Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantitation of synthetic cannabinoids of the aminoalkylindole type and methanandamide in serum and its application to forensic samples

After the discovery of synthetic cannabimimetic substances in 'Spice'-like herbal mixtures marketed as 'incense' or 'plant fertilizer' the active compounds have been declared as controlled substances in several European countries. As expected, a monitoring of new herbal mixtures which continue to appear on the market revealed that shortly after control measures have been taken by legal authorities, other compounds were added to existing mixtures and to new products. Several compounds of the aminoalkylindole type have been detected so far in herbal mixtures but still their consumption cannot be detected by commonly used drug-screening procedures, encouraging drug users to substitute cannabis with those products. There is a increasing demand on the part of police authorities, hospitals and psychiatrists for detection and quantification of synthetic cannabinoids in biological samples originating from psychiatric inpatients, emergency units or assessment of fitness to drive. Therefore, a liquid chromatography-tandem mass spectrometry method after liquid-liquid extraction for the quantitation of JWH-015, JWH-018, JWH-073, JWH-081, JWH 200, JWH-250, WIN 55,212-2 and methanandamide and the detection of JWH-019 and JWH-020 in human serum has been developed and fully validated according to guidelines for forensic toxicological analyses. The method was successfully applied to 101 serum samples from 80 subjects provided by hospitals, detoxification and therapy centers, forensic psychiatric centers and police authorities. Fifty-seven samples or 56.4% were found positive for at least one aminoalkylindole. JWH-019, JWH-020, JWH-200, WIN 55,212-2 and methanandamide were not detected in any of the analyzed samples.

Chromatographic analysis and survey studies to evaluate the emerging drugs of synthetic cannabinoids in Scotland and Saudi Arabia

Synthetic cannabinoid receptor agonists or more commonly known as synthetic cannabinoids (SCs) were originally created to obtain the medicinal value of THC but they are an emerging social problem. SCs are mostly produced coated on herbal materials or in powder form and marketed under a variety of brand names, e.g. “Spice”, “K2”. Despite many SCs becoming controlled under drug legislation, many of them remain legal in some countries around the world. In Scotland, SCs are controlled under the Misuse of Drugs Act 1971 and Psychoactive Substances Act 2016 that only cover a few early SCs. In Saudi Arabia, even fewer are controlled. The picture of the SCs-problem in Scotland is vague due to insufficient prevalence data, particularly that using biological samples. Whilst there is evidence of increasing use of SCs throughout the world, in Saudi Arabia, there is currently no data regarding the use of products containing SCs among Saudi people. Several studies indicate that SCs may cause serious toxicity and impairment to health therefore it is important to understand the scale of use within society. A simple and sensitive method was developed for the simultaneous analysis of 10 parent SCs (JWH-018, JWH-073, JWH-250, JWH-200, AM-1248, UR-144, A-796260, AB-FUBINACA, 5F-AKB-48 and 5F-PB-22) in whole blood and 8 corresponding metabolites (JWH-018 4-OH pentyl, JWH-073 3-OH butyl, JWH-250 4-OH pentyl, AM-2201 4-OH pentyl, JWH-122 5-OH pentyl, JWH-210 5-OH pentyl, 5F-AKB-48 (N-4 OH pentyl), 5F-PB-22 3-carboxyindole)in urine using LLE and LC-MS/MS. The method was validated according to the standard practices for method validation in forensic toxicology (SWGTOX, May 2013). All analytes gave acceptable precision, linearity and recovery for analysing blood and urine samples. The method was applied to 1,496 biological samples, a mixture of whole blood and urine. Blood and/or urine samples were analysed from 114 patients presenting at Accident and Emergency in Glasgow Royal Infirmary, in spring 2014 and JuneDecember 2015. 5F-AKB-48, 5F-PB-22 and MDMB-CHMICA were detected in 9, 7 and 9 cases respectively. 904 urine samples from individuals admitted to/liberated from Scottish prisons over November 2013 were tested for the presence of SCs. 5F-AKB-48 (N-4 OH pentyl) was detected in 10 cases and 5F-PB-22 3-carboxyindole in 3 cases. Blood and urine samples from two post-mortem cases in Scotland with suspected ingestion of SCs were analysed. Both cases were confirmed positive for 5F-AKB-48. A total of 463 urine samples were collected from personnel who presented to the Security Forces Hospital in Ryiadh for workplace drug testing as a requirement for their job during July 2014. The results of the analysis found 2 samples to be positive for 5F-PB-22 3carboxyindole. A further study in Saudi Arabia using a questionnaire was carried out among 3 subpopulations: medical professionals, members of the public in and around smoking cafes and known drug users. With regards to general awareness of Spice products, 16%, 11% and 22% of those participants of medical professionals, members of the public in and around smoking cafes and known drug users, respectively, were aware of the existence of SCs or Spice products. The respondents had an overall average of 4.5% who had a friend who used these Spice products. It is clear from the results obtained in both blood and urine testing and surveys that SCs are being used in both Scotland and Saudi Arabia. The extent of their use is not clear and the data presented here is an initial look into their prevalence. Blood and urine findings suggest changing trends in SC use, moving away from JWH and AM SCs to the newer 5F-AKB-48, 5-F-PB-22 and MDMBCHMICA compounds worldwide. In both countries 5F-PB-22 was detected. These findings clarify how the SCs phenomenon is a worldwide problem and how the information of every country regarding what SCs are seized can help and is not specific for that country. The analytes included in the method were selected due to their apparent availability in both countries, however it is possible that some newer analytes have been used and these would not have been detected. For this reason it is important that methods for testing SCs are updated regularly and evolve with the ever-changing availability of these drugs worldwide. In addition, there is little published literature regarding the concentrations of these drugs found in blood and urine samples and this work goes some way towards understanding these.

A systematic review of adverse events arising from the use of synthetic cannabinoids and their associated treatment

Saccharomyces cerevisiae as a toxicological model to study synthetic cannabinoids and its pyrolysis products

Poster presented at the 7th European Academy of Forensic Science Conference. Prague, 6-11 September 2015

Cannabinoids impact on pregnancy: effects in trophoblast cells

Cannabinoids (CBs) can be classified as: phytocannabinoids, the constituents of the Cannabis sativa plant; synthetic cannabinoids lab-synthesized and the endocannabinoids that are endogenous lipid mediators. Cannabinoid compounds activate cannabinoid receptors – CB1 and CB2. The most prevalent psychoactive phytocannabinoid is Δ9tetrahydrocannabinol (THC), but more than 60 different CBs were already identified in the plant. The best characterized endocannabinoids (eCBs) are anandamide (AEA) and 2arachidonoylglycerol (2-AG), that are involved in several physiological processes including synaptic plasticity, pain modulation, energy homeostasis and reproduction. On the other hand, some synthetic cannabinoids that were initially designed for medical research, are now used as drugs of abuse. During the period of placental development, highly dynamic processes of remodeling occur, involving proliferation, apoptosis, differentiation and invasion of trophoblasts. It is known that a tight control of eCBs levels is required for normal pregnancy progression and that eCBs are involved in trophoblast cells turnover. Therefore, by sharing activation of the same receptors, exposure to exocannabinoids either by recreational or medicinal use may lead to alterations in the eCBs levels and in the endocannabinoid system homeostasis In this work, it was studied the impact of CBs in BeWo trophoblastic cells and in primary cultures of human cytotrophoblasts. Cells were treated for 24 hours with different concentrations of THC, the synthetic cannabinoid WIN‐55,212 (WIN) and 2-AG. Treatment with THC did not affect BeWo cells viability while WIN and 2-AG caused a dose-dependent viability loss. Morphological studies together with biochemical markers indicate that 2-AG is able to induce apoptosis in cytotrophoblasts. On the other hand, morphological studies after acridine orange staining suggest that autophagy may take part in WIN-induced loss of cell viability. All cannabinoids caused a decrease in mitochondrial membrane potential (Δψm) but only 2-AG led to ROS/RNS generation, though no changes in glutathione levels were observed. In addition, ER-stress may be involved in the 2-AG induced-oxidative stress, as preliminary results point to an increase in CCAAT-enhancer-binding protein homologous protein (CHOP) expression. Besides the decrease in cell viability, alterations in cell cycle progression were observed. WIN treatment induced a cell cycle arrest in G0/G1 phase, whereas 2-AG induced a cell cycle arrest in G2/M phase. Here it is reinforced the relevance of cannabinoid signaling in fundamental processes of cell proliferation and cell death in trophoblast cells. Since cannabis-based drugs are the most consumed illicit drugs worldwide and some of the most consumed recreational drugs by pregnant women, this study may contribute to the understanding of the impact of such substances in human reproduction.

Effets motivationnels des cannabinoïdes dans un modèle animal de la schizophrénie

Depuis quelques décennies, la consommation de cannabis et son usage thérapeutique sont le sujet de nombreux débats. Le cannabis est la drogue illicite la plus consommée au monde et cette consommation se trouve dix fois plus élevée chez les patients atteints de schizophrénie que dans la population générale. L’hypothèse d’une automédication initialement proposée afin d’expliquer la consommation élevée de cannabis chez les patients atteints de schizophrénie est maintenant remise en question. En effet, les rapports indiquant une aggravation des symptômes plutôt qu’une amélioration suite à une consommation à long terme sont de plus en plus nombreux. Sachant que le cannabis peut induire des effets soit plaisants soit aversifs, la question se pose à savoir si une prédominance de la valence motivationnelle positive ou une diminution de la valence négative du cannabis peut expliquer la consommation élevée parmi les individus ayant un diagnostic de schizophrénie? Bien qu’un grand nombre de recherches pré-cliniques aient été menées chez l’animal normal pour évaluer l’effet motivationnel du Δ9-tétrahydrocannabinol (THC) et autres cannabinoïdes synthétiques, aucune n’a abordé cette problématique dans un modèle animal de la schizophrénie. Cette lacune nous a donc amené à étudier la valence motivationnelle du THC et de l’agoniste cannabinoïde WIN55,212-2 (WIN) dans un modèle animal de la schizophrénie: la lésion néonatale de l’hippocampe ventral (NVHL). Dans le premier article, nous présentons les résultats de quatre expériences. Une première avait pour objectif de déterminer si la procédure expérimentale que nous avons utilisée permettait de reproduire des signes distinctifs du modèle animal de la schizophrénie. Par la suite, nous avons évalué i) l’effet d’une dose de WIN sur l’activité locomotrice spontanée et ii) la valence motivationnelle du THC (0.5 mg/kg, i.p) et du WIN (1 mg/kg, i.p) chez les rats adolescents (jour post-natal 28-40, PD28-40) et adultes (PD56) au moyen du paradigme de préférence de place conditionnée (PPC). Tel qu’attendu, la réponse locomotrice à l’amphétamine (0.75 et 1.5 mg/kg) chez les rats NVHL adultes était supérieure à celle des rats contrôles (test distinctif du modèle). Le THC a induit une tendance aversive chez les rats contrôles adultes. Enfin, le WIN a stimulé l’activité locomotrice et induit une aversion significative chez les rats adultes NVHL. Dans un deuxième article, nous avons évalué la valence motivationnelle du THC (0.5 mg/kg), du WIN (1 et 3 mg/kg) et l’effet de l’amphétamine au moyen du paradigme d’autostimulation électrique intracérébrale (ASI). Les résultats montrent que : i) l’effet amplificateur de l’amphétamine sur l’ASI était de plus courte durée chez les rats NVHL; ii) le THC produit une légère atténuation de la récompense chez les rats contrôles tandis que le WIN a produit une atténuation plus prononcée de la récompense chez les rats NVHL, un effet qui a été bloqué par l’antagoniste aux récepteurs CB1, le AM251 (3 mg/kg). Pour la première fois les résultats suggèrent une altération du système endocannabinoïde dans un modèle animal de la schizophrénie. Ils indiquent qu’une exposition aigüe conduit à une prédominance de la valence négative. Bien qu’en apparente contradiction avec les études cliniques, ces résultats soulignent l’importance du contexte socio-environnemental pour expliquer les effets du cannabis chez les patients. De plus ils encouragent les futures études à évaluer cette valence sur un modèle d’exposition chronique.

Bioanalysis of new designer drugs

Since the late 1990s the illicit drug market has undergone considerable change: along with the traditional drugs of abuse that still dominate, more than 100 psychotropic substances designed to bypass controlled substances legislation have appeared and led to intoxications and fatalities. Starting from the huge class of phenylalkylamines, containing many subgroups, the spectrum of structures has grown from tryptamines, piperazines, phenylcyclohexyl derivates and pyrrolidinophenones to synthetic cannabinoids and the first synthetic cocaine. Due to the small prevalence and high number of unknown substances, the detection of new designer drugs is a challenge for clinical and forensic toxicologists. Standard screening procedures might fail because a recently discovered or yet unknown substance has not been incorporated in the library used. Nevertheless, many metabolism studies, case reports, screening methods and substance-profiling papers concentrating on single compounds have been published. This review provides an overview of the developed bioanalytical and analytical methods, the matrices used, sample-preparation procedures, concentration of analytes in case of intoxication and also gives a résumé of immunoassay experiences. Additionally, six screening methods for biological matrices with a larger spectrum of analytes are described in more detail.

Chemistry and Analysis of Phytocannabinoids and Other Cannabis Constituents

The Cannabis plant and its products consist of an enormous variety of chemicals. Some of the 483 compounds identified are unique to Cannabis, for example, the more than 60 cannabinoids, whereas the terpenes, with about 140 members forming the most abundant class, are widespread in the plant kingdom. The term “cannabinoids” [note: “ ” represents a group of C21 terpenophenolic compounds found until now uniquely in Cannabis sativa L. (1). As a consequence of the development of synthetic cannabinoids (e.g., nabilone [2], HU-211 [dexanabinol; ref. (3), or ajulemic acid [CT-3; ref. 4]) and the discovery of the chemically different endogenous cannabinoid receptor ligands (“endocannabinoids,” e.g., anandamide, 2-arachidonoylglycerol) (5,6), the term ’“phytocannabinoids’” was proposed for these particular Cannabis constituents (7).

Toxicological impact of JWH-018 and its phase I metabolite N-(3-hydroxypentyl) on human cell lines

"The emergence and abuse of synthetic cannabinoids has been increasing as an alternative to cannabis, mainly among youth. As their appearance on the drug market has been recent, the pharmacological and toxicological profiles of these psychoactive substances are poorly understood. Current studies suggest that they have stronger effects compared to their natural alternatives and their metabolites retain affinity towards CB1 receptors in CNS. Since studies on its toxicological properties are scarce, the effects of the drug in human derived cell lines were investigated. The present study was designed to explore the toxicological impact of parent drug versus phase I metabolites of synthetic cannabinoids on human cells with and without CB1 receptor. The human cell line of neuroblastoma SH-SY5Y and human kidney cell line HEK-293T were exposed to JWH-018 and to its N-(3-hydroxypentyl) metabolite. Cell toxicity was evaluated using the MTT and LDH assay. Additionally, a dual staining methodology with fluorescent Annexin V-FITC and propidium iodide was performed to address the question of whether JWH-018 N-(3-hydroxypentyl) metabolite is inducing cell death through apoptosis or necrosis, in HEK293T and SH-SY5Y cell lines. The obtained results show that JWH-018 does not cause a statistically significant decrease in cell viability, in contrast to its N-(3-hydroxypentyl) metabolite, which at ≥25μM causes a significant decrease in cell viability. Both cell lines are affected by JWH-018 metabolite. Our results point to higher toxicity of JWH-018 metabolite when compared to its parent drug, suggesting a non-CB1 receptor mediated toxicological mechanism. Comparing the results from Annexin V/PI with MTT and LDH assays of SH-SY5Y and HEK293T in the presence of the synthetic cannabinoid metabolite, emerges the picture that cellular viability decreases and associated death is occurring through necrosis."

Thermal decomposition of the synthetic hydrotalcite iowaite

The thermal stability and thermal decomposition pathways for synthetic iowaite have been determined using thermogravimetry in conjunction with evolved gas mass spectrometry. Chemical analysis showed the formula of the synthesised iowaite to be Mg6.27Fe1.73(Cl)1.07(OH)16(CO3)0.336.1H2O and X-ray diffraction confirms the layered structure. Dehydration of the iowaite occurred at 35 and 79°C. Dehydroxylation occurred at 254 and 291°C. Both steps were associated with the loss of CO2. Hydrogen chloride gas was evolved in two steps at 368 and 434°C. The products of the thermal decomposition were MgO and a spinel MgFe2O4. Experimentally it was found to be difficult to eliminate CO2 from inclusion in the interlayer during the synthesis of the iowaite compound and in this way the synthesised iowaite resembled the natural mineral.

The Behavior of Hydroxyl Units of Synthetic Goethite and its Dehydroxylated Product Hematite

The behavior of the hydroxyl units of synthetic goethite and its dehydroxylated product hematite was characterized using a combination of Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) during the thermal transformation over a temperature range of 180-270 degrees C. Hematite was detected at temperatures above 200 degrees C by XRD while goethite was not observed above 230 degrees C. Five intense OH vibrations at 3212-3194, 1687-1674, 1643-1640, 888-884 and 800-798 cm(-1), and a H2O vibration at 3450-3445 cm(-1) were observed for goethite. The intensity of hydroxyl stretching and bending vibrations decreased with the extent of dehydroxylation of goethite. Infrared absorption bands clearly show the phase transformation between goethite and hematite: in particular. the migration of excess hydroxyl units from goethite to hematite. Two bands at 536-533 and 454-452 cm(-1) are the low wavenumber vibrations of Fe-O in the hematite structure. Band component analysis data of FTIR spectra support the fact that the hydroxyl units mainly affect the a plane in goethite and the equivalent c plane in hematite.

An Improved Synthetic Route to the Potent Angiogenesis Inhibitor Benzyl Manα(1→3)-Manα(1→3)-Manα(1→3)-Manα(1→2)-Man Hexadecasulfate

An improved synthetic route to α(1→3)/α(1→2)-linked mannooligosaccharides has been developed and applied to a more efficient preparation of the potent anti-angiogenic sulfated pentasaccharide, benzyl Manα(1→3)-Manα(1→3)-Manα(1→3)-Manα(1→2)-Man hexadecasulfate, using only two monosaccharide building blocks. Of particular note are improvements in the preparation of both building blocks and a simpler, final deprotection strategy. The route also provides common intermediates for the introduction of aglycones other than benzyl, either at the building block stage or after oligosaccharide assembly. The anti-angiogenic activity of the synthesized target compound was confirmed via the rat aortic assay.

Simultaneous determination of three synthetic glucocorticoids by differential pulse stripping voltammetry with the aid of chemometrics

A novel voltammetric method for simultaneous determination of the glucocorticoid residues prednisone, prednisolone, and dexamethasone was developed. All three compounds were reduced at a mercury electrode in a Britton-Robinson buffer (pH 3.78), and well-defined voltammetric waves were observed. However, the voltammograms of these three compounds overlapped seriously and showed nonlinear character, and thus, it was difficult to analyze the compounds individually in their mixtures. In this work, two chemometrics methods, principal component regression (PCR) and partial least squares (PLS), were applied to resolve the overlapped voltammograms, and the calibration models were established for simultaneous determination of these compounds. Under the optimum experimental conditions, the limits of detection (LOD) were 5.6, 8.3, and 16.8 µg l-1 for prednisone, prednisolone, and dexamethasone, respectively. The proposed method was also applied for the determination of these glucocorticoid residues in the rabbit plasma and human urine samples with satisfactory results.

Raman spectroscopic study of the uranyl carbonate mineral čejkaite and its comparison with synthetic trigonal Na4[UO2(CO3)3]

Raman and infrared spectroscopies were used to characterise two samples of triclinic ejkaite Na4[UO2(CO3)3] and its synthetic trigonal analogue. The v3 (UO2)2+ mode is not Raman active, whereas both the v3 and v1 (UO2)2+ modes are infrared active. U--O bond lengths in uranyls were calculated from the spectra obtained and compared with bond lengths derived from crystal structure analyses. From the higher number of bands related to the uranyl and carbonate vibrations, the presence of symmetrically distinct (UO2)2+ and (CO3)2- units in both structures is proposed.