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.

Identification and Analytical Characterization of a Novel Synthetic Cannabinoid-Type Substance in Herbal Material in Europe.

The rapid diffusion of new psychoactive substances (NPS) presents unprecedented challenges to both customs authorities and analytical laboratories involved in their detection and characterization. In this study an analytical approach to the identification and structural elucidation of a novel synthetic cannabimimetic, quinolin-8-yl-3-[(4,4-difluoropiperidin-1-yl) sulfonyl]-4-methylbenzoate (2F-QMPSB), detected in seized herbal material, is detailed. An acid precursor 4-methyl-3-(4,4-difluoro-1-piperidinylsulfonyl) benzoic acid (2F-MPSBA), has also been identified in the same seized material. After extraction from the herbal material the synthetic cannabimimetic, also referred to as synthetic cannabinoid receptor agonists or "synthetic cannabinoids", was characterized using gas chromatography-mass spectrometry (GC-MS), 1H, 13C, 19F and 15N nuclear magnetic resonance (NMR) and high-resolution tandem mass spectrometry (HR-MS/MS) combined with chromatographic separation. A cheminformatics platform was used to manage and interpret the analytical data from these techniques.

Cumyl-CBMICA: A new synthetic cannabinoid receptor agonist containing a cyclobutyl methyl side chain.

Since the beginning of the phenomenon of new psychoactive substances (NPS), synthetic cannabinoid receptor agonists (SCRAs) have been the largest and most prevalent subclass of these drugs in Europe. Many countries implemented specific legislation scheduling classes of substances defined on the basis of their chemical structure to reduce supply. We describe the identification and analytical characterization within the EU project ADEBAR plus of 1-(cyclobutylmethyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide which resulted in the formal notification through the Early Warning System of the European Monitoring Centre for Drug and Drug Addiction (EMCDDA). This is the first identification of this new SCRA worldwide and the analytical data was distributed (inter-)nationally right after identification in 2019. First, the substance was isolated from the herbal material using preparative high-performance liquid chromatography (HPLC). Structure elucidation and analytical characterization were performed using gas chromatography-mass spectrometry (GC-MS), gas chromatography-solid state infrared spectroscopy (GC-sIR), liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry (LC-ESI-qToF-MS), Raman spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. The new compound contains a cyclobutyl methyl group as a side chain and has not been described in any patent to our knowledge. Based on the semisystematic nomenclature of SCRAs, we propose Cumyl-CBMICA as a short name for the compound.

Analysis of supplements available to UK consumers purporting to contain selective androgen receptor modulators.

Selective androgen receptor modulators (SARMs) are compounds with specific androgenic properties investigated for the treatment of conditions such as muscle wasting diseases. The reported androgenic properties have resulted in their use by athletes, and consequently they have been on the World Anti-Doping Agency prohibited list for more than a decade. SARMs have been investigated by pharmaceutical companies as potential drug candidates, but to date no SARM has demonstrated sufficient safety and efficacy to gain clinical approval by either the European Medicines Agency or the U.S. Food and Drug Administration. Despite their lack of safety approval, SARMs are often illegally marketed as dietary supplements, available for consumers to buy online. In this study, a range of supplement products marketed as SARMs were purchased and analyzed using high resolution accurate mass - mass spectrometry to evaluate the accuracy of product claims and content labeling. This study found discrepancies ranging from a supplement in which no active ingredients were found, to supplements containing undeclared prohibited analytes. Where SARMs were detected, discrepancies were observed between the concentrations measured and those detailed on the product packaging. The outcome of this experiment highlights the high risk of such supplement products to consumers. The inaccurate product claims give rise to uncertainty over both the dose taken and the identity of any of these unapproved drugs. Even for supplements for which the product labeling is correct, the lack of complete toxicity data, especially for combinations of SARMs taken as stacks, means that the safety of these supplements is unknown.

Trends using biological target-based assays for drug detection in complex sample matrices.

In vivo, drug molecules interact with their biological targets (e.g., enzymes, receptors, ion channels, transporters), thereby eliciting therapeutic effects. Assays that measure the interaction between drugs and bio-targets may be used as drug biosensors, which are capable of broadly detecting entire drug classes without prior knowledge of their chemical structure. This Trends article covers recent developments in bio-target-based screening assays for detecting drugs associated with the following areas: illicit products marketed as dietary supplements, food-producing animals, and bodily fluids. General challenges and considerations associated with using bio-target assays are also presented. Finally, future applications of these assays for drug detection are suggested based upon current needs.

Quantification of Herbal Mixtures Containing Cumyl-PEGACLONE-Is Inhomogeneity Still an Issue?

Synthetic cannabinoid receptor agonizts (SCRAs), also known as synthetic cannabinoids, are mostly consumed in the form of herbal mixtures available in online shops. These herbal mixtures are produced by soaking dried, crushed plant material in a solution of SCRAs or by spraying the solution on the plant material. Inhomogeneity in the distribution of the active ingredient can occur during the production process and pose a serious health risk for consumers of these drugs. In the present study 20 herbal mixtures containing Cumyl-PEGACLONE, one of the most prevalent SCRAs in Germany in 2017, were quantitatively analyzed by high-performance liquid chromatography with diode array detection (HPLC-DAD) after an initial screening by gas chromatography mass spectrometry. All investigated herbal mixtures were purchased in online shops during a systematic product monitoring carried out in the frame of the EU project "SPICE Profiling". The complete content of the packages was divided into aliquots without homogenization and extracted three times with methanol under ultrasonication. The combined extracts of each aliquot were filtered and quantified with a fully validated HPLC-DAD method using a 7-point calibration curve (1-50 µg/mL). The Cumyl-PEGACLONE content in the analyzed material ranged from 8.6 to 146 mg/g (median 29.4 mg/g, mean 38.5 mg/g). The intrapackage concentration variability was mostly below 10% RSD. Analyzed concentrations roughly correlated with product advisory (e.g., "strong") on the websites, if available. Aliquots at the bottom of a package generally tended to show higher levels of Cumyl-PEGACLONE than the upper aliquots. Packages of the same brand with different date of order did not always show the same mean concentrations. Compared to former studies, the SCRA concentrations are generally lower and the risk of extreme variation of intrapackage SCRA contents seems to have dropped.

Music festivals and drugs: Wastewater analysis.

Drug consumption in individual cities, regions, and at various music events and festivals across the EU has generally been monitored via questionnaires, patients' medical data, and police reports. However, an overview of drug consumption obtained from these methods can be negatively affected by various subjective factors. We aimed to investigate an association between levels of target drugs in wastewater, music genres, and festival courses. The occurrence of illicit drugs, their metabolites, and psychoactive compounds was investigated in the influent of six wastewater treatment plants in the Czech and Slovak Republic during seven large-scale music festivals from different music genres: metal, rock, pop, country and folk, ethnic, multi-genre, dance, and trance. The total number of participants included >130,000 active festival attendees. The association between music genre and illicit drug and/or psychoactive pharmaceutical consumptions is discussed on the basis of the results obtained through wastewater analyses. The observed trend was similar to worldwide published data with a specific local phenomenon of methamphetamine prevalence that did not significantly change between music events. Increased specific loads of cocaine (measured as its metabolite benzoylecgonine) and Ecstasy, along with some cannabis, were mainly observed during pop/rock and dance music festivals. However, there was no significant increase observed in the specific loads of all monitored psychoactive pharmaceuticals. This study demonstrates that the abuse of some illicit drugs is closely associated with specific music preferences.

Ion mobility spectrometry as a fast screening tool for synthetic cannabinoids to uncover drug trafficking in jail via herbal mixtures, paper, food, and cosmetics.

The greatest challenge for European drug policies is how to effectively respond to the dynamic and constantly changing market for new psychoactive substances (NPS). Even small modifications in the chemical structure of substances often allow circumventing existing laws. Also in prison, the consumption of NPS is rising and there is growing evidence that NPS are responsible for a large share of drug-related problems. Ion mobility spectrometry (IMS) is the technique of choice for trace analysis of illicit drugs or explosives at security points, for example airports. Currently, databases of the reduced mobility (K0 ) values are limited to classical drugs and should be completed with data of emerging NPS. In this article, K0 values, LODs (0.7-3.6 ng) and drift times of 25 synthetic cannabinoids were evaluated. The data were added to existing databases of IMS which were then applied for fast screening in prison. The detection capability of the portable IMS technique was evaluated by the determination of intra-day (0.089%) and inter-day precision (0.004% to 0.14%), systematic error (0.19%), and separation capability for structurally related NPS. The applicability of the methodology was demonstrated by the successful analysis of 12 different pieces of paper impregnated with synthetic cannabinoids, 7 different cosmetics, and 5 food samples (liquids), spiked with a mixture of narcotic drugs and a synthetic cannabinoid. In addition, 14 herbal mixtures and 36 different casework samples from prisons were analyzed provided by the State Office of Criminal Investigation Rhineland-Palatinate (Germany).

Characterization of a new illicit phosphodiesterase-type-5 inhibitor identified in the softgel shell of a dietary supplement.

A new sildenafil analog has been identified in the softgel shell of a dietary supplement. The compound was investigated by UV spectroscopy and high-resolution MS analysis, leading to the proposed structure 1-methyl-5-{5-[2-(4-methylpiperazin-1-yl)acetyl]-2-propoxyphenyl}-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one. A synthetic reference compound with the proposed structure was prepared, and the two sets of analytical data were compared, confirming the structure of the new compound. The compound was named propoxyphenyl noracetildenafil from its structure and similarity with the known compound.

Use of DART-TOF-MS for Screening Drugs of Abuse.

Screening is an integral component of an analytical scheme to identify the presence of controlled substances in submissions to the crime laboratory. Many techniques are utilized, including color tests, thin-layer chromatography, and ultraviolet spectroscopy. While these are useful techniques to guide the examiner, all will, at best, categorize the material into a broad group of compounds. Direct Analysis in Real Time (DART), coupled with a time-of-flight (TOF) mass spectrometer, is an emerging technique that yields highly definitive screening data leading to the identity of controlled substances present in a case sample. Sample preparation is quick and simple and run times are typically only a few minutes. Collected data will allow the examiner to determine appropriate standards for confirmation, making the overall analysis much more efficient. Presented here is a guide to using this technique for the screening of case submissions for controlled substances.

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.

Occurrence of illicit drugs in two wastewater treatment plants in the South of Italy.

In this study the occurrence and the behavior of illicit drugs and their metabolites have been investigated for two wastewater treatment plants (WWTPs) (namely, WWTP-1 and WWTP-2) located in Sicily (island of Italy). Samples were analyzed for methamphetamine, cocaine (COC), 3,4-methylenedioxymethamphetamine (MDMA), methadone (METH), 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), 3,4-methylenedioxy amphetamine (MDA); 3,4-methylenedioxy ethylamphetamine (MDEA), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) and Benzoylecgonine (BEG). The BEG, COC, MOR and THC-COOH were found at the highest concentration in both WWTPs. The Wastewater-based epidemiology calculation for BEG, COC, cannabinoids and THC-COOH was performed. On average, for both plants, population consumes 1.6 and 23.4 dose 1000 inh-1 day-1 of cocaine and cannabis, respectively. For WWTP-1 negative removals of illicit drugs were observed. For WWTP-2 the following average removal efficiencies were obtained: BEG (77.85%), COC (92.34%), CODEINE (64.75%), MOR (90.16%) and THC-COOH (68.64%).

Qualitative Analysis and Detection of the Pyrolytic Products of JWH-018 and 11 Additional Synthetic Cannabinoids in the Presence of Common Herbal Smoking Substrates.

Synthetic cannabinoids have become a ubiquitous challenge in forensic toxicology and seized drug analysis. Thermal degradation products have yet to be identified and evaluated for toxicity in comparison to parent and metabolic compounds. An investigation into these pyrolytic products, as the major route of ingestion is inhalation, may produce additional insight to understand the toxicity of synthetic cannabinoids. The pyrolysis of JWH-018 and 11 additional synthetic cannabinoids and six herbal plant substrates were conducted using an in-house constructed smoking simulator. After pyrolysis of herbal material alone, the plant substrate was spiked with the drug compounds to 2-5% w/w concentrations. Samples were collected, filtered, evaporated under nitrogen gas, reconstituted in methanol, and analyzed via gas chromatograph-mass spectrometer. Pyrolysis of the plant material alone produced 10 consistently observed compounds between the six plant species. The pyrolysis of the synthetic cannabinoids produced a total of 52 pyrolytic compounds, where 32 were unique to a particular parent compound and the remaining 20 were common products between multiple cannabinoids. The thermal degradation followed three major pathways that are outlined to assist in producing a predictive model for new synthetic cannabinoids that may arise in case samples. The observed pyrolytic products are also viable options for analysis in post mortem samples and the evaluation of toxicity.

Identification of Eight Synthetic Cannabinoids, Including 5F-AKB48 in Seized Herbal Products Using DART-TOF-MS and LC-QTOF-MS as Nontargeted Screening Methods.

Synthetic cannabinoids are sprayed onto plant material and smoked for their marijuana-like effects. Clandestine manufacturers modify synthetic cannabinoid structures by creating closely related analogs. Forensic laboratories are tasked with detection of these analog compounds, but targeted analytical methods are often thwarted by the structural modifications. Here, direct analysis in real time coupled to accurate mass time-of-flight mass spectrometry (DART-TOF-MS) in combination with liquid chromatography quadruple time-of-flight mass spectrometry (LC-QTOF-MS) are presented as a screening and nontargeted confirmation method, respectively. Methanol extracts of herbal material were run using both methods. Spectral data from four different herbal products were evaluated by comparing fragmentation pattern, accurate mass and retention time to available reference standards. JWH-018, JWH-019, AM2201, JWH-122, 5F-AKB48, AKB48-N-(4-pentenyl) analog, UR144, and XLR11 were identified in the products. Results demonstrate that DART-TOF-MS affords a useful approach for rapid screening of herbal products for the presence and identification of synthetic cannabinoids.

Occurrence and fate of illicit drugs and pharmaceuticals in wastewater from two wastewater treatment plants in Costa Rica.

Chemical analysis of raw wastewater in order to assess the presence of biological markers entering a wastewater treatment plant can provide objective information about the health and lifestyle of the population connected to the sewer system. This work was performed in a tropical country of Central America, Costa Rica, with the aim of extending this knowledge to new world regions. This work is the first to report wastewater-based epidemiological data on the use of illicit drugs in this region of the world. Composite wastewater samples from the influents of two different wastewater treatment facilities and surface water samples from surrounding areas were collected applying the best practice protocol and analysed to investigate the occurrence and fate of selected illicit drugs of abuse and pharmaceuticals. Results showed the presence of chemical indicators of the classic drugs cocaine and cannabis at high concentration levels, besides the moderate presence of the opiates codeine and morphine. Neither the worldwide commonly used psychoactive substances of abuse such as synthetic phenethylamines, nor pharmaceuticals from the family of benzodiazepines were detected, demonstrating the spatial differences in drug use among different world regions. In addition, effluent wastewater samples were analysed and compared to influent concentrations in order to evaluate the decrease in concentration of the targeted analytes through two treatment technologies. As a final step, a wide-scope qualitative screening, including hundreds of suspect compounds, was applied in order to have a better knowledge on the presence of pharmaceuticals in waters and to assess the potential impact of the treated wastewater into the receiving aquatic ecosystems.

Quantification of synthetic cannabinoids in herbal smoking blends using NMR.

Herbal smoking blends containing synthetic cannabinoids have become popular alternatives to marijuana. These products were previously sold in pre-packaged foil bags, but nowadays seizures usually contain synthetic cannabinoid powders together with unprepared plant materials. A question often raised by the Swedish police is how much smoking blend can be prepared from certain amounts of banned substance, in order to establish the severity of the crime. To address this question, information about the synthetic cannabinoid content in both the powder and the prepared herbal blends is necessary. In this work, an extraction procedure compatible with direct NMR quantification of synthetic cannabinoids in herbal smoking blends was developed. Extraction media, time and efficiency were tested for different carrier materials containing representative synthetic cannabinoids. The developed protocol utilizes a 30 min extraction step in d4 -methanol in presence of internal standard allowing direct quantitation of the extract using NMR. The accuracy of the developed method was tested using in-house prepared herbal smoking blends. The results showed deviations less than 0.2% from the actual content, proving that the method is sufficiently accurate for these quantifications. Using this method, ten synthetic cannabinoids present in sixty-three different herbal blends seized by the Swedish police between October 2012 and April 2015 were quantified. Obtained results showed a variation in cannabinoid contents from 1.5% (w/w) for mixtures containing MDMB-CHMICA to over 5% (w/w) for mixtures containing 5F-AKB-48. This is important information for forensic experts when making theoretical calculations of production quantities in legal cases regarding "home-made" herbal smoking blends. Copyright © 2016 John Wiley & Sons, Ltd.

Pharmaceutical doses of the banned stimulant oxilofrine found in dietary supplements sold in the USA.

Oxilofrine (4-[1-hydroxy-2-(methylamino)propyl]phenol) is a pharmaceutical stimulant prescribed in dosages of 16 to 40 mg to stimulate the heart and increase blood pressure. It has never been approved for use in the USA as a prescription drug or as a dietary supplement. Several athletes, however, have been banned from sport for testing positive for oxilofrine and have claimed that they inadvertently consumed oxilofrine in sports supplements. Consumption of supplements containing oxilofrine may also pose serious health risks. For example, one brand of supplements containing oxilofrine has been linked to serious adverse events including vomiting, agitation, and cardiac arrest. We designed our study to determine the presence and quantity of oxilofrine in dietary supplements sold in the USA. A validated ultra-high performance liquid chromatography-quadrupole time of flight-mass spectrometry method was developed for the identification and quantification of oxilofrine. The separation was achieved using a reversed phase column, mass spectrometry detection, and a water/acetonitrile gradient as the mobile phase. The presence of oxilofrine was confirmed using a reference standard. We analyzed 27 brands of supplements labelled as containing a synonym of oxilofrine ('methylsynephrine') and found that oxilofrine was present in 14 different brands (52%) at dosages ranging from 0.0003 to 75 mg per individual serving. Of the supplements containing oxilofrine, 43% (6/14) contained pharmaceutical or greater dosages of oxilofrine. Following instructions on the label, consumers could ingest as much as 250 mg of oxilofrine per day. The drug oxilofrine was found in pharmacological and greater dosages in supplements labelled as containing methylsynephrine. Copyright © 2016 John Wiley & Sons, Ltd.

Development and evaluation of an immunochromatographic strip for rapid screening of sildenafil-type compounds as illegal additives in functional foods.

Sildenafil is a phosphodiesterase-5 inhibitor (PDE-5) for the treatment of erectile dysfunction. Undeclared sildenafil and related analogues adulterated in functional foods are a threat to public health. To screen these illegal drugs rapidly in herbal samples, an immunochromatographic (IC) assay was developed based on polyclonal antibodies specific to both sildenafil and its analogues. A group that is pharmacological necessary for sildenafil and its analogues was employed as a representative hapten for the generation antibodies against the target compounds. The desired antisera showed satisfactory specificities to sildenafil and major analogues with IC50 values ranging from 19.3 to 34.6 ng ml(-1) in a referring enzyme-linked immunosorbent assay (ELISA). The optimised IC assay showed detection thresholds in the range 5.0-20 µg g(-1) for sildenafil and major analogues in herbal samples. Sixty herbal food supplements were screened and six were found to be positive using the IC strip. It was confirmed by ELISA and UPLC-PDA-MS/MS that positive samples contain target illegal additives in levels of 10-40 mg g(-1) (1-4%). In this range, sensitivity of the IC strip is adequate to screen sildenafil-type compounds in herbal commodities under a dilution ratio of 1:10(3). Thus, the current IC assay is a suitable tool for screening sildenafil and its analogues as illegal additives in herbal food supplements.

Wastewater-based assessment of regional and temporal consumption patterns of illicit drugs and therapeutic opioids in Croatia.

A comprehensive study of spatial and temporal consumption patterns of the selected illicit drugs (heroin, cocaine, amphetamine, MDMA, methamphetamine, cannabis) and therapeutic opioids (codeine, methadone) has been performed in six Croatian cities by applying wastewater-based epidemiology. The investigated cities (Bjelovar, Vinkovci, Varazdin, Karlovac, Zadar and Zagreb) varied widely in the population size (27,000-688,000 inhabitants) as well as in the number of registered drug consumers included in compulsory and voluntary medical treatment and rehabilitation programs (30-513 persons/100,000 inhabitants of age 15-64). The most consumed illicit drugs were cannabis (10-70doses/day/1000 inhabitants), heroin (<0.2-10doses/day/1000 inhabitants) and cocaine (0.2-8.7doses/day/1000 inhabitants), while the consumption of amphetamine-type drugs was much lower (<0.01-4.4doses/day/1000 inhabitants). Enhanced consumption of illegal drugs was generally associated with larger urban centers (Zagreb and Zadar) however comparatively high consumption rate of cocaine, MDMA and methadone was determined in some smaller cities as well. The overall average dose number of 3 major illegal stimulants (cocaine, MDMA, amphetamine) was rather similar to the number of corresponding heroin doses, which is in disagreement with a comparatively much higher proportion of heroin users in the total number of registered drug users in Croatia. Furthermore, the illicit drug consumption pattern in the large continental city (Zagreb) was characterized by a significant enhancement of the consumption of all stimulants during the weekend, which could not be confirmed neither for the coastal city of Zadar nor for the remaining small continental cities. On the other hand, the city of Zadar exhibited a significant increase of stimulant drug usage during summer vacation period, as a result of pronounced seasonal changes of the population composition and lifestyle in coastal tourist centers. The obtained results represent a valuable complementary data source for the optimisation and implementation of strategies to combat drug abuse in Croatia.