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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.

This study aimed at evaluating the extent of benzodiazepine (BZD) consumption among nursing home residents and examining additional mental health problems. Urine samples were collected in nursing homes in a border region between Austria and Germany and analyzed for BZD using a liquid chromatographic-tandem mass spectrometric method (LC–MS/MS). In addition, a questionnaire was applied to the residents including the ICD-10 symptom rating scale; nursing staff filled out a further questionnaire on residents’ consumption of addictive drugs. Of 301 urine samples collected in 33 nursing homes, 103 (34.2%) were BZD positive. BZD users showed significantly higher values for depression and anxiety. Nursing staff reported consumption of addictive drugs in 29 (37.2% of BZD positives) with almost entirely BZD use. In nearly 4% of BZD users, nursing staff indicated drug abuse/dependence. BZD use appears to be rather common in nursing homes and nurses apparently do not recognize the addictive potential of BZD sufficiently. Interventions should aim at a reduction of BZD prescriptions by sensitizing primary care physicians and nursing staff.

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 (cmax ) of 210 ng/mL was reached 2.5 hours (tmax ) 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.

Synthetic cannabinoid receptor agonists, commonly referred to as ‘synthetic cannabinoids’ (SCs), gained popularity as recreational drugs due to their cannabis-like effects. The subclass of valine or tert-leucine-derived SCs has dominated the ‘designer drug’ market in recent years and has been associated with several severe intoxication cases. Most SCs are highly lipophilic compounds and are extensively metabolized prior to renal excretion. Hence, for drug detection in urine samples, the major metabolites of new compounds have to be identified first. The aim of this study was to elucidate structure-metabolism relationships (SMRs) of valine and tert-leucine-derived SCs enabling in-depth understanding of their phase I biotransformation and facilitating the prediction of suitable analytical targets for urine analysis. After incubation of 32 different valine/tert-leucine-derived SCs with pooled human liver microsomes (pHLM), the phase I metabolite profile of each compound was characterized using liquid chromatography–quadrupole time-of-flight mass spectrometry. By comparing chemical-structural analogs with the relative abundances of their metabolites, SMRs were studied. The terminal functionality (amide vs. methyl ester), the amino acid side chain (valine vs. tert-leucine), the core ring system (indole vs. indazole), and the N-alkyl side chain (cyclohexyl methyl vs. pentyl vs. 5-fluoropentyl vs. 4-fluorobenzyl) showed distinct differences of metabolic dehalogenation, dehydrogenation, formation of dihydrodiols, hydrolysis, hydroxylation, and N-dealkylation. The presented pHLM approach proved to be an effective tool for systematic investigation of SMRs. The information gained from this work may be useful for predicting potential SC metabolites for urine analysis.

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.

Since their first appearance on the Internet in 2012, designer benzodiazepines established as an additional, quickly growing compound class among new psychoactive substances. Data regarding pharmacokinetic parameters, metabolism, and detectability for new compounds are limited or often not available. One of these compounds, flubromazolam (8-bromo-6-(2-fluorophenyl)-1-methyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine), the triazolo-analogue of flubromazepam, has been offered on the Internet from 2014 on. The purpose of the present study was to assess the period of detectability in biological samples along with preliminary basic pharmacokinetic parameters of the designer benzodiazepine flubromazolam. To investigate these, one of the authors ingested a capsule containing 0.5 mg of the drug. Metabolism studies and suitability tests for the detection with immunochemical assays were performed with the samples obtained from the self-experiment and five authentic case samples. Flubromazolam and its mono-hydroxylated metabolite were detectable by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in urine for up to 6.5 and 8 days, respectively (lower limit of quantification (LLOQ) flubromazolam: 0.1 ng/mL). Peak serum concentrations were as low as 8 ng/mL (8 h post ingestion). Glucuronides were also detected. The terminal elimination half-life could be estimated in the range of 10-20 h. Immunochemical assays yielded negative results for serum samples and positive results for urine samples for up to five days post ingestion. The presented data demonstrate the detectability of a single uptake of 0.5 mg of flubromazolam in hair samples collected two weeks after drug uptake by LC-MS3 (cmax 0.6 pg/mg; LOD 0.01 pg/mg). The detected metabolites were in good agreement with those described in other studies. Copyright © 2017 John Wiley & Sons, Ltd.

The betel quid is one of the most commonly consumed psychoactive substances in the world. By archaeological evidence like the occurrence of areca nuts in archaeological sites, the typical overall reddish-brown staining on prehistoric human teeth or specific artifacts linked with the habit, it is assumed that this tradition reaches back to prehistoric times. Since this kind of evidence is indirect, it is frequently doubted. The present study provides the earliest direct analytical indication of betel nut chewing in human history. A typical stained tooth from an Iron Age skeleton (site Go O Chua in Southern Vietnam, 400–100 BC) was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-high-resolution mass spectrometry (LC-HR-ToF-MS) and the alkaloid arecoline which is specific for Areca catechu L. (Arecaceae) was detected.

Designer benzodiazepines represent an emerging class of new psychoactive substances. While other classes of new psychoactive substances such as cannabinoid receptor agonists and designer stimulants are mainly consumed for hedonistic reasons, designer benzodiazepines may also be consumed as ‘self-medication’ by persons suffering from anxiety or other psychiatric disorders or as stand-by ‘antidote’ by users of stimulant and hallucinogenic drugs. In the present study, five benzodiazepines (adinazolam, cloniprazepam, fonazepam, 3-hydroxyphenazepam and nitrazolam) and one thienodiazepine (metizolam) 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 nuclear magnetic resonance spectroscopy, gas chromatography–mass spectrometry (MS), liquid chromatography MS/MS and liquid chromatography quadrupole time-of-flight MS analysis. The detected in vitro phase I metabolites of adinazolam were N-desmethyladinazolam and N-didesmethyladinazolam. Metizolam showed a similar metabolism to other thienodiazepines comprising monohydroxylations and dihydroxylation. Cloniprazepam was metabolized to numerous metabolites with the main metabolic steps being N-dealkylation, hydroxylation and reduction of the nitro function. It has to be noted that clonazepam is a metabolite of cloniprazepam, which may lead to difficulties when interpreting analytical findings. Nitrazolam and fonazepam both underwent monohydroxylation and reduction of the nitro function. In the case of 3-OH-phenazepam, no in vitro phase I metabolites were detected. Formation of licensed benzodiazepines (clonazepam after uptake of cloniprazepam) and the sale of metabolites of prescribed benzodiazepines (fonazepam, identical to norflunitrazepam, and 3-hydroxyphenazepam) present the risk of incorrect interpretation of analytical findings. Copyright © 2016 John Wiley & Sons, Ltd.

Introduction Abstinence from ethanol is necessary in various situations. Among these are jail terms. Nevertheless, it is a matter of fact that ethanol is illegally produced and ingested in prisons. So far, data regarding drug prevalence in jail have mainly been collected by questionnaires. To get an objective database for the prevalence of ethanol consumption in jail, a cross-sectional study was performed. Methods Inmates of two German prisons (Offenburg and Freiburg) were asked to give a urine sample at an unknown and random point of time. Participation was voluntary and did lead to neither negative consequences nor benefits. All samples were anonymized. Using the consumption markers ethyl glucuronide (EtG) and ethyl sulfate (EtS), the urine samples were tested for previous ethanol consumption. Analyses were performed by a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Results In total 676 male inmates participated in this study. The participation rate was 70–75 % of all permanent inmates in Offenburg and 30.6 % in Freiburg. Ten of the 555 (1.8 %) samples from Offenburg and 1 of the 121 (0.8 %) samples from Freiburg were positive for ethanol consumption markers with concentrations ranging from trace amounts to 1400 ng/mL for EtG and up to 510 ng/mL for EtS, respectively. Conclusions The number of participants in this study was rather high, so that the results represent a good cross section, at least for Offenburg, the jail with the higher number of positive samples.

In 2012, the first designer benzodiazepines were offered in Internet shops as an alternative to prescription-only benzodiazepines. Soon after these compounds were scheduled in different countries, new substances such as clonazolam, deschloroetizolam, flubromazolam, and meclonazepam started to emerge. This article presents the characterization of these four designer benzodiazepines using nuclear magnetic resonance spectroscopy, gas chromatography–electron ionization-mass spectrometry, liquid chromatography–tandem mass spectrometry, liquid chromatography–quadrupole time-of-flight-mass spectrometry, and infrared spectroscopy. The major in vitro phase I metabolites of the substances were investigated using human liver microsomes. At least one monohydroxylated metabolite was identified for each compound. Dihydroxylated metabolites were found for deschloroetizolam and flubromazolam. For clonazolam and meclonazepam, signals at mass-to-charge ratios corresponding to the reduction of the nitro group to an amine were observed. Desalkylations, dehalogenations, or carboxylations were not observed for any of the compounds investigated. Furthermore, for clonazolam and meclonazepam, no metabolites formed by a combination of reduction and mono-/dihydroxylation were detected. This knowledge will help to analyze these drugs in biological samples.

The stimulant designer drug 3,4-methylenedioxypyrovalerone (MDPV) was first synthesized by Boehringer Ingelheim in 1969 and introduced on the black market in 2006. Only a small number of fatal intoxication cases have been reported in the literature, all with significant blood MDPV concentrations. In this report, we describe one fatality attributed to an idiosyncratic reaction to MDPV. The victim displayed agitation, violent behavior and delirium followed by cardiac arrest. Hyperthermia was observed at the hospital. The MDPV cardiac and femoral blood concentrations were 6 ng/mL. The presence of excited delirium syndrome and MDPV, a drug with a pharmacology similar to cocaine, leads to the conclusion that the victim suffered a fatal adverse reaction to MDPV. This is the first published case of idiosyncratic reaction to MDPV.

The number of new psychoactive substances (NPS) that have emerged on the European market has been rapidly growing in the last 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 CB1 and CB2 and efficacy in a cAMP accumulation assay was 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 also 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.

Drugs deposited in human teeth are well preserved; the spectrum of toxicological investigations may therefore be supplemented by an analysis method for drugs in teeth. A liquid chromatography–electrospray ionization tandem mass spectrometry assay for the detection and quantification of basic drugs of abuse in bovine dentin samples was developed and validated. The drugs and metabolites amphetamine, methamphetamine, methylenedioxymethylamphetamine, methylenedioxyethylamphetamine, codeine, morphine, cocaine and benzoylecgonine were extracted from 50 mg ground dentin powder by ultrasonication for 60 min in methanol 3 times. The extracts were analyzed on a triple-quadrupole mass-spectrometer in multiple reaction monitoring mode. The method was validated and proved to be accurate, precise, selective, specific and stable with good linearity within the calibration range and a lower limit of quantification of 10 to 20 pg/mg. To artificially load bovine dentin samples with drugs, the natural process of de- and remineralization in the oral cavity was mimicked by a pH-cycling experiment. The artificially drug-loaded dentin samples showed drug concentrations of 20 to 80 pg/mg. The method can be applied in further in vitro experiments as well as in post-mortem cases, especially where limited sample tissue is available. Copyright © 2014 John Wiley & Sons, Ltd.

The appearance of pyrazolam in Internet shops selling ‘research chemicals’ in 2012 marked the beginning of designer benzodiazepines being sold as recreational drugs or ‘self medication’. With recent changes in national narcotics laws in many countries, where two uncontrolled benzodiazepines (phenazepam and etizolam), which were marketed by pharmaceutical companies in some countries, were scheduled, clandestine laboratories seem to turn to poorly characterized research drug candidates as legal substitutes. Following the appearance of pyrazolam, it comes with no surprise that recently, flubromazepam (7-bromo-5-(2-fluorophenyl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one), a second designer benzodiazepine, was offered on the market. In this article, this new compound was characterized using nuclear magnetic resonance, gas chromatography-mass spectrometry (GC–MS), liquid chromatography–mass spectrometry (LC–MS/MS) and liquid chromatography quadrupole time-of-flight MS (LC–Q–ToF–MS). Additionally, a study was carried out, in which one of the authors consumed 4 mg of flubromazepam to gain preliminary data on the pharmacokinetic properties and the metabolism of this compound. For this purpose, serum as well as urine samples were collected for up to 31 days post-ingestion and analyzed applying LC–MS/MS and LC–Q-ToF-MS techniques. On the basis of this study, flubromazepam appears to have an extremely long elimination half-life of more than 100 h. One monohydroxylated compound and the debrominated compound could be identified as the predominant metabolites, the first allowing a detection of a consumption for up to 28 days post-ingestion when analyzing urine samples in our case. Additionally, various immunochemical assays were evaluated, showing that the cross-reactivity of the used assay seems not to be sufficient for safe detection of the applied dose in urine samples, bearing the risk that it could be misused in drug-withdrawal settings or in other circumstances requiring regular drug testing. Furthermore, it may be used in drug-facilitated crimes without being detected. Copyright © 2013 John Wiley & Sons, Ltd.

In 2012, online shops selling so-called research chemicals started offering pyrazolam, a new benzodiaze- pine that differs from phenazepam and etizolam, which have also recently appeared on the ''gray market'', in that it is not marketed by pharmaceutical companies anywhere in the world. This article describes the characterization of pyrazolam (8-bromo-1-methyl-6-pyridin-2-yl-4H-(1,2,4) triazolo(4,3-a)(1, 4)benzodiazepine) using gas chroma- tography-mass spectrometry, liquid chromatography- tandem mass spectrometry (LC-MS-MS), liquid chroma- tography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS), and nuclear magnetic resonance spec- troscopy. In addition, a study was carried out in which one of the authors ingested two 0.5-mg pyrazolam tablets. Serum and urine samples were then obtained to investigate the metabolism of pyrazolam and to obtain preliminary results for the elimination half-life and the detectability of a 1-mg dose in serum and urine using a highly sensitive LC-MS-MS method and immunoassays. The results showed an elimination half-life of about 17 h and no detectable metabolism. The parent compound was detected with the described LC-MS-MS method in serum for more than 50 h and in urine for approximately 6 days. Immu- noassays showed cross-reactivity, but poor detection in the study samples demonstrated that consumption or adminis- tration of this presumably potent drug could go undetected unless instrumental analytical techniques are also used.

Objectifs In 2012 benzodiazepine-type compounds started to extend the group of New Psychoactive Substances (NPS) sold on the internet as alleged legal recreational drugs. Initially, benzodiazepines like phenazepam and etizolam - which are still prescribed in some countries - were sold on the internet as recreational drugs. Subsequently, the group of so-called designer benzodiazepines was enlarged by compounds that either are active metabolites (e.g. norfludiazepam) or precursors (e.g. diclazepam) of known benzodiazepines or combine structural properties of different classical benzodiazepines (e.g. flubromazolam). Patents and scientific literature describe the synthesis and detailed results of animal model studies for more than a hundred different benzodiazepines, so it can be assumed that this sub-group of NPS will extend quickly in the future. The aim of this project was to develop an easy-to-use screening-method to detect and identify prescription and designer benzodiazepines and additionally obtain semi-quantitative information in a single run. Methods 1 mL serum sample was extracted using an established liquid-liquid extraction (LLE) protocol. Chromatographic separation was performed using a 13-minute formic acid/acetonitrile LC gradient and a Dionex Acclaim RLSC C18 100 × 2 mm column. An amaZon speed ion trap MS (Toxtyper ® , Bruker Daltonik) operating in ESI positive mode was used to generate MS2 and MS3 spectra according to a scheduled precursor list (SPL) triggered acquisition process. Compounds were identified by an in-house generated spectral library, containing retention time, MS and MS 2 /MS 3 information of currently 58 benzodiazepines. The linear range of the method was evaluated by fortifying blank human serum with benzodiazepine standards from 5 to 500 ng/mL. Semi-quantitative results were obtained using a single-point calibration (c = 50 ng/mL). Results The current spectral library contains 19 designer benzodiazepines, prescription benzodiazepines and metabolites. The method can easily be extended once new designer benzodiazepines emerge on the drug market or according to specific needs of the user. The LOD was 5 ng/mL except for six compounds only detected at concentrations above 10 ng/mL. Nifoxipam, known to have a poor ESI response and being highly instable in serum, was the only compound that could not be detected at practically relevant concentrations in serum. For each analyte, a linear calibration range (calLow to calHigh) was determined and calculated concentrations within this range are reported as semi-quantitative result in the automatically generated report. Findings below or above the linear range are reported as ‘ calHigh’, respectively. Semi-quantitative results were found to vary between ± 20 and ± 40% at the lower end of the calibration range and ± 10 to ± 25% at medium and high concentrations. Conclusion The presented method allows automated identification and semi-quantitative determination of 58 benzodiazepines, including 18 designer benzodiazepines. LOD in serum allow the detection below the desired or at the concentration needed for the majority of medical benzodiazepines, making the screening applicable for clinical research and forensic analysis. Semi-quantitative analysis enables a quick toxicological research evaluation of the results and helps to decide on the analytical strategy in case work. Although this approach requires a more time consuming sample preparation when compared to routine immunoassays, unambiguous identification and semi-quantitative determination of compounds also offers more detailed information.

A drinking experiment with participants suffering from Gilbert's syndrome was performed to study the possible influence of this glucuronidation disorder on the formation of ethyl glucuronide (EtG). Gilbert's syndrome is a rather common and, in most cases, asymptomatic congenital metabolic aberration with a prevalence of about 5 %. It is characterized by a reduction of the enzyme activity of the uridine diphosphate glucuronosyltransferase (UGT) isoform 1A1 up to 80 %. One of the glucuronidation products is EtG, which is formed in the organism following exposure to ethanol. EtG is used as a short-term marker for ethyl alcohol consumption to prove abstinence in various settings. After 2 days of abstinence from ethanol and giving a void urine sample, 30 study participants drank 0.1 L of sparkling wine (9 g ethanol). 3, 6, 12, and 24 h after drinking, urine samples were collected. 3 hours after drinking, an additional blood sample was taken, in which liver enzyme activities, ethanol, hematological parameters, and bilirubin were measured. EtG and ethyl sulfate (EtS), another short-term marker of ethanol consumption, were determined in the urine samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS); creatinine was measured photometrically. In all participants, EtG and EtS were detected in concentrations showing a wide range (EtG: 3 h sample 0.5-18.43 mg/L and 6 h sample 0.67-13.8 mg/L; EtS: 3 h sample 0.87-6.87 mg/L and 6 h sample 0.29-4.48 mg/L). No evidence of impaired EtG formation was found. Thus, EtG seems to be a suitable marker for ethanol consumption even in individuals with Gilbert's syndrome.

Objectives New synthetic cannabinoids (SC) frequently appear on the markets challenging toxicologists. Perceived as a legal alternative to cannabis a growing number of SCs is available. Consequently, analytical methods have to be adapted frequently to include newly emerging compounds. Since most SC are metabolized extensively metabolite identification prior to analyzing urine samples is inevitable. The presented study aimed at the development and implementation of a workflow allowing for the identification of the major in vitro phase I metabolites of new compounds by using dedicated software to mine analytical data of pooled human liver microsome (pHLM) incubations. The identified metabolites and their respective MSMS fragment information are utilized to update analytical methods for the reliable identification of SC uptake in human urine samples. Methods The performed pHLM assay was according to an in-house protocol. The MS was operated in positive ESI with data-dependent MSMS fragmentation. MassMetaSite software (Molecular Discovery) was used to analyze HPLC-MSMS datasets of the incubations sampled at time points zero and one hour. Precursor and fragment information of the identified metabolites as well as their retention times were collected and added to a database used for LC-QToF-MS screening of urine samples for SC uptake. To allow for retrospective data evaluation authentic urine samples were analyzed in data independent broad-band-CID mode (bbCID). Results The highly potent and prevalent synthetic cannabinoid MDMB-CHMICA was chosen as a model compound. Analysis of the pHLM incubations of MDMB-CHMICA with MassMetaSite revealed 10 metabolites with at least two fragment masses each. Although in-depth manual data evaluation lead to the identification of 15 phase I metabolites, all main in vitro phase I metabolites were detected with the software. Authentic forensic case samples were screened after enzymatic cleavage of glucuronides and processed with the updated TASQ method. Despite varying relative abundances of the detected metabolites, the in vitro and in vivo data showed good agreement with respect to the chosen MDMB-CHMICA metabolites. Conclusion Using MassMetaSite software and the described workflow proved to be a suitable procedure to identify metabolites and subsequently update analytical method to account for the rapid fluctuation of compounds among the group of new psychoactive substances (NPS). The here described approach can be helpful for updating screening methods with metabolite information. This is necessary whenever dealing with analytes that are extensively metabolized such as SCs. With other NPS the identification of metabolites along with the parent compound can serve as a plausibility check and may help in estimating the time of the last drug uptake. The use of dedicated software to help with metabolite identification is less time consuming thus providing a useful alternative for the rapid method update in routine forensic toxicology.

In a seizure of German customauthorities two 3-([1,3]-thiazol-2-yl)indoles ( N -(2-methoxyethyl), N - iso -propyl-2-(1-pentyl-1 H -indol-3-yl)-4-thiazolemethanamine ( 1 ) and N , N -diethyl-2-(1-pentyl-1 H -indol-3-yl)-4-thiazolemethanamine ( 2 )) and one benzimidazole (1-(cyclohexylmethyl)-2-[(4-ethoxyphenyl)methyl]- N , N -diethyl-1 H -benzimidazole-5-carboxamide ( 6 )) were seized as pure compounds. The compounds have been detected in Germany for the first time, and no analytical data had been previously published. Mass spectrometric (MS), infrared (IR) spectroscopic, and nuclear magnetic resonance (NMR) spectroscopic data are presented and the way of the structure elucidation of these rather uncommon compounds is discussed.

In the field of forensic toxicology, numerous strategies using different types of LC-MS platforms have been developed to set up an ultimate comprehensive screening method. Despite all this research, the question for the detection of a dedicated set of substances arises quite often in daily routine work. In this project, a screening method for the detection of psychotropic drugs based on the open library concept of a recently developed LC-MS(n) screening approach was developed and the effectiveness of a heated ESI-source was evaluated. To set up an individual spectral library all available data of psychotropics from the Toxtyper™ library was transferred to a new library format and complemented by MS, MS(2) and MS(3) data of additional psychotropic compounds. Precursor masses and retention time information of the library were used to trigger data dependent acquisition of MS(n)-spectra. Serum samples were analysed after alkaline liquid-liquid extraction on a Dionex RSLC (Acclaim™ C18 100×2.1C) coupled to a Bruker amaZon speed ion trap. A conventional ESI-source and an ionBooster™ source (IB) were used for ionization. All other LC and MS parameters were adopted from the original screening approach. Identification and result reporting was carried out by a fully automated software script. This screening method finally contains the individual precursor mass and retention time of 105 psychotropic substances and metabolites. Method evaluation was performed using pooled serum samples fortified with 12 different mixtures containing a total of 99 compounds at low therapeutic concentrations (cLOW and 2×cLOW). The customized method (ESI/IB) led to a higher rate of identifications (92%) - especially at low concentration levels (cLOW) - as the comprehensive screening approach (87%). Results from routine analysis with known intake of psychotropic drugs were confirmed with positive findings, if the concentration range was above or around the assumed limit of detection from this evaluation study. The Toxtyper open library concept enables fast and easy generation of new screening methods. The generated screening method is a fast and robust tool for the detection and identification of 105 psychotropics in human serum. Use of the ionBooster source led to a significant increase of the ionization efficiency within this sort of substance class. Evaluation in spiked human serum samples showed detection of low therapeutic levels for the majority of compounds, making the screening applicable for clinical and forensic samples (intoxication and post mortem cases).

Considering the vast variety of synthetic cannabinoids and herbal mixtures - commonly known as 'Spice' or 'K2' - on the market and the resulting increase of severe intoxications related to their consumption, there is a need in clinical and forensic toxicology for comprehensive up-to-date screening methods. The focus of this project aimed at developing and implementing an automated screening procedure for the detection of synthetic cannabinoids in serum using a liquid chromatography-ion trap-MS (LC-MS(n)) system and a spectra library-based approach, currently including 46 synthetic cannabinoids and 8 isotope labelled analogues. In the process of method development, a high-temperature ESI source (IonBooster(TM), Bruker Daltonik) and its effects on the ionization efficiency of the investigated synthetic cannabinoids were evaluated and compared to a conventional ESI source. Despite their structural diversity, all investigated synthetic cannabinoids benefitted from high-temperature ionization by showing remarkably higher MS intensities compared to conventional ESI. The employed search algorithm matches retention time, MS and MS(2)/MS(3) spectra. With the utilization of the ionBooster source, limits for the automated detection comparable to cut-off values of routine MRM methods were achieved for the majority of analytes. Even compounds not identified when using a conventional ESI source were detected using the ionBooster-source. LODs in serum range from 0.1 ng/ml to 0.5 ng/ml. The use of parent compounds as analytical targets offers the possibility of instantly adding new emerging compounds to the library and immediately applying the updated method to serum samples, allowing the rapid adaptation of the screening method to ongoing forensic or clinical requirements. The presented approach can also be applied to other specimens, such as oral fluid or hair, and herbal mixtures and was successfully applied to authentic serum samples. Quantitative MRM results of samples with analyte concentrations above the determined LOD were confirmed as positive findings by the presented method.

Objectif La sensibilite est un parametre important pour la plupart des applications MS, en particulier lors de la recherche de traces en toxicologie medico-legale. Cette etude a pour objectif d’evaluer l’impact d’une nouvelle source ESI haute temperature sur l’efficacite d’ionisation de certains analytes d’interet (Cannabinoides de synthese, psychotropes, benzodiazepines, medicaments basiques, marqueurs de consommation alcoolique…) Methodes L’evaluation experimentale a ete basee sur des spectrometres de masse type Trappe ionique (amaZon speed™ Bruker) et type Q-TOF (maXis impact™ Bruker) avec respectivement, une source standard ESI et une source ESI haute temperature (Ion Booster™, Bruker). Les echantillons dopes par differents composes presentaient la gamme de concentration suivante (10ng/ml a 100 ng/ml). Ces derniers ont ete analyses en mode positif ou en mode negatif en variant les temperatures de vaporisation pour la source Ion booster™. La separation chromatographique a ete realisee via une methode UHPLC. Resultats Cette evaluation a demontre un gain significatif en sensibilite pour la majorite des composes testes. Un facteur d’a peu pres fois 10 a ete constate par rapport a une source ESI conventionnelle. En particulier, la cocaine et ses metabolites (benzoylecgonine et ecgonine methyl ester) ont presente la plus nette et la plus importante amelioration de signal. En effet, avec une temperature de vaporisation a 400 °C, des augmentations de sensibilite d’un facteur 50, 30 et 100 ont ete respectivement constatees. Aucun des composes analyses n’a montre de diminution de sensibilite. Cependant, cette amelioration de sensibilite varie en fonction des differentes proprietes chimiques des composes, de leurs comportements d’ionisation et surtout de leurs thermolabilites. Conclusion L’amelioration globale du signal en utilisant une source ESI haute temperature pourrait apporter un grand benefice pour certains echantillons medico-legaux non thermolabiles avec peu de materiel disponible et une concentration analytique tres basse (Cheveux par exemple).

Aim Screening for xenobiotics is a crucial part of post-mortem toxicological analysis. Vitreous humor is an easy-to-handle body fluid only little affected by putrefaction and therefore a smart alternative to urine or blood. Due to the blood-retinal barrier, interpretation of quantitative results in vitreous humor is quite difficult but is a suitable matrix for qualitative screening analysis. When analyzing body fluids, liquid chromatography - mass spectrometry has become the method of choice for a wide range of analytical questions. In this project, a previously developed automated LC-MSn approach was evaluated for the detection of drugs and drugs of abuse in vitreous humor to extend its application to post-mortem analysis. Methods Twenty-four samples (vitreous humor) autopsy cases were worked-up by a two-step SPE extraction (Isolute HCX5 ® ), and analyzed by LC-MSn. The LC-MSn system used is the Toxtyper™ (LC-IonTrap Amazon Speed Bruker and UHPLC RSLC 3000 Dionex-Thermo). The screening method is based on alternative polarity (Zero Delay), ultrascan mode (70–800 Da at 32,000 Da/s) and Data Dependend Acquisition (With Scheduled Precursor List). Acquired data (full scan MS, MS2 and MS3) were searched against the Toxtyper library (900 compounds). Results In total, 57 substances distributed to six mixtures were analyzed at three different concentrations (low, med, high). Medium concentration levels were adjusted to vitreous humor concentrations reported in the literature and cut-offs of other established screening methods. Approximately 94% of the compounds could be detected and identified correctly at each concentration level investigated. Mirtazepine could only be detected at the medium concentration level (10 ng/mL) while tramadol and olanzapine could only be identified correctly at high concentration levels (25 and 150 ng/mL). LSD was the only compound that could not be detected at all, probably due to its’ sensitivity to light. Limit of detection was set to the lowest concentration level that could be identified correctly in duplicate determination. The results from real cases were in good agreement with the findings from routine post-mortem analysis. In vitreous humor of 24 autopsy cases (c1–c24), 76% of the active agent consumed by the deceased could be identified in accordance to routine post-mortem analysis. For final method evaluation a confirmatory analysis to determine vitreous humor concentrations of at least the most commcon substances found in post-mortem analysis is mandatory. Conclusion The applied screening approach is a suitable tool for the detection of xenobiotics in vitreous humor. Besides the known physiological limitations of the matrix itself, the obtained limits of detection seem to be adequate for forensic casework. The easy handling of vitreous humor when compared to sample preparation of tissue samples combined with the LC-MSn analysis and automated data evaluation of the Toxtyper present a time- and cost-effective screening alternative for post-mortem cases if no urine and/or only a limited volume of blood is available.

Objectif Il existe un reel besoin en toxicologie clinique et medico-legale en termes de technologies automatisees, robustes et specifiques en vue des limitations bien connues des methodes actuelles (GC-MS, LC-UV, Immuno dosages). La chromatographie liquide couplee a la spectrometrie de masse en tandem est une technologie emergente pour les criblages toxicologiques. Ce poster decrit une nouvelle solution simple et robuste, le Toxtyper™, pour la detection et l’identification des drogues et medicaments. La reproductibilite MS/MS inter-sites de cette solution a ete evaluee. Methodes Apres une extraction liquide-liquide (LLE) des echantillons seriques, une rapide separation UHPLC de 11 minutes a ete mise en oeuvre avec un gradient acide formique/acetonitrile et une colonne analytique Dionex Acclaim RSLC C18 100 x 2 mm. Un spectrometre de masse type trappe ionique, l’amaZon speed™ Bruker a ete utilise pour generer des spectres MS2 et MS3 selon un processus AutoMSn de fragmentation automatique SmartFrag™ base sur une liste d’ions precurseurs MS (SPL, Scheduled Precursor List). Afin de proceder aux identifications des composes analyses, une base de donnees de 839 drogues, medicaments et metabolites est interrogee (Temps de retention, informations MS et MS2/MS3). Le traitement des donnees et l’elaboration du rapport de resultats sont automatises via un logiciel convivial le Compass Open Access (COA). Un rapport PDF peut etre envoye automatiquement par e-mail aux destinataires prealablement definis. Resultats Trois melanges de composes toxicologiques d’interet (Antidepresseurs, benzodiazepines et hypnotiques) ont ete testes de facon aveugle sur sept systemes Toxtyper™, repartis a cinq endroits differents. Cette nouvelle approche de criblage toxicologique a presente une reproductibilite inter-sites exceptionnelle entre les differents instruments et laboratoires. Conclusion La methode de screening Toxtyper™ presentee offre un outil d’identification rapide, fiable et accessible a du personnel qualifie ou non experts en MS pour les analyses cliniques et medico-legales. La combinaison des spectres MS2/MS3 et des temps de retention permet une identification robuste des drogues et des medicaments. Le haut niveau d’automatisation et de standardisation est idealement adapte pour une utilisation en routine clinique 24 heures sur 24 et 7 jours sur 7.