Bjoern Moosmann

Characteristics of the designer drug and synthetic cannabinoid receptor agonist AM-2201 regarding its chemistry and metabolism.

Aminoalkylindoles, a subclass of synthetic cannabinoid receptor agonists, show an extensive and complex metabolism in vivo, and due to their structural similarity, they can be challenging in terms of unambiguous assignment of metabolic patterns in urine samples to consumed substances. The situation may even be more complicated as these drugs are usually smoked, and the high temperature exposure may lead to formation of artifacts. Typical metabolites of JWH-018 (Naphthalen-1-yl(1-pentyl-1H-indol-3-yl)methanone) were reportedly detected not only in urine samples collected after consumption of JWH-018 but also after AM-2201 (1-(5-fluoropentyl-1H-indol-3-yl)-(naphthalene-1-yl)methanone) use. The aim of the presented study was to evaluate if typical JWH-018 metabolites can be formed metabolically in humans and if JWH-018 may be formed artifactually during smoking of AM-2201. Therefore, one of the authors ingested 5 mg of pure AM-2201, and serum as well as urine samples were analyzed subsequently. Additionally, the smoke condensate from a cigarette laced with pure AM-2201 was investigated. In addition, urine samples of patients after known consumption of AM-2201 or JWH-018 were evaluated. The results of the study prove that typical metabolites of JWH-018 and JWH-073 are built in humans after ingestion of AM-2201. However, the N-(4-hydroxypentyl) metabolite of JWH-018, which is the major metabolite after JWH-018 use, was not detected after the self-experiment. In the smoke condensate, small amounts of JWH-018 and JWH-022 (Naphthalen-1-yl[1-(pent-4-en-1-yl)-1H-indol-3-yl]methanone) were detected. Nevertheless, the results of our study suggest that the amounts absorbed by smoking do not significantly influence the metabolic pattern in urine samples. Therefore, the N-(4-hydroxypentyl) metabolite of JWH-018 can serve as a valuable marker to distinguish consume of products containing AM-2201 from JWH-018 use.

Hair analysis for THCA-A, THC and CBN after passive in vivo exposure to marijuana smoke

Condensation of marijuana smoke on the hair surface can be a source of an external contamination in hair analysis and may have serious consequences for the person under investigation. Δ9-tetrahydrocannabinolic acid A (THCA-A) is found in marijuana smoke and in hair analysis, but is not incorporated into the hair through the bloodstream. Therefore it might be a promising marker for external contamination of hair and could facilitate a more accurate interpretation of analytical results. In this study, three participants were exposed to the smoke of one joint every weekday over three weeks. Inhalation was excluded by an alternative breathing source. Hair samples were obtained up to seven weeks after the last exposure and analyzed for THCA-A, Δ9-tetrahydrocannabinol (THC) and cannabinol (CBN) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Additionally 30 hair samples from various regions of the head were obtained seven weeks after the exposure from one participant. The obtained results show that the degree of contamination depends on the hair length, with longer hair resulting in higher THC and CBN concentrations (1300 pg/mg and 530 pg/mg at the end of the exposure period) similar to the ones typically found after daily cannabis consumption. THCA-A could be detected in relatively low concentrations. Analysis of the distribution of the contamination showed that the posterior vertex region was affected most. The relatively low THCA-A concentrations in the samples suggest that most of the THCA-A found in forensic hair samples is not caused by sidestream marijuana smoke, but by other sources.

Designer benzodiazepines: A new challenge

In the February 2015 issue of World Psychiatry, Schifano et al (1) gave an overview of novel psychoactive substances and their potentially harmful effects. They highlighted that in the last couple of years the number of drugs offered via Internet shops has increased dramatically and that benzodiazepines are often used to treat intoxications with these drugs in the clinical setting. We would like to point out that designer benzodiazepines have become a rapidly growing class of drugs of abuse in their own right in the last two years. We believe that mental health professionals should be aware of this new development. The first designer benzodiazepines available online were diclazepam, flubromazepam and pyrazolam (2–4). Recently, five others became readily available (i.e., clonazolam, deschloroetizolam, flubromazolam, nifoxipam and meclonazepam), none of which has been approved for medicinal use in any country. Nearly all of these compounds have been synthesized as drug candidates by pharmaceutical companies and their syntheses, as well as basic animal testing data, are described in the literature along with many more potential successors (5). Typical formulations are tablets, capsules or blotters in various doses. Furthermore, the drugs are also offered as pure powders with prices as low as 5-10 US cents per dose. Immunochemical tests applied in clinical settings and drug rehabilitation detect most of the designer benzodiazepines with sufficient sensitivity. However, the mass spectrometric methods needed for confirmation do not regularly cover the latest designer benzodiazepines, due to lack of reference materials. Practitioners should be aware of this limitation and carefully assess seemingly “false-positive” results. Due to their high potency, compounds like clonazolam or flubromazolam can cause strong sedation and amnesia at oral doses as low as 0.5 mg. Such low doses are extremely difficult to measure for users handling bulk materials, and tablets often vary greatly in the content of the active ingredient. This can lead to unintended overdosing, and could also be of concern in drug facilitated crimes (6). Designer benzodiazepines are often taken as “self-medication” by users of stimulant and hallucinogenic drugs, leading to “upper downer cycles” (7) and risk of severe addiction in people frequenting the party scene. Persons with anxiety disorders also tend to self-medicate on these drugs if a medical prescription cannot be obtained (8). The high availability of these drugs via online vendors and the low price may facilitate development of addiction in this population. Many “classical” benzodiazepines are listed in Schedule 4 of the 1971 United Nations Convention. They are also in Schedule IV of the U.S. Controlled Substances Act, but it is unclear if designer benzodiazepines are covered by the Controlled Substances Analogue Enforcement Act, 1986. Similar legal problems exist in most other countries in the world, making it difficult to reduce availability of these dangerous new drugs.

Characterization of the four designer benzodiazepines clonazolam, deschloroetizolam, flubromazolam, and meclonazepam, and identification of their in vitro metabolites

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.

Phase I metabolism of the highly potent synthetic cannabinoid MDMB-CHMICA and detection in human urine samples

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 CB1 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

Finding cannabinoids in hair does not prove cannabis consumption.

Hair analysis for cannabinoids is extensively applied in workplace drug testing and in child protection cases, although valid data on incorporation of the main analytical targets, ∆9-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-THC (THC-COOH), into human hair is widely missing. Furthermore, ∆9-tetrahydrocannabinolic acid A (THCA-A), the biogenetic precursor of THC, is found in the hair of persons who solely handled cannabis material. In the light of the serious consequences of positive test results the mechanisms of drug incorporation into hair urgently need scientific evaluation. Here we show that neither THC nor THCA-A are incorporated into human hair in relevant amounts after systemic uptake. THC-COOH, which is considered an incontestable proof of THC uptake according to the current scientific doctrine, was found in hair, but was also present in older hair segments, which already grew before the oral THC intake and in sebum/sweat samples. Our studies show that all three cannabinoids can be present in hair of non-consuming individuals because of transfer through cannabis consumers, via their hands, their sebum/sweat, or cannabis smoke. This is of concern for e.g. child-custody cases as cannabinoid findings in a child’s hair may be caused by close contact to cannabis consumers rather than by inhalation of side-stream smoke.

Metabolites of synthetic cannabinoids in hair—proof of consumption or false friends for interpretation?

AbstractThe 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. Graphical AbstractDegradation processes leading to artefacts identical with main metabolites of synthetic cannabinoids

Stability of 11 prevalent synthetic cannabinoids in authentic neat oral fluid samples: glass versus polypropylene containers at different temperatures.

Although synthetic cannabinoids have been intensively investigated in recent years and oral fluid testing is becoming increasingly popular in suspected driving under the influence of drugs cases, only scarce data on their stability in authentic neat oral fluid (nOF) samples are yet available. However, especially for these new psychoactive drugs, investigations focusing on stability issues are necessary as inappropriate storage conditions may lead to considerable analytical problems. Since it has been shown for Δ(9) -tetrahydrocannabinol that adsorption to plastic surfaces may lead to considerable drug loss, we aimed to evaluate whether adsorption also has to be taken into account for synthetic cannabinoids in nOF samples. In this paper, the results of investigations on the recovery of 11 prevalent synthetic cannabinoids from authentic nOF samples stored over 72 h in RapidEASE (high quality borosilicate glass) and Sciteck Saliva Split Collector (polypropylene) tubes at 4 and 25 °C are presented. Our findings clearly demonstrate that lipophilic synthetic cannabinoids present in nOF samples adsorb to the surface of polypropylene containers when stored at room temperature, leading to considerable drug loss. Hence, when using polypropylene tubes, samples should be shipped cooled in order to avoid a substantial decrease of the analyte concentration during transportation.

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

Abstract 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.

Hair analysis for Δ 9 -tetrahydrocannabinolic acid A (THCA-A) and Δ 9 -tetrahydrocannabinol (THC) after handling cannabis plant material

A previous study has shown that Δ(9) -tetrahydrocannabinolic acid A (THCA-A), the non-psychoactive precursor of Δ(9) -tetrahydrocannabinol (THC) in the cannabis plant does not get incorporated in relevant amounts into the hair through the bloodstream after repeated oral intake. However, THCA-A can be measured in forensic hair samples in concentrations often exceeding the detected THC concentrations. To investigate whether the handling of cannabis plant material prior to consumption is a contributing factor for THC-positive hair results and also the source for THCA-A findings in hair, a study comprising ten participants was conducted. In this study, the participants rolled a marijuana joint on five consecutive days and hair samples of each participant were obtained. Urine samples were taken to exclude cannabis consumption prior to and during the study. THCA-A and THC could be detected in the hair samples from all participants taken at the end of the exposure period (concentration range: 15-1800 pg/mg for THCA-A and < 10-93 pg/mg for THC). Four weeks after the first exposure, THCA-A could still be detected in the hair samples of nine participants (concentration range: 4-57 pg/mg). Furthermore, THC could be detected in the hair samples of five participants (concentration range: < 10-17 pg/mg). Based on these results, it can be concluded that at least parts of the THC as well as the major part of THCA-A found in routine hair analysis derives from external contamination caused by direct transfer through contaminated fingers. This finding is of particular interest in interpreting THC-positive hair results of children or partners of cannabis users, where such a transfer can occur due to close body contact. Analytical findings may be wrongly interpreted as a proof of consumption or at least passive exposure to cannabis smoke. Such misinterpretation could lead to severe consequences for the people concerned.