Rainer Lindigkeit

Identification and quantification of synthetic cannabinoids in 'spice-like' herbal mixtures: a snapshot of the German situation in the autumn of 2012.

Synthetic compounds mimicking cannabis-like effects are a recent trend. Currently, these so-called synthetic cannabinoids are the largest and fastest growing class of newly appearing designer drugs. Many national authorities are continuously adapting their regulations to keep pace with the permanently changing variety of compounds. We have analyzed eight herbal smoking blends containing synthetic cannabinoids. Altogether, nine compounds could be identified, namely AM-2201, AM-2201-pMe (MAM-2201), AM-1220, AM-1220-azepane, UR-144, XLR-11, JWH-122-pentenyl, AM-2232, and STS-135. Newly appearing compounds were isolated by column chromatography and their structures elucidated by 1D- and 2D-nuclear magnetic resonance (NMR) experiments. In addition, the compounds were investigated by electron ionization-mass spectrometry (EI-MS) and electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to complete the physicochemical dataset. Based on the purified compounds a universal gas chromatography-mass spectrometry (GC-MS) method was developed for the identification and quantification of these compounds in commercial smoking blends. By applying this method, up to five different compounds could be found in such products showing total concentrations from 72 to 303 mg/g smoking blend while individual compounds ranged from 0.4 to 303 mg/g. (1)H NMR spectra of the chiral compounds AM-1220 and its azepane-isomer recorded in the presence of 1 equivalent of (R)-(+)-α-methoxy-α-trifluoromethylphenylacetic acid (MTPA, Moshers acid) showed them to be racemic mixtures.

Synthetic cannabinoids in “spice-like” herbal blends: First appearance of JWH-307 and recurrence of JWH-018 on the German market

Herbal smoking blends, available on the German market were analyzed and several known synthetic cannabinoids were identified (JWH-122 and JWH-018). In addition, we isolated a new active ingredient by silica gel column chromatography and elucidated the structure by nuclear magnetic resonance (NMR) methods. The compound was identified as JWH-307, a synthetic cannabinoid of the phenyl-pyrrole subclass with known in vitro binding affinities for cannabinoid receptors. To date, this is the first appearance of this subclass of cannabimimetics in such products. JWH-307 has been further characterized by gas chromatography accurate mass spectrometry (GC-HRMS), electrospray tandem mass spectrometry (ESI-MS/MS), ultraviolet (UV) and infrared (IR) spectroscopy. JWH-018 was among the first compounds banned by many countries world-wide including Germany. The identification of JWH-018 was striking, since this is the first report where JWH-018 recurred on the German market thus violating existing laws. A generic method was established to quantify synthetic cannabinoids in herbal smoking blends. Quantification was achieved using an isotopically labeled standard (JWH-018-D(3)). JWH-018 was found at a level of 150 mg/g while JWH-122 and JWH-307 occurred as a mixture at a total level of 232 mg/g.

Detoxification of pyrrolizidine alkaloids by the harvestman Mitopus morio (Phalangidae) a predator of alkaloid defended leaf beetles

Summary.The harvestman Mitopus morio (Phalangidae) is a generalist predator. It is known to prey on larvae of the chrysomelid leaf beetle Oreina cacaliae defended by plant acquired pyrrolizidine alkaloids (PAs). Tracer feeding experiments were performed to determine how harvestmen tolerate protoxic PAs. Minced meat containing either [14C]senecionine or [14C]senecionine N-oxide was fed to M. morio and subsequently feces and bodies were analyzed. Labeled alkaloid N-oxide remained stable and was eliminated almost unaltered with the feces; only 10% was recovered as tertiary PA. In contrast, approximately 80% of labeled tertiary alkaloid (senecionine) ingested with the diet was N-oxidized and eliminated; the remaining 20% consisted of unchanged senecionine and a polar metabolite of unknown structure. Harvestmen process their diet by excreting digestive juice, indicated by bleaching of the meat color. Analysis of the processed diet revealed some N-oxidation of [14C]senecionine, suggesting the gut as the site of Noxidation. Analysis of the bodies of harvestmen 80 hours after the tracer feeding pulse revealed only trace amounts of the polar metabolite. Neither senecionine nor its N-oxide could be detected in the body extracts. The results are discussed in relation to the strategies of PA adapted insects to avoid accumulation of tertiary PAs in living tissues.

Incidence of Pyrrolizidine Alkaloids in Herbal Medicines from German Retail Markets: Risk Assessments and Implications to Consumers

The occurrence of potentially toxic pyrrolizidine alkaloids (PAs) in herbal medicines (HMs) is currently intensely being discussed in Europe. Pyrrolizidine alkaloids, particularly the 1,2‐unsaturated PAs, are undesired compounds in HMs due to their potential hepatotoxic and carcinogenic properties. In this study, 98 widely patronized HMs from six popular German retail supermarkets/drugstores, as well as from pharmacies, were analyzed by high‐performance liquid chromatography–electrospray ionization–tandem mass spectrometry for the presence of PAs. The results showed that about 63% of the HMs were PA positive, whereas the average PA concentration of the samples was 201 μg/kg, the highest concentration of PAs (3270 μg/kg) was attributed to a product that was purchased from the pharmacy and contained Hypericum perforatum L. (St. Johns Wort) as an active ingredient. In addition, H. perforatum‐containing products were frequently contaminated with PAs from Echium spp., while both Cynara cardunculus L. products and fixed‐combination products of Gentiana lutea L., Rumex acetosa L., Verbena officinalis L., Sambucus nigra L., and Primula veris L. products were commonly contaminated with PAs of Senecio spp. The study showed that H. perforatum, C. cardunculus, Urtica dioica L., and fixed‐combination products were frequently contaminated with PA levels above the recommended values of both the German and European Medicines Agencies. Copyright

Determination of cross-reactivity of poly- and monoclonal antibodies for synthetic cannabinoids by direct SPR and ELISA

One of the main reasons for the rise in popularity of synthetic cannabinoids (SCs) is their ability to remain unrecognized in conventional drug screenings. Due to their structural diversity, caused by the constant introduction of new substances to circumvent legal regulation, antibodies with a wide range of cross-reactivity are necessary for the establishment of a reliable immunological based drug test. Therefore, high-quality binding data are needed to select promising antibody candidates for further development. In this study, we carried out a direct surface plasmon resonance (SPR) method and evaluated its suitability for the characterization of antibody-SC interactions. The cross-reactivity of 22 SCs with three polyclonal antibodies, raised against JWH-018 haptens with different attachment positions of the linker, and two commercial available monoclonal antibodies were determined. These results were compared with the commonly used competitive enzyme-linked immunosorbent assay (ELISA). It could be demonstrated, that direct SPR and competitive ELISA show comparable specificity results for the majority of the measured compounds. However, the reduced manual labor, the real-time analysis and the high information content about the binding events of SPR compared to ELISA, showed that SPR is a valuable tool during the development of antibodies against synthetic cannabinoids, currently the largest group of new psychoactive substances.