Monitoring the use of novel psychoactive substances in Australia by wastewater-based epidemiology.

Users of novel psychoactive substances (NPS) are at risk, due to limited information about the toxicity and unpredictable effects of these compounds. Wastewater-based epidemiology (WBE) has been used as a tool to provide insight into NPS use at the population level. To understand the preferences and trends of NPS use in Australia, this study involved liquid chromatography mass spectrometry analysis of wastewater collected from Australian states and territories from February 2022 to February 2023. In total, 59 different NPS were included across two complementary analytical methods and covered up to 57 wastewater catchments over the study. The NPS detected in wastewater were 25-B-NBOMe, buphedrone, 1-benzylpiperazine (BZP), 3-chloromethcathinone, N,N-dimethylpentylone (N,N-DMP), N-ethylheptedrone, N-ethylpentylone, eutylone, 4F-phenibut, 2-fluoro deschloroketamine, hydroxetamine, mephedrone, methoxetamine, methylone, mitragynine, pentylone, phenibut, para-methoxyamphetamine (PMA), alpha-pyrrolidinovalerophenone (α-PVP) and valeryl fentanyl. The detection frequency for these NPS ranged from 3 % to 100 % of the sites analysed. A noticeable decreasing trend in eutylone detection frequency and mass loads was observed whilst simultaneously N,N-DMP and pentylone increased over the study period. The emergence of some NPS in wastewater pre-dates other sources of monitoring and provides further evidence that WBE can be used as an additional early warning system for alerting potential NPS use.

Highly sensitive screening and analytical characterization of synthetic cannabinoids in nine different herbal mixtures.

The popularity of new psychoactive substances among drug users has become a public health concern worldwide. Among them, synthetic cannabinoids (SCs) represent the largest, most diversified and fastest growing group. Commonly known as 'synthetic marijuana' as an alternative to cannabis, these synthetic compounds are easily accessible via the internet and are sold as 'herbal incenses' under different brand names with no information about the chemical composition. In the present work, we aim to integrate gas chromatography-tandem mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) data as useful strategy for the identification and confirmation of synthetic cannabinoids present in nine seized herbal incenses. The analysis of all samples allowed the initial identification of 9 SCs, namely 5 napthoylindoles (JWH-018, JWH-073, JWH-122, JWH-210, MAM-2201), APINACA, XLR-11 and CP47,497-C8 and its enantiomer. JWH-018 was the most frequently detected synthetic compound (8 of 9 samples), while APINACA and XLR-11 were only identified in one herbal product. Other non-cannabinoid drugs, including oleamide, vitamin E and vitamin E acetate, have also been detected. Oleamide and vitamin E are two adulterants, frequently added to herbal products to mask the active ingredients or added as preservatives. However, to our knowledge, no analytical data about vitamin E acetate was reported in herbal products, being the first time that this compound is identified on this type of samples. The integration data obtained from the used analytical technologies proved to be useful, allowing the preliminary identification of the different SCs in the mixture. Furthermore, the examination of mass spectral fragment ions, as well as the results of both 1D and 2D NMR experiments, enabled the identification and confirmation of the molecular structure of SCs.

Direct and Fast Screening of New Psychoactive Substances Using Medical Swabs and Atmospheric Solids Analysis Probe Triple Quadrupole with Data-Dependent Acquisition.

New psychoactive substances (NPS) have become a serious public health problem, as they are continuously changing their structures and modifying their potency and effects on humans, and therefore, novel compounds are unceasingly appearing. One of the major challenges in forensic analysis, particularly related to the problem of NPS, is the development of fast screening methodologies that allow the detection of a wide variety of compounds in a single analysis. In this study, a novel application of the atmospheric solids analysis probe (ASAP) using medical swabs has been developed. The swab-ASAP was coupled to a triple quadrupole mass analyzer working under a data-dependent acquisition mode in order to perform a suspect screening of NPS in different types of samples as well as on surfaces. The compounds were automatically identified based on the observed fragmentation spectra using an in-house built MS/MS spectra library. The developed methodology was applied for the identification of psychoactive substances in research chemicals and herbal blends. The sensitivity of the method, as well as its applicability for surface analysis, was also assessed by identifying down to 1 µg of compound impregnated onto a laboratory table. Another remarkable application was the identification of cathinones and synthetic cannabinoids on the fingers of potential consumers. Interestingly, our data showed that NPS could be identified on the fingers after being in contact with the product and even after cleaning their hands by shaking off with a cloth. The methodology proposed in this paper can be applied for routine analyses of NPS in different matrix samples without the need to establish a list of target compounds prior to analysis.

Using Sesame Seed Oil to Preserve and Preconcentrate Cannabinoids for Paper Spray Mass Spectrometry.

Cannabinoids present a unique set of analytical challenges. An increasing number of states have voted to decriminalize recreational marijuana use, creating a need for new kinds of rapid testing. At the same time, synthetic compounds with activity similar to THC, termed synthetic cannabinoids, have become more prevalent and pose significant health risks. A rapid method capable of detecting both natural and synthetic cannabinoids would be useful in cases of driving under the influence of drugs, where it might not be obvious whether the suspect consumed marijuana, a synthetic cannabinoid, or both. Paper spray mass spectrometry is an ambient ionization technique which allows for the direct ionization of analyte from a biofluid spot on a piece of paper. Natural cannabinoids like THC, however, are labile and rapidly disappear from dried sample spots, making it difficult to detect them at clinically relevant levels. Presented here is a method to concentrate and preserve THC and synthetic cannabinoids in urine and oral fluid on paper for analysis by paper spray mass spectrometry. Sesame seed oil was investigated both as a means of preserving THC and as part of a technique, termed paper strip extraction, wherein urine or oral fluid is flowed through an oil spot on a strip of paper to preconcentrate cannabinoids. This technique preserved THC in dried biofluid samples for at least 27 days at room temperature; paper spray MS/MS analysis of these preserved dried spots was capable of detecting THC and synthetic cannabinoids at low ng/mL concentrations, making it suitable as a rapid screening technique. The technique was adapted to be used with a commercially available autosampler.

Biomedical analysis of New Psychoactive Substances (NPS) of natural origin.

New psychoactive substances (NPS) can be divided into two main groups: synthetic molecules and active principles of natural origin. With respect to this latter group, a wide range of alkaloids contained in plants, mainly from Asia and South America, can be included in the class of NPS of natural origin. The majority NPS of natural origin presents stimulant and/or hallucinogenic effects (e.g. Catha edulis and Ayahuasca, respectively) while few of them show sedative and relaxing properties (e.g. kratom). Few information is available in relation to the analytical identification of psychoactive principles contained in the plant material. Moreover, to our knowledge, scarce data are present in literature, about the characterization and quantification of the parent drug in biological matrices from intoxication and fatality cases. In addition, the metabolism of natural active principles has not been yet fully investigated for most of the psychoactive substances from plant material. Consequently, their identification is not frequently performed and produced metabolites are often unknown. To fill this gap, we reviewed the currently available analytical methodologies for the identification and quantification of NPS of natural origin in plant material and, whenever possible, in conventional and non-conventional biological matrices of intoxicated and dead subjects. The psychoactive principles contained in the following plants were investigated: Areca catechu, Argyreia nervosa, Ayahuasca, Catha edulis, Ipomoea violacea, Mandragora officinarum, Mitragyna speciosa, Pausinystalia yohimbe, Piper methisticum, Psilocybe, Rivea corymbosa, Salvia divinorum, Sceletium tortuosum, Lactuca virosa. From the results obtained, it can be evidenced that although several analytical methods for the simultaneous quantification of different molecules from the same plants have been developed and validated, a comprehensive method to detect active compounds from different natural specimens both in biological and non-biological matrices is still lacking.

Evaluating novel silica hydride-based stationary phases for the analysis of phytocannabinoids and other psychoactive drugs.

Three silica hydride based novel chromatographic phases chemically-bonded with allyloxy-DL-alpha-tocopherol, allylpentafluorophenyl, and 1-eicosene moieties were evaluated as separation media for selected phytocannabinoids and other substances of abuse. In order to assess column selectivity, a series of reference standards was analyzed and detected by using liquid chromatography with mass spectrometry. Further, quantitative detections of cannabidiol and tetrahydrocannabinol were attempted for the extracts of cannabis plants and cannabidiol gummy formulation. For potential bioanalytical applications, the columns were evaluated for substance screening in a human urine matrix. In summary, the newly developed columns are functional and effective for the analysis of phytocannabinoids and various psychoactive drugs with or without the presence of biological matrices.

Detection of Diagnostic Plant-Derived Psychoactive Biomarkers in Fingerprints by MALDI-SpiralTOF-Mass Spectrometry Imaging.

One of the current challenges in forensics is establishment of a connection between an individual and substances to which they have been exposed, and which might have relevance in crime scene investigation. An example of a situation in which this arises is when an individual has handled, and is under the influence of any one of a large number of currently unscheduled plant-based mind-altering substances. In such instances as a medical emergency or a crime scene investigation , one way to establish a connection between an individual and their exposure to such substances is to take advantage of the high information content of their fingerprint. The fingerprint pattern not only establishes the identity of the individual, but also contains rarely exploited chemical information about molecules to which they have been exposed that might have a bearing on a crime. If the fingerprint image is based on the spatial distribution of diagnostic molecular markers indicative of a substance, then an individual's identity can be definitively tied to exposure to the substance. The fingerprint image derived from the spatial distribution of diagnostic molecules can be obtained by mass spectrometry imaging (MSI). Here, we demonstrate how the handling by an individual of a plant-derived psychoactive brew called ayahuasca can be established through determination, by matrix-assisted laser desorption ionization (MALDI) MSI, of ion images featuring biomarkers from the plants from which the brew is made.

Forensic applications of supercritical fluid chromatography - mass spectrometry.

Achievements of supercritical fluid chromatography with mass spectrometric detection made in the field of forensic science during the last decade are reviewed. The main topics include analysis of traditional drugs of abuse (e.g. cannabis, methamphetamine) as well as new psychoactive substances (synthetic cannabinoids, cathinones and phenethylamines), doping agents (anabolic steroids, stimulants, diuretics, analgesics etc.) and chemical warfare agents. Control of food authenticity, detection of adulteration and identification of toxic substances in food are also pointed out. Main aspects of an analytical workflow, such as sample preparation, separation and detection are discussed. A special attention is paid to the performance characteristics and validation parameters of supercritical fluid chromatography-mass spectrometric methods in comparison with other separation techniques.

Analysis of cannabinoids in commercial hemp seed oil and decarboxylation kinetics studies of cannabidiolic acid (CBDA).

Hemp seed oil from Cannabis sativa L. is a very rich natural source of important nutrients, not only polyunsaturated fatty acids and proteins, but also terpenes and cannabinoids, which contribute to the overall beneficial effects of the oil. Hence, it is important to have an analytical method for the determination of these components in commercial samples. At the same time, it is also important to assess the safety of the product in terms of amount of any psychoactive cannabinoid present therein. This work presents the development and validation of a highly sensitive, selective and rapid HPLC-UV method for the qualitative and quantitative determination of the main cannabinoids, namely cannabidiolic acid (CBDA), tetrahydrocannabinolic acid (THCA), cannabidiol (CBD), tetrahydrocannabinol (THC), cannabinol (CBN), cannabigerol (CBG) and cannabidivarin (CBDV), present in 13 commercial hemp seed oils. Moreover, since decomposition of cannabinoid acids generally occurs with light, air and heat, decarboxylation studies of the most abundant acid (CBDA) were carried out in both open and closed reactor and the kinetics parameters were evaluated at different temperatures in order to evaluate the stability of hemp seed oil in different storage conditions.

Mitragynine concentrations in two fatalities.

Two cases of fatalities are reported of which the recreational use of Mitragyna speciosa ("kratom") could be confirmed. One of these cases presents with one of the highest postmortem mitragynine concentrations published to date. Our results show that even extremely high mitragynine blood concentrations following the consumption of kratom do not necessarily have to be the direct cause of death in such fatalities as a result of an acute overdose. The two cases are compared with regard to the differences in mitragynine concentrations detected and the role of mitragynine in the death of the subjects. Irrespective of the big differences in mitragynine concentrations in the postmortem blood samples, mitragynine was not the primary cause of death in either of the two cases reported here. Additionally, by rough estimation, a significant difference in ratio of mitragynine to its diastereomers in the blood and urine samples between the two cases could be seen.

Evolutionary Considerations on the Emerging Subculture of the E-psychonauts and the Novel Psychoactive Substances: A Comeback to the Shamanism?

BACKGROUND: Evolutionary research on drug abuse has hitherto been restricted to proximate studies, considering aetiology, mechanism, and ontogeny. However, in order to explain the recent emergency of a new behavioral pattern (e.g. 'the e-psychonaut style') of novel psychoactive substances' (NPS) intake, a complementary evolutionary model may be needed. OBJECTIVE: A range of evolutionary interpretations on the 'psychonaut style' and the recent emergency of NPS were here considered. METHOD: The PubMed database was searched in order to elicit evolutionary theory-based documents commenting on NPS/NPS users/e-psychonauts. RESULTS: The traditional 'shamanic style' use of entheogens/plant-derived compounds may present with a range of similarities with the 'e-psychonauts' use of mostly of hallucinogen/psychedelic NPS. These users consider themselves as 'new/technological' shamans. CONCLUSION: Indeed, a range of evolutionary mechanisms, such as: optimal foraging, costly signaling, and reproduction at the expense of health may all cooperate to explain the recent spread and diffusion of the NPS market, and this may represent a reason of concern.

Alpha-PVP as an active component of herbal highs in Poland between 2013 and 2015.

Alpha-PVP (alpha-pyrrolidinovalerophenone, α-PVP) is a synthetic derivative of cathinone. It has been one of the most frequently detected new psychoactive substances (NPS) available on the drug market in recent years in Poland. The usual routes of administration of the drug include oral, insufflation, and injection. Unexpectedly, we dealt with a great number of herbal samples that turned out to contain α-PVP. A total number of 352 herbal samples from 19 cases in which we detected synthetic cathinones, were investigated in the Institute of Forensic Research (IFR) from 2013 to 2015. The seized products that were received by our laboratory were first screened by gas chromatography coupled to mass spectrometry (GC-MS). Quantification of α-PVP and other cathinones was performed by ultra-performance liquid chromatography with photodiode array detection (UPLC-PDA). Of the samples, 84% contained only α-PVP. Other groups of products were those containing only α-PVT, α-PVP and α-PVT, α-PVP and synthetic cannabinoid A-834, 735, and α-PVP and cannabis. In one herbal sample, α-PVP was detected along with caffeine and tadalafil. The herbal products present on the market containing only α-PVP usually had a mass of 0.3 to 0.6 g, and concentration range in this group of samples was 3.0-44.0% (content: 13.0-222.0 mg per package). The amount of α-PVP in samples below 0.30 g was in a range 9-18 mg whiles in samples above 0.60 g it was in the range 30-716 mg. There were also products containing a mixture of α-PVP and α-PVT. In those samples, α-PVP concentrations were: 3.0-6.0% (amount: 15.0-34.0 mg). Copyright © 2016 John Wiley & Sons, Ltd.

APPLICATION OF CHEMOMETRICS FOR IDENTIFICATION OF PSYCHOACTIVE PLANTS.

Drug market changes dynamically causing many analytical challenges for police experts. Among illicit substances there are synthetic designer products but also herbal material. Plant material is usually in fine-cut or powdered form, thus difficult to identify. For such fragmented material classic taxonomical identification methods using anatomical and morphological features of the plant cannot be employed. The aim of the study was to develop an identification method of the powdered material with employment of multidimensional data analysis techniques. Principal Component Analysis (PCA) was chosen as a method of data exploration. The study was conducted on four plants controlled in Poland: Salvia divinorum, Mitragyna speciosa, Psychotria viridis and Calea zacatechichi. The compatibility of grouping features of selected species was compared in two variants: chemical and elemental composition. In a first variant, GC-MS chromatograms of extracts were analyzed and in the second, elements composition with the AAS and the ICP-MS techniques. The GC-MS method, based on the qualitative interpretation of results, allows for clear differentiation of samples with regard to their species affiliation. Even the plants belonging to the same family Rubiaceae, P. viridis and M. speciosa formed homogeneous and clearly separated clusters. Additionally, the cluster analysis was performed, as a method confirming sample grouping.

Light microscopy can reveal the consumption of a mixture of psychotropic plant and fungal material in suspicious death.

Light microscopical examination of plant and fungal remains in the post mortem gut may be capable of demonstrating the ingestion of unexpected natural psychotropic materials. This is demonstrated here in a case in which a 'shaman' was accused of causing the death of a young man. The deceased had participated in a ceremony which involved the drinking of ayahuasca in order to induce a psychotropic experience. Ayahuasca is an infusion of Banisteriopsis caapi (ayahuasca vine), which produces a monoamine oxidase inhibitor, and one or more additional tropical plants, generally Psychotria viridis (chacruna) which produces dimethyltryptamine (DMT). The monoamine oxidase inhibitor prevents DMT from being broken down in the gut, so enabling its passage into the bloodstream and across the blood/brain barrier. Toxicological tests for DMT demonstrated the presence of this compound in the body. The deceased was reported to be in the habit of using Psilocybe semilanceata (liberty cap). This fungus (popularly called magic mushroom) contains psilocybin which is hydrolysed in the gut to psilocin; this compound mimics a serotonin uptake inhibitor, and also invokes psychotropic experiences. Microscopical examination established that the ileum and colon contained spores of Psilocybe and, in addition, pollen of Cannabis sativa and seeds of Papaver cf. somniferum (opium poppy). Both the plant species yield psychotropic substances. Palynological and mycological analysis of containers from the deceased person's dwelling also yielded abundant trace evidence of pertinent pollen and spores. The police had requested analysis for DMT but there was no screening for other psychotropic substances. Investigators were surprised that a mixture of hallucinogenic materials had been consumed by the deceased. The charge was modified from manslaughter to possession of a 'Class A' drug as the deceased had been consuming psychotropic substances not administered by the 'shaman'. Where death involving drugs from plants or fungi is suspected, microscopical examination of samples from the gut can provide a rapid and effective method for assessing, in a temporal context, the presence of ingested materials that may not have been previously suspected. The example presented here also demonstrates the need for caution in interpreting toxicological results where screening for unusual compounds has been limited.

Rapid detection by direct analysis in real time-mass spectrometry (DART-MS) of psychoactive plant drugs of abuse: the case of Mitragyna speciosa aka "Kratom".

Mitragyna speciosa, also known commonly as "Kratom" or "Ketum", is a plant with psychoactive properties that have been attributed to the presence of various indole alkaloids such as mitragynine and 7-hydroxymitragynine. M. speciosa use is gaining popularity internationally as a natural and legal alternative to narcotics. As a drug of abuse, its detection and identification are not straightforward, since M. speciosa plant material is not particularly distinctive. Here, we show that direct analysis in real time-mass spectrometry (DART-MS) can be used not only to rapidly identify M. speciosa plant material and distinguish it from other plants, but also to distinguish between M. speciosa plant varieties, based on differences between their chemical profiles. The method is rapid and the analysis expeditious. Plant material such as that found at a crime scene can be analyzed directly with no sample pre-preparation steps. Furthermore, we show that the basis set of principal components that permit characterization of the plant material can be used to positively identify M. speciosa.

Simultaneous quantification of delta-9-THC, THC-acid A, CBN and CBD in seized drugs using HPLC-DAD.

An HPLC-DAD method for the quantitative analysis of Δ(9)-tetrahydrocannabinol (THC), Δ(9)-tetrahydrocannabinolic acid-A (THCA-A), cannabidiol (CBD), and cannabinol (CBN) in confiscated cannabis products has been developed, fully validated and applied to analyse seized cannabis products. For determination of the THC content of plant material, this method combines quantitation of THCA-A, which is the inactive precursor of THC, and free THC. Plant material was dried, homogenized and extracted with methanol by ultrasonication. Chromatographic separation was achieved with a Waters Alliance 2695 HPLC equipped with a Merck LiChrospher 60 RP-Select B (5µm) precolumn and a Merck LiChroCart 125-4 LiChrospher 60 RP-Select B (5µm) analytical column. Analytes were detected and quantified using a Waters 2996 photo diode array detector. This method has been accepted by the public authorities of Switzerland (Bundesamt für Gesundheit, Federal Office of Public Health), and has been used to analyse 9092 samples since 2000. Since no thermal decarboxylation of THCA-A occurs, the method is highly reproducible for different cannabis materials. Two calibration ranges are used, a lower one for THC, CBN and CBD, and a higher one for THCA-A, due to its dominant presence in fresh plant material. As provider of the Swiss proficiency test, the robustness of this method has been tested over several years, and homogeneity tests even in the low calibration range (1%) show high precision (RSD≤4.3%, except CBD) and accuracy (bias≤4.1%, except CBN).

Characterization of four new designer drugs, 5-chloro-NNEI, NNEI indazole analog, α-PHPP and α-POP, with 11 newly distributed designer drugs in illegal products.

Our continuous survey of illegal products in Japan revealed the new distribution of 15 designer drugs. We identified four synthetic cannabinoids, i.e., NNEI (1), 5-fluoro-NNEI (2), 5-chloro-NNEI (3) and NNEI indazole analog (4), and seven cathinone derivatives, i.e., MPHP (5), α-PHPP (6), α-POP (7), 3,4-dimethoxy-α-PVP (8), 4-fluoro-α-PVP (9), α-ethylaminopentiophenone (10) and N-ethyl-4-methylpentedrone (11). We also determined LY-2183240 (12) and its 2'-isomer (13), which were reported to inhibit endocannabinoid uptake, a methylphenidate analog, 3,4-dichloromethylphenidate (14), and an MDA analog, 5-APDB (15). No chemical and pharmaceutical data for compounds 3, 4, 6 and 7 had been reported, making this the first report on these compounds.