Insights into the metabolism of CH-PIATA-A novel synthetic cannabinoid featuring an acetamide linker.

The recent change from the popular carboxamide to an acetamide (ATA) linker scaffold in synthetic cannabinoid receptor agonists (SCRAs) can be interpreted as an attempt to circumvent legal regulations, setting new analytical challenges. Metabolites of N-cyclohexyl-2-(1-pentyl-1 H-indol-3-yl)acetamide: CH-PIATA, the second ATA type SCRA detected in the EU, were investigated in urine and serum samples by LC-HRMS-MS and LC-MS-MS. Two different in vitro models, a pHLM assay and HepG2-cells, as well as an in silico prediction by GLORYx freeware assisted in metabolite formation/identification. CH-PIATA was extensively metabolized, leading to metabolites formed primarily by mono- and dihydroxylation. For urine and serum specimens, monohydroxylation at the indole core or the methylene spacer of the acetamide linker (M1.8), carboxylic acid formation at the N-pentyl side chain (M3.1) and degradation of the latter leading to a tentatively identified N-propionic acid metabolite (M5.1) are suggested as reliable markers for substance intake. The N-propionic acid metabolite could not be confirmed in the in vitro assays as it includes multiple consecutive metabolic reactions. Furthermore, CH-PIATA could be detected as parent substance in blood samples, but not in urine. Both in vitro assays and the in silico tool proved suitable for predicting metabolites of CH-PIATA. Considering effort and costs, pHLM incubations seem to be more effective for metabolite prediction in forensic toxicology than HepG2 cells. The highlighted Phase I metabolites serve as reliable urinary targets for confirming CH-PIATA use. The in silico approach is advantageous when reference material is unavailable.

A fatal case of aspiration due to consumption of the hallucinogenic tryptamine derivative dipropyltryptamine (DPT).

BACKGROUND AND AIM: This case involves a 20-year-old man with prior hallucinogen-use experience, who sniffed an unknown amount of dipropyltryptamine in an apartment. Dipropyltryptamine, a hallucinogenic compound belonging to the tryptamine class is recognized for inducing effects similar to dimethyltryptamine (DMT) but with a longer duration. Ten to fifteen minutes later he experienced visual hallucinations, followed by increasing apathy. Two hours post consumption he developed abdominal pain, leading to collapse, seizure, and vomiting. Despite emergency medical resuscitation on site, transport to hospital 2.5 hours post consumption and extracorporeal life support he died 21 hours later. Relevant toxicological and morphological findings are presented. METHODS: A serum sample was collected four hours post consumption. Autopsy was performed six days after death. Antemortem serum, as well as postmortem cardiac blood and urine were analyzed for alcohol and psychoactive drugs by systematic toxicological analyses employing gas chromatography-mass spectrometry (Maurer/Pfleger/Weber library among others), liquid chromatography-ion trap mass spectrometry (LC-MSn, Toxtyper™), and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Dipropyltryptamine was quantified by LC-MS/MS after solid-phase extraction. RESULTS: Autopsy revealed a state after deep aspiration of gastric contents with consecutive brain edema due to oxygen deprivation. Dipropyltryptamine concentrations were approximately 210 ng/ml, 110 ng/ml and 180 ng/ml in antemortem serum, postmortem cardiac blood and urine, respectively. To the best of our knowledge, these are the first reported concentrations of dipropyltryptamine in a fatal case. CONCLUSION: Unlike typical tryptamine overdose reports, this case did not present with agitation, hyperthermia, or tachycardia. Despite the individual's prior experience with tryptamines and the generally low toxicity associated with this class of hallucinogens, death in this case was an indirect consequence of the nasal consumption of a high dose of dipropyltryptamine.

Detection of methadone and buprenorphine in urine samples from inmates of German prisons.

The use of non-prescribed opioid substitution drugs is a serious public health problem, involving general population as well as vulnerable populations such as prisoners. The estimation of the prevalence of opioid substitution drug misuse in prisoners is crucial to suggest strategies to contrast this phenomenon and reduce the associated morbidity and mortality. The present study aimed to provide an objective estimation of the prevalence of illicit use of methadone and buprenorphine in two German prisons. Urine samples were collected from inmates of Freiburg and Offenburg prisons at random times and tested for the detection of methadone, buprenorphine and their metabolites. Analyses were performed by a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. In total 678 inmates participated in this study. The participation rate was about 60% of all permanent inmates. Of the 675 samples suitable for the analysis, 70 samples (10.4%) tested positive for methadone, 70 samples (10.4%) for buprenorphine, and 4 samples (0.6%) for both drugs. At least 100 samples (14.8%) were not associated with reported prescribed-opioid substitution treatment (OST). Buprenorphine was the most common illicitly used drug. In one of the prisons, buprenorphine was brought in from the outside. The present cross-sectional experimental study was able to provide reliable information regarding the illicit use of opioid substitution drugs in prisons.

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

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

Method validation and preliminary pharmacokinetic studies on the new designer stimulant 3-fluorophenmetrazine (3-FPM).

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.

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

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

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

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

Structural characterization and pharmacological evaluation of the new synthetic cannabinoid CUMYL-PEGACLONE.

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 CB1 and CB2 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.

Flubromazolam - Basic pharmacokinetic evaluation of a highly potent designer benzodiazepine.

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.

A case of fatal idiosyncratic reaction to the designer drug 3,4-methylenedioxypyrovalerone (MDPV) and review of the literature.

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.

Betel Nut Chewing in Iron Age Vietnam? Detection of Areca catechu Alkaloids in Dental Enamel.

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 Gò Ô Chùa 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.

Characterization and in vitro phase I microsomal metabolism of designer benzodiazepines - an update comprising adinazolam, cloniprazepam, fonazepam, 3-hydroxyphenazepam, metizolam and nitrazolam.

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.

Detection of the ethanol consumption markers ethyl glucuronide and ethyl sulfate in urine samples from inmates of two German prisons.

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.

Influence of Gilbert's syndrome on the formation of ethyl glucuronide.

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.

'Psychotropics caught in a trap' - adopting a screening approach to specific needs.

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).

A comprehensive library-based, automated screening procedure for 46 synthetic cannabinoids in serum employing liquid chromatography-quadrupole ion trap mass spectrometry with high-temperature electrospray ionization.

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.

Detection and identification of the designer benzodiazepine flubromazepam and preliminary data on its metabolism and pharmacokinetics.

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.