNA migration in an electric field and enable the detection of single- and double-strand breaks and apurinic sites. Furthermore, we found that both drugs induce micronuclei which are formed as a consequence of chromosomal aberrations. The lack of effects in SCGE experiments with lesion-specific enzymes (FPG, Endo III) shows that the DNA damage is not caused by formation of oxidatively damaged bases; experiments with liver enzyme homogenates and bovine serum albumin indicate that the drugs are not converted enzymatically to DNA-reactive intermediates. Furthermore, results with buccal- and lung-derived human cells show that gaseous treatment of the cells under conditions which reflect the exposure situation in drug users may cause damage of the genetic material in epithelia of the respiratory tract. Since DNA instability is involved in the etiology of cancer, these findings can be taken as an indication that consumption of the SCs may cause tumors in the respiratory tract of consumers., Competing Interests: The authors state that they have no conflict of interest.

Since 2009, more than 140 different synthetic cannabinoids (SC) have been identified in herbal mixtures consumed as recreational drugs. Knowledge of the acute toxicity of each individual compound remains sparse. Here we present a retrospective observational case series of patients presenting to emergency departments with analytically confirmed intake of JWH-210 as the only SC detected in serum samples. Cases were selected from a poison centre database from March 2011 to June 2014. In total, 22 patients were included (aged 12-25 years, median 17.5; 18 males 4 female). JWH-210 was identified in the serum samples in concentrations ranging from 0.18 to 90 ng/mL. Tachycardia, nausea, somnolence, hypokalemia, hypertension, restlessness, and/or agitation were most frequently reported. Diplopia, seizures, syncope, and ECG changes such as T-wave inversion and bradycardia were also noted. Acute adverse effects of JWH-210 typically include central nervous system depression or cerebral seizures, but also signs of sympathomimetic toxicity. Nausea was reported in 80% and typically shows a sudden onset shortly after inhalation, suggesting a central nervous effect possibly mediated by CB 1 receptors. Cardiovascular effects are reported in up to 80% of the patients and might not only include alterations in blood pressure and heart rate, but also changes in the electrocardiogram (ECG). JWH-210 as a representative of a strong CB 1 receptor agonist confirms previous reports about adverse effects of SC, but shows a distinct quantitative pattern of symptoms, compared to several other SC. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Synthetic cannabinoids have become an integral part of the drugs of abuse market since many years. The most frequent form of consumption for this class of substances is smoking of herbal mixtures purchased via the Internet. In this article the identification and structure elucidation of a new synthetic cannabinoid, [1-(cyclohexylmethyl)-1H-indol-3-yl](naphthalen-1-yl)methanone, is described. The compound was found along with 5F-ADB in a 'herbal mixture' called 'Jamaican Gold Extreme', which was sent to our laboratory in the context of a suspected intoxication. For isolation of the substance a flash chromatography separation was applied. Structure elucidation was performed using gas chromatography-mass spectrometry (GC-MS), gas chromatography solid-state infrared (GC-sIR) and nuclear magnetic resonance (NMR) analysis. The new compound can be described as the cyclohexyl methyl derivative of the first generation synthetic cannabinoid JWH-018, and the authors suggest to use "NE-CHMIMO" as a semisystematic name., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Synthetic cathinones (SCAs) are consumed worldwide as psychostimulants and are increasingly marketed as surrogates of classical illicit drugs via the internet. The genotoxic properties of most of these drugs have not been investigated. Results of earlier studies show that amphetamines which are structurally closely related to these compounds cause damage to the genetic material. Therefore, we tested the genotoxic properties of two widely consumed SCAs, namely, 3-MMC (2-(methylamino)-1-(3-methylphenyl) propan-1-one) and 4-MEC (2-(ethylamino)-1-(4-methylphenyl) propan-1-one) in a panel of genotoxicity tests. We found no evidence for induction of gene mutations in Salmonella /microsome assays, but both drugs caused positive results in the single cell gel electrophoresis (SCGE) assay which detects single and double strand breaks of DNA in a human derived buccal cell line (TR146). 3-MMC induced similar effects as 4-MEC and also caused significant induction of micronuclei which are formed as a consequence of structural and chromosomal aberrations. Negative results obtained in SCGE experiments with lesion specific enzymes (FPG and Endo III) show that these drugs do not cause oxidative damage of DNA. However, moderate induction of TBARS (which leads to the formation of DNA-reactive substances) was observed with 4-MEC, indicating that the drug causes lipid peroxidation while no clear effect was detected with 3-MMC. Results obtained with liver homogenate in SCGE-experiments show that phase I enzymes do not lead to the formation of DNA reactive metabolites. Taken together, our findings indicate that consumption of certain SCAs may cause adverse health effects in users as a consequence of damage to the genetic material.

Novel psychoactive substances (NPS) are easily accessible and the consumption has increased in recent years. New compounds as well as compounds derived from pharmaceutical research or the patent literature are provided, mostly without any declaration. As a consequence, severe adverse reactions may occur after consumption of unknown doses of these drugs, in particular after mixed intake of different psychoactive substances or co-medication. The toxic effects in such cases are not predictable. We report cases of rhabdomyolysis in patients after consumption of desoxipipradrol in combination with other NPS. Particularly in case of synergistic serotonergic effects a distinct stimulation of 5-HT2A-receptors (or 5-HT1A-receptors) should be considered which may lead to serotonergic syndrome., (© Georg Thieme Verlag KG Stuttgart · New York.)

The detection of drug metabolites in hair is widely accepted as a proof for systemic uptake of the drug, unless the metabolites can be formed as artefacts. However, regarding synthetic cannabinoids, not much is known about mechanisms of incorporation into hair. For a correct interpretation concerning hair findings of these compounds and their metabolites, it is necessary to identify the different routes of incorporation and to assess their contribution to analytical findings. This study presents the results of the LC-ESI-MS/MS analysis of an authentic hair sample taken from a patient with a known history of heavy consumption of synthetic cannabinoids. In the authentic hair sample, 5F-PB-22 and AB-CHMINACA as well as their main metabolites 5F-PB-22 3-carboxyindole, PB-22 5-OH-pentyl, and AB-CHMINACA valine were detected in all segments, comprising segments grown in a time period where the substances had not been distributed on the 'legal high' market. To enable interpretation of the results regarding the distribution of the detected analytes along the hair shaft, the stability of 5F-PB-22 and AB-CHMINACA in hair matrix and under thermal stress was assessed. The stability tests revealed that the three 'metabolites' are also formed in externally contaminated hair after storage of the samples under different conditions. In addition, 5F-PB-22 3-carboxyindole and AB-CHMINACA valine were identified as degradation products in smoke condensate. Therefore, interpretation of 'metabolite' findings of compounds comprising chemically labile amide/ester bonds or 5-fluoro-pentyl side chains should be carried out with utmost care, taking into account the different mechanisms of formation and incorporation into hair.

Background: New psychoactive substances (NPS) have become increasingly prevalent and are sold in internet shops as 'bath salts' or 'research chemicals' and comprehensive bioanalytical methods are needed for their detection., Methodology: We developed and validated a method using LC and MS/MS to quantify 56 NPS in blood and urine, including amphetamine derivatives, 2C compounds, aminoindanes, cathinones, piperazines, tryptamines, dissociatives and others. Instrumentation included a Synergi Polar-RP column (Phenomenex) and a 3200 QTrap mass spectrometer (AB Sciex). Run time was 20 min., Conclusion: A novel method is presented for the unambiguous identification and quantification of 56 NPS in blood and urine samples in clinical and forensic cases, e.g., intoxications or driving under the influence of drugs.

The continuing emergence of designer drugs imposes high demands on the scope and sensitivity of toxicological drug screening procedures. An ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) method was developed for screening and simultaneous confirmation of both designer drugs and other drugs of abuse in urine samples in a single run. The method covered selected synthetic cannabinoids and cathinones, amphetamines, natural cannabinoids, opioids, cocaine and other important drugs of abuse, together with their main urinary metabolites. The database consisted of 277 compounds with molecular formula and exact monoisotopic mass; retention time was included for 192 compounds, and primary and secondary qualifier ion exact mass for 191 and 95 compounds, respectively. Following a solid-phase extraction, separation was performed by UHPLC and mass analysis by HR-TOFMS. MS, and broad-band collision-induced dissociation data were acquired at m/z range 50-700. Compound identification was based on a reverse database search with acceptance criteria for retention time, precursor ion mass accuracy, isotopic pattern and abundance of qualifier ions. Mass resolving power in spiked urine samples was on average FWHM 23,500 and mass accuracy 0.3 mDa. The mean and median cut-off concentrations determined for 75 compounds were 4.2 and 1 ng/mL, respectively. The range of cut-off concentrations for synthetic cannabinoids was 0.2-60 ng/mL and for cathinones 0.7-15 ng/mL. The method proved to combine high sensitivity and a wide scope in a manner not previously reported in drugs of abuse screening. The method's feasibility was demonstrated with 50 authentic urine samples.

Cph2 from the cyanobacterium Synechocystis sp. PCC 6803 is a hybrid photoreceptor that comprises an N-terminal module for red/far-red light reception and a C-terminal module switching between a blue- and a green-receptive state. This unusual photoreceptor exerts complex, light quality-dependent control of the motility of Synechocystis sp. PCC 6803 cells by inhibiting phototaxis towards blue light. Cph2 perceives blue light by its third GAF domain that bears all characteristics of a cyanobacteriochrome (CBCR) including photoconversion between green- and blue-absorbing states as well as formation of a bilin species simultaneously tethered to two cysteines, C994 and C1022. Upon blue light illumination the CBCR domain activates the subsequent C-terminal GGDEF domain, which catalyses formation of the second messenger c-di-GMP. Accordingly, expression of the CBCR-GGDEF module in Δcph2 mutant cells restores the blue light-dependent inhibition of motility. Additional expression of the N-terminal Cph2 fragment harbouring a red/far-red interconverting phytochrome fused to a c-di-GMP degrading EAL domain restores the complex behaviour of the intact Cph2 photosensor. c-di-GMP was shown to regulate flagellar and pili-based motility in several bacteria. Here we provide the first evidence that this universal bacterial second messenger is directly involved in the light-dependent regulation of cyanobacterial phototaxis., (© 2012 Blackwell Publishing Ltd.)