Identification and quantification of synthetic cannabinoids in 'spice-like' herbal mixtures: Update of the German situation in 2018.

In June 2018, 15 'Spice-like' herbal products from German language internet shops were analyzed. In total, three different synthetic cannabinoids (SCs) were identified by gas chromatography-mass spectrometry (GC-MS). Two of the active ingredients were identified as the recently described 5F-ADB and Cumyl-PeGaClone. The third compound was identified as the so far unknown SC 5F-Cumyl-PeGaClone. 5F-Cumyl-PeGaClone was subject to an in-depth characterization by nuclear magnetic resonance spectroscopy (NMR), electron ionization mass spectrometry (EI-MS), electrospray ionization tandem mass spectrometry (ESI-MS/MS), infrared and uItraviolet-visible spectroscopy (IR and UV/Vis). In addition, all SCs in all products were quantified by a GC-MS method using JWH-018 as internal standard and corresponding response factors. While Cumyl-PeGaClone and 5F-ADB were detected in one, respectively two products, the newly identified 5F-Cumyl-PeGaClone was detected as the only active ingredient in the remaining twelve products. The SC content ranged from 14.7 to 76.2mg/g (average: 32.1mg/g).

Identification and quantification of synthetic cannabinoids in 'spice-like' herbal mixtures: Update of the German situation in early 2017.

In February 2017, eleven "Spice-like" products (14 individual packages including replicates) from German language internet shops were analyzed. In total, three different synthetic cannabinoids (SCs) were identified by gas-chromatography-mass spectrometry (GC-MS), namely MDMB-CHMICA and two, so far only partially described compounds, 5F-Cumyl-P7AICA and Cumyl-PeGACLONE. All analyzed products contained only one synthetic cannabinoid as active ingredient. 5F-Cumyl-P7AICA and Cumyl-PeGACLONE were subject to an in-depth characterization by nuclear magnetic resonance spectroscopy (NMR), electron ionization mass spectrometry (EI-MS), electrospray ionization tandem mass spectrometry (ESI-MS/MS), infrared and ultraviolet-visible spectroscopy (IR and UV/Vis). Cumyl-PeGACLONE shows a rather unexpected structure compared to conventional SCs of the past. Hence a global minima calculation was conducted to demonstrate structural similarity of Cumyl-PeGACLONE to JWH-018, a classical SC. In addition, all SCs were quantified by a GC-MS method using JWH-018 as internal standard and corresponding response factors. While MDMB-CHMICA was detected in six out of 14 tested products (ranging from 6 to 20mg/g; average 10mg/g), 5F-Cumyl-P7AICA and Cumyl-PeGACLONE were detected in three (109-153mg/g; average 131mg/g) and five products (15-74mg/g; average 39mg/g), respectively.

Identification and quantification of synthetic cannabinoids in "spice-like" herbal mixtures: Update of the German situation for the spring of 2016.

In February 2016, nine "spice-like" products from German language internet shops were analyzed. In total, eight different synthetic cannabinoids were identified by gas chromatography-mass spectrometry (GC-MS), namely THJ-018, THJ-2201, MAB-CHMINACA, 5F-ADB, 5Cl-AKB48 (syn.: 5C-AKB48), 4-pentenyl-AKB48, MDMB-CHMICA and 5F-AB-PINACA. For the majority of products only one synthetic cannabinoid was identified as the active ingredient, while two products contained 2 and 5 compounds, respectively. For some of the identified cannabinoids (MAB-CHMINACA, 5Cl-AKB48 and 4-pentenyl-AKB48) no or only insufficient physico-chemical data were available in literature. To our knowledge 5Cl-AKB48 and 4-pentenyl-AKB48 were found for the first time in commercially available products, hence an in-depth characterization of these compounds by NMR, EI-MS, ESI-MS/MS, IR- and UV spectroscopy was conducted. In addition, all synthetic cannabinoids were quantified by a GC-MS method using JWH-018 as internal standard and the corresponding response factors to calculate the total amount of all synthetic cannabinoids in the commercial smoking mixtures. The content of synthetic cannabinoids in the investigated products ranged from 23 to 120mg/g (average: 57mg/g), while individual compounds ranged from 1 to 120mg/g.

Molecular Cloning and Characterization of a Xanthone Prenyltransferase from Hypericum calycinum Cell Cultures.

In plants, prenylation of metabolites is widely distributed to generate compounds with efficient defense potential and distinct pharmacological activities profitable to human health. Prenylated compounds are formed by members of the prenyltransferase (PT) superfamily, which catalyze the addition of prenyl moieties to a variety of acceptor molecules. Cell cultures of Hypericum calycinum respond to elicitor treatment with the accumulation of the prenylated xanthone hyperxanthone E. A cDNA encoding a membrane-bound PT (HcPT) was isolated from a subtracted cDNA library and transcript preparations of H. calycinum. An increase in the HcPT transcript level preceded hyperxanthone E accumulation in cell cultures of H. calycinum treated with elicitor. The HcPT cDNA was functionally characterized by expression in baculovirus-infected insect cells. The recombinant enzyme catalyzed biosynthesis of 1,3,6,7-tetrahydroxy-8-prenylxanthone through regiospecific C-8 prenylation of 1,3,6,7-tetrahydroxyxanthone, indicating its involvement in hyperxanthone E formation. The enzymatic product shared significant structural features with the previously reported cholinesterase inhibitor γ-mangostin. Thus, our findings may offer a chance for semisynthesis of new active agents to be involved in the treatment of Alzheimer's disease.

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, Mosher's acid) showed them to be racemic mixtures.

Cinnamate:CoA ligase initiates the biosynthesis of a benzoate-derived xanthone phytoalexin in Hypericum calycinum cell cultures.

Although a number of plant natural products are derived from benzoic acid, the biosynthesis of this structurally simple precursor is poorly understood. Hypericum calycinum cell cultures accumulate a benzoic acid-derived xanthone phytoalexin, hyperxanthone E, in response to elicitor treatment. Using a subtracted complementary DNA (cDNA) library and sequence information about conserved coenzyme A (CoA) ligase motifs, a cDNA encoding cinnamate:CoA ligase (CNL) was isolated. This enzyme channels metabolic flux from the general phenylpropanoid pathway into benzenoid metabolism. HcCNL preferred cinnamic acid as a substrate but failed to activate benzoic acid. Enzyme activity was strictly dependent on the presence of Mg²âº and K⁺ at optimum concentrations of 2.5 and 100 mM, respectively. Coordinated increases in the Phe ammonia-lyase and HcCNL transcript levels preceded the accumulation of hyperxanthone E in cell cultures of H. calycinum after the addition of the elicitor. HcCNL contained a carboxyl-terminal type 1 peroxisomal targeting signal made up by the tripeptide Ser-Arg-Leu, which directed an amino-terminal reporter fusion to the peroxisomes. Masking the targeting signal by carboxyl-terminal reporter fusion led to cytoplasmic localization. A phylogenetic tree consisted of two evolutionarily distinct clusters. One cluster was formed by CoA ligases related to benzenoid metabolism, including HcCNL. The other cluster comprised 4-coumarate:CoA ligases from spermatophytes, ferns, and mosses, indicating divergence of the two clades prior to the divergence of the higher plant lineages.

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.

Analysis of synthetic cannabinoids in "spice-like" herbal highs: snapshot of the German market in summer 2011.

In this study, seven commercial "spice-like" products available on the German market were analyzed. They all contained significant amounts of synthetic cannabinoids and had distinctly different compositions of these adulterants. All synthetic cannabinoids were extracted and purified by different chromatographic techniques from the respective product. The structures of all compounds were elucidated by nuclear magnetic resonance spectroscopy and further characterized by mass spectrometry (MS) and ultraviolet and infrared spectroscopy to generate a full data set of each compound. Altogether, eight compounds were identified, and one deuterium-labeled cannabinoid was used as internal standard. Four products contained only one individual compound, while three products contained mixtures of two compounds. Among the eight isolated compounds, six were already known from recent publications (JWH-081, JWH-210, JWH-122, AM2201, RCS-4, and JWH-203), but the published data were not always complete. In addition, two unknown compounds (AM2201-pMe, RCS-4-(N-Me)) were isolated. Overall, compounds from three distinct classes of synthetic cannabinoids could be identified, characterized, and compared. The MS data of the different subclasses allowed the postulation of some general key fragmentations to distinguish between these subclasses. In addition, we established a general method using an isotopically labeled internal standard (JWH-018-D(3)) to quantify synthetic cannabinoids in herbal mixtures. The total content of the synthetic cannabinoids ranged from 77.5 to 202 mg/g, while individual compounds were detected from 19.3 to 202 mg/g in these products. The spectroscopic data for all compounds mentioned here were collected and added en bloc as Electronic supplementary material to this manuscript.

Identification and characterization of JWH-122 used as new ingredient in "Spice-like" herbal incenses.

Recently, several cases across Germany were reported where teenagers were hospitalized showing severe side effects after consumption of a new "Spice-like" herbal incense called "Lava red". The active component of "Lava red", obtained from German internet shops, was isolated by silica gel column chromatography and the structure was elucidated by NMR methods. It is a known N-alkyl-3-(1-naphthoyl)indole (CAS No.: 619294-47-2) JWH-122 which was synthesized recently as model component for in vitro drug testing. The structure is related to compounds that were used two years ago (JWH-018, JWH-073) as synthetic cannabimimetics in similar incense products which are now banned as illegal drugs in Germany and several other European countries. The concentration of JWH-122 was estimated to be 82mg/g product. The isolated compound was further analyzed by different spectroscopic and mass spectrometry techniques to obtain a complete dataset of the physico-chemical properties of the molecule. The data presented here is useful for easy and fast detection of JWH-122 in trace amounts in complex matrices.