Exposure to Synthetic Psychoactive Substances: A Potential Cause for Increased Human Hepatotoxicity Markers.

BACKGROUND: Approximately 30 million people worldwide consume new psychoactive substances (NPS), creating a serious public health issue due to their toxicity and potency. Drug-induced liver injury is the leading cause of liver disease, responsible for 4% of global deaths each year. CONTENT: A systematic literature search revealed 64 case reports, in vitro and in vivo studies on NPS hepatotoxicity. Maximum elevated concentrations of aspartate aminotransferase (136 to 15 632 U/L), alanine transaminase (121.5 to 9162 U/L), total bilirubin (0.7 to 702 mg/dL; 0.04 to 39.03 mmol/L), direct (0.2-15.1 mg/dL; 0.01-0.84 mmol/L) and indirect (5.3 mg/dL; 0.29 mmol/L) bilirubin, alkaline phosphatase (79-260 U/L), and gamma-glutamyltransferase (260 U/L) were observed as biochemical markers of liver damage, with acute and fulminant liver failure the major toxic effects described in the NPS case reports. In vitro laboratory studies and subsequent in vivo NPS exposure studies on rats and mice provide data on potential mechanisms of toxicity. Oxidative stress, plasma membrane stability, and cellular energy changes led to apoptosis and cell death. Experimental studies of human liver microsome incubation with synthetic NPS, with and without specific cytochrome P450 inhibitors, highlighted specific enzyme inhibitions and potential drug-drug interactions leading to hepatotoxicity. SUMMARY: Mild to severe hepatotoxic effects following synthetic NPS exposure were described in case reports. In diagnosing the etiology of liver damage, synthetic NPS exposure should be considered as part of the differential diagnosis. Identification of NPS toxicity is important for educating patients on the dangers of NPS consumption and to suggest promising treatments for observed hepatotoxicity.

Molecular Insights of New Psychoactive Substances (NPS) 2.0.

The New Psychoactive Substances (NPS) phenomenon represents an ever-changing global issue, with a number of new molecules entering the illicit market every year in response to international banning laws [...].

Synthetic Cathinones and Neurotoxicity Risks: A Systematic Review.

According to the EU Early Warning System (EWS), synthetic cathinones (SCs) are the second largest new psychoactive substances (NPS) class, with 162 synthetic cathinones monitored by the EU EWS. They have a similar structure to cathinone, principally found in Catha Edulis; they have a phenethylamine related structure but also exhibit amphetamine-like stimulant effects. Illegal laboratories regularly develop new substances and place them on the market. For this reason, during the last decade this class of substances has presented a great challenge for public health and forensic toxicologists. Acting on different systems and with various mechanisms of action, the spectrum of side effects caused by the intake of these drugs of abuse is very broad. To date, most studies have focused on the substances' cardiac effects, and very few on their associated neurotoxicity. Specifically, synthetic cathinones appear to be involved in different neurological events, including increased alertness, mild agitation, severe psychosis, hyperthermia and death. A systematic literature search in PubMed and Scopus databases according to PRISMA guidelines was performed. A total of 515 studies published from 2005 to 2022 (350 articles from PubMed and 165 from Scopus) were initially screened for eligibility. The papers excluded, according to the criteria described in the Method Section (n = 401) and after full text analyses (n = 82), were 483 in total. The remaining 76 were included in the present review, as they met fully the inclusion criteria. The present work provides a comprehensive review on neurotoxic mechanisms of synthetic cathinones highlighting intoxication cases and fatalities in humans, as well as the toxic effects on animals (in particular rats, mice and zebrafish larvae). The reviewed studies showed brain-related adverse effects, including encephalopathy, coma and convulsions, and sympathomimetic and hallucinogenic toxidromes, together with the risk of developing excited/agitated delirium syndrome and serotonin syndrome.

Sweat Testing for the Detection of Methylone after Controlled Administrations in Humans.

The aim of this study was to determine the excretion of methylone and its metabolites in sweat following the ingestion of increasing controlled doses of 50, 100, 150 and 200 mg of methylone to twelve healthy volunteers involved in a clinical trial. Methylone and its metabolites 4-hydroxy-3-methoxy-N-methylcathinone (HMMC) and 3,4-methylenedioxycathinone (MDC) were analyzed in sweat patches by liquid chromatography-tandem mass spectrometry. Methylone and MDC were detected in sweat at 2 h and reached their highest accumulation (Cmax) at 24 h after the administration of 50, 100, 150 and 200 mg doses. In contrast, HMMC was not detectable at any time interval after each dose. Sweat proved to be a suitable matrix for methylone and its metabolites' determination in clinical and toxicological studies, providing a concentration that reveals recent drug consumption.

Methylone and MDMA Pharmacokinetics Following Controlled Administration in Humans.

The aim of this study is to define, for the first time, human methylone and HMMC plasma pharmacokinetics following controlled administration of 50-200 mg methylone to 12 male volunteers. A new LC-MS/MS method was validated to quantify methylone, MDMA, and their metabolites in plasma. The study was a randomized, cross-over, double-blinded and placebo-controlled study, with a total of 468 plasma samples collected. First, 10 µL of MDMA-d5, MDA-d5 and methylone-d3 internal standards were added to 100 µL of plasma. Two mL of chloroform and ethyl acetate 9:1 (v/v) were then added, mixed well and centrifuged. The supernatant was fortified with 0.1 mL acidified methanol and evaporated under nitrogen. Samples were reconstituted with a mobile phase and injected into the LC-MS/MS instrument. The method was fully validated according to OSAC guidelines (USA). Methylone plasma concentrations increased in a dose-proportional manner, as demonstrated by the increasing maximum concentration (Cmax) and area under the curve of concentrations (AUC). Methylone Cmax values were reported as 153, 304, 355 and 604 ng/mL, AUC0-24 values were reported as 1042.8, 2441.2, 3524.4 and 5067.9 h·ng/mL and T1/2 values as 5.8, 6.4, 6.9 and 6.4 h following the 50, 100, 150 and 200 mg doses, respectively. Methylone exhibited rapid kinetics with a Tmax of 1.5 h for the 50 mg dose and 2 h approximately after all the other doses. HMMC exhibited faster kinetics compared to methylone, with a Cmax value that was 10-14-fold lower and an AUC0-24 value that was 21-29-fold lower. Methylone pharmacokinetics was linear across 50-200 mg oral doses in humans, unlike the previously described non-linear oral MDMA pharmacokinetics. An LC-MS/MS method for the quantification of methylone, MDMA and their metabolites in human plasma was achieved. Methylone exhibited linear pharmacokinetics in humans with oral doses of 50-200 mg.

Production of melatonin and other tryptophan derivatives by Oenococcus oeni under winery and laboratory scale.

Malolactic fermentation (MLF) in Valtellina Superiore DOCG red wine was monitored in 4 cellars and the final products were analysed to determine the content of melatonin (MEL) and other tryptophan (TRP) derivatives, including tryptophan ethyl ester (TEE) and MEL isomers (MISs), and to isolate predominant O. oeni strains. MEL and TEE significantly increased in wines after MLF from two cellars out of four. Six strains were isolated during the MLF of red wines and under laboratory scale, in rich and synthetic wine cultural media, together with other four O. oeni strains able to trigger the MLF. Results showed that the presence of stressful growth factors, like ethanol and acid pH, has a pivotal role in triggering the release of TEE by oenococci. Indeed, all the strains became capable to produce also MEL and MISs, together with TEE. under harsh growth conditions, as in a synthetic wine medium. The production of these compounds was strain-dependent and a maximum amount of 0.0078 ±â€¯0.0023 ngT/mL (UMB472) and 619.85 ±â€¯196.16 ngT/mL (UMB436) of MEL and TEE was obtained, respectively. In particular, different MISs were detected under oenological and laboratory scale suggesting that other factors (i.e. technological and/or physico-chemical) could affect the synthesis of TRP derivatives.

Toxicology and Analysis of Psychoactive Tryptamines.

Our understanding of tryptamines is poor due to the lack of data globally. Tryptamines currently are not part of typical toxicology testing regimens and their contribution to drug overdoses may be underestimated. Although their prevalence was low, it is increasing. There are few published data on the many new compounds, their mechanisms of action, onset and duration of action, toxicity, signs and symptoms of intoxication and analytical methods to identify tryptamines and their metabolites. We review the published literature and worldwide databases to describe the newest tryptamines, their toxicology, chemical structures and reported overdose cases. Tryptamines are 5-HT2A receptor agonists that produce altered perceptions of reality. Currently, the most prevalent tryptamines are 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DiPT), 5-methoxy-N,N- diallyltryptamine (5-MeO-DALT) and dimethyltryptamine (DMT). From 2015 to 2020, 22 new analytical methods were developed to identify/quantify tryptamines and metabolites in biological samples, primarily by liquid chromatography tandem mass spectrometry. The morbidity accompanying tryptamine intake is considerable and it is critical for clinicians and laboratorians to be informed of the latest data on this public health threat.

Medicinal Cannabis and Synthetic Cannabinoid Use.

Cannabis products have been used for centuries by humans for recreational and medical purposes. Resent research, proposed the promising therapeutic potential of cannabis and related cannabinoids for a wide range of medical conditions, including psychiatric and neurological diseases. This Special Issue presents the latest updates on medicinal cannabis and synthetic cannabinoids pharmacology, toxicology and new analytical methods to identify and quantify these compounds in conventional and non-conventional biological matrices. Moreover, it provides current data regarding their adverse effects, safety, application for medical purposes and their harmful effects.

Separation of undeuterated and partially deuterated enantioisotopologues of some amphetamine derivatives on achiral and polysaccharide-based chiral columns in high-performance liquid chromatography.

The different behavior of enantiomers of chiral compounds in non-isotropic environments (among them in living organism) is well known. On the other hand, the importance of a kinetic isotope effect in the biomedical field has become evident during past few decades. Thus, separation of both, enantiomers and isotopologues is now critical. Only very few published studies have attempted the simultaneous separation of enantioisotopologues. In this article we report baseline separation of partially deuterated isotopologues of a few amphetamine derivatives in high-performance liquid chromatography (HPLC) using achiral columns. In addition, the simultaneous separations of enantiomers and isotopologues (i.e. enantioisotopologues) were attempted on polysaccharide-based chiral columns. For several compounds the isotope effect was tunable and could be switched from a "normal" to "inverse" by making changes to the mobile-phase composition. A stronger isotope effect was observed in acetonitrile-containing mobile phases compared to methanol-containing ones with both chiral and achiral columns. In a separation system where both "normal" and "inverse" isotope effects were observed the "normal" isotope effect was favored in polar organic solvents while increasing content of the aqueous component in the reversed-phase (RP) mobile phase favored an "inverse" isotope effect. This observation indicates that polar, hydrogen bonding-type noncovalent interactions are involved in the "normal" isotope effect, while apolar hydrophobic-type interactions are mostly responsible for the "inverse" isotope effect.

Optimization of enantioselective high-performance liquid chromatography-tandem mass spectrometry method for the quantitative determination of 3,4-methylenedioxy-methamphetamine (MDMA) and its phase-1 metabolites in human biological fluids.

Recently we published in this journal an enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of 3,4-methylenedioxymethamphetamine (MDMA) and its major phase-1 metabolites, 4-hydroxy-3-methoxyamphetamine (HMA), 4-hydroxy-3-methoxymethamphetamine (HMMA) and 3,4-methylenedioxyamphetamine (MDA) in human plasma, sweat, oral fluid and urine. Since we did not achieve simultaneous enantioseparation of all 4 compounds with a single chiral column, two amylose-based chiral columns were used alternatively. Further optimization of the mobile phase in the present study enabled baseline separation of all four pairs of enantiomers on a single Lux AMP column. In addition, by optimization of the column dimension and applied flow-rate it became possible to complete the separation within 6 minutes. These new methods were applied to the analysis of human plasma, oral fluid and urine. While results on the concentration of MDMA and its metabolites in various biological fluids were reported in our recent publication, in the present study an attempt was made to hydrolyze glucuronides in urine samples by using alternatively, hydrochloric acid or glucuronidase and to evaluate the effect of hydrolysis on the concentration and enantiomeric distribution of hydroxy metabolites of MDMA such as HMA and HMMA.

Separation of isotopologues of amphetamine with various degree of deuteration on achiral and polysaccharide-based chiral columns in high-performance liquid chromatography.

Hydrogen/deuterium (H/D) isotope effects are not unusual in chromatography and such phenomena have been observed in both gas- and liquid-phase separations. Despite the numerous reports on this topic, the understanding of mechanisms and the underlying noncovalent interactions at play remains rather challenging. In our recent study, we reported baseline separation of isotopologoues of some amphetamine (AMP) derivatives on achiral and polysaccharide-based chiral columns, as well as some correlations between the degree of separation of enantiomers and isotopologues on (the same) polysaccharide-based chiral column(s). Following our previous findings on isotope effects in high-performance liquid chromatography, we report herein a comparative study on the isotope effects observed with AMP and methamphetamine (MET). The impact of some pivotal factors such as the number of deuterium atoms part of AMP isotopologues, the structure of its isotopomers, the chemical structure of the achiral and chiral stationary phases used in this study, and the use of methanol- vs acetonitrile-containing mobile phases on the isotope effects was examined and discussed. Quantitative correlations between the observed isotope effects and the enantioselectivity of the chiral columns used are also shortly discussed. Furthermore, considering the chromatographic results as benchmark experimental data, we attempted to elucidate the molecular bases of the observed phenomena using quantum mechanics calculations.

Simultaneous chemo- and enantio-separation of 2-, 3- and 4-chloro-methcatinones by high-performance liquid chromatography-tandem mass spectrometry and its application to oral fluid samples.

A method of analysis was developed for the simultaneous chemo- and enantioseparation of 2-, 3-, and 4-chloromethcathinones by high-performance liquid chromatography tandem mass-spectrometry. The fast method enables the reliable identification of positional isomers of chloromethcathinones in biological samples. In addition, the same method can be used for the enantioselective quantitative determination of one of these compounds and its major phase-1 metabolites in biological fluids. The developed method was applied to oral fluid samples collected by police during routine random traffic control in Belgium from January to November, 2023. It was found that 3-CMC was more frequently abused compared to 4-CMC. Although some differences were observed between the concentrations of enantiomers in OF, most likely the drugs were abused in the racemic form. No abuse of 2-CMC was detected at the timepoint of sample collection.

Challenges encountered in the enantioselective analysis of new psychoactive substances exemplified by clephedrone (4-CMC).

In this study we report on efforts to develop an enantioselective method for the detection of the drug of abuse clephedrone (1-(4-chlorophenyl)-2-(methylamino)-1-propanone (4-chloromethcathinone, also known as 4-CMC or para-chloro-methcathinone)) and its phase-1 metabolites in human biological fluids. The major goal is not to only report results, but primarily to emphasize the various challenges encountered when developing a reliable analytical method for the detection and quantification of novel psychoactive substances (NPS) and their metabolites in the matrix of interest. Such challenges start with the lack of chemical stability of some NPS in biological matrices. Additionally, most often metabolites are unavailable in pure form to serve as analytical standards, just as deuterated standards for native drugs and metabolites are frequently not commercially available. Furthermore, if the NPS is chiral, enantiomerically pure standards with known absolute stereochemistry are required, as well as a stereochemical stability of a drug and its metabolites becomes an issue. In addition, the chirality of a NPS significantly increases the number of species to be detected in the sample and thus challenges the development of an adequate separation method. These issues are shortly addressed, and some solutions offered in this manuscript.

Disposition of Hexahydrocannabinol Epimers and Their Metabolites in Biological Matrices following a Single Administration of Smoked Hexahydrocannabinol: A Preliminary Study.

In 2023, hexahydrocannabinol (HHC) attracted the attention of international agencies due to its rapid spread in the illegal market. Although it was discovered in 1940, less is known about the pharmacology of its two naturally occurring epimers, 9(R)-HHC and 9(S)-HHC. Thus, we aimed to investigate the disposition of hexahydrocannabinol epimers and their metabolites in whole blood, urine and oral fluid following a single controlled administration of a 50:50 mixture of 9(R)-HHC and 9(S)-HHC smoked with tobacco. To this end, six non-user volunteers smoked 25 mg of the HHC mixture in 500 mg of tobacco. Blood and oral fluid were sampled at different time points up to 3 h after the intake, while urine was collected between 0 and 2 h and between 2 and 6 h. The samples were analyzed with a validated HPLC-MS/MS method to quantify 9(R)-HHC, 9(S)-HHC and eight metabolites. 9(R)-HHC showed the highest Cmax and AUC0-3h in all the investigated matrices, with an average concentration 3-fold higher than that of 9(S)-HHC. In oral fluid, no metabolites were detected, while they were observed as glucuronides in urine and blood, but with different profiles. Indeed, 11nor-9(R)-HHC was the most abundant metabolite in blood, while 8(R)OH-9(R) HHC was the most prevalent in urine. Interestingly, 11nor 9(S) COOH HHC was detected only in blood, whereas 8(S)OH-9(S) HHC was detected only in urine.

New Psychoactive Substances Intoxications and Fatalities during the COVID-19 Epidemic.

In January 2020, the World Health Organization (WHO) issued a Public Health Emergency of International Concern, declaring the COVID-19 outbreak a pandemic in March 2020. Stringent measures decreased consumption of some drugs, moving the illicit market to alternative substances, such as New Psychoactive Substances (NPS). A systematic literature search was performed, using scientific databases such as PubMed, Scopus, Web of Science and institutional and government websites, to identify reported intoxications and fatalities from NPS during the COVID-19 pandemic. The search terms were: COVID-19, SARS-CoV-2, severe acute respiratory syndrome coronavirus 2, coronavirus disease 2019, intox*, fatal*, new psychoactive substance, novel psychoactive substance, smart drugs, new psychoactive substance, novel synthetic opioid, synthetic opioid, synthetic cathinone, bath salts, legal highs, nitazene, bath salt, legal high, synthetic cannabinoid, phenethylamine, phencyclidine, piperazine, novel benzodiazepine, benzodiazepine analogue, designer benzodiazepines, tryptamine and psychostimulant. From January 2020 to March 2022, 215 NPS exposures were reported in Europe, UK, Japan and USA. Single NPS class intoxications accounted for 25, while mixed NPS class intoxications represented only 3 cases. A total of 130 NPS single class fatalities and 56 fatalities involving mixed NPS classes were published during the pandemic. Synthetic opioids were the NPS class most abused, followed by synthetic cathinones and synthetic cannabinoids. Notably, designer benzodiazepines were frequently found in combination with fentalogues. Considering the stress to communities and healthcare systems generated by the pandemic, NPS-related information may be underestimated. However, we could not define the exact impacts of COVID-19 on processing of toxicological data, autopsy and death investigations.

UHPLC-MS-MS Determination of THC, CBD and Their Metabolites in Whole Blood of Light Cannabis Smokers.

"Light cannabis" is a product legally sold in Europe with Δ9-tetrahydrocannabinol (THC) concentration <0.2% and variable cannabidiol (CBD) content. In this study, we aimed to assess the time courses of THC and metabolites (11-nor-9-carboxy-THC and 11-hydroxy-THC) and CBD and metabolites (CBD-7-oic acid, 7-hydroxy-CBD, 6α-hydroxy-CBD and 6ß-hydroxy-CBD) in whole blood of 10 healthy participants after smoking one or four light cannabis cigarettes (0.16% THC and 5.8% CBD). Blood samples were collected 0.5-4 h after administration. Blood analysis was performed by reversed-phase ultra-performance liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode after glucuronide hydrolysis and liquid-liquid extraction in basic and acidic conditions. The method was validated following the most recent guidelines in toxicology: the method was linear, accurate, precise and sensitive (lower limits of quantification ranged from 0.005 to 0.01 ng/mL); carryover, matrix effect, recovery, process efficiency and dilution integrity were also assessed. As previously reported, the main metabolites of THC were THC-COOH and then 11-OH-THC, and the main metabolites of CBD were 7-OH-CBD and then 7-COOH-CBD. The time of the first collection, which likely occurred after the maximal concentration of most of the analytes, and the short monitoring time, up to 4 h after smoking, limited the evaluation of the pharmacokinetic parameters.

Development of an enantioselective high-performance liquid chromatography-tandem mass spectrometry method for the quantitative determination of methorphan and its O-demethylated metabolite in human blood and its application to post-mortem samples.

In the present work an isocratic enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the separation and quantitative determination of dextro - and levo -methorphan and their pharmacologically relevant metabolites, dextrorphan and levorphanol, respectively, in human blood samples. The separation of enantiomers of methorphan and metabolites was performed on the polysaccharide-based chiral column Lux AMP in combination with acetonitrile and 5 mM aqueous ammonium bicarbonate pH 11 in the ratio 50:50 (%, v/v) as mobile phase with the flow rate 1 mL/min. The mass spectrometer was operated in scheduled multiple reaction monitoring (MRM) mode, with four transitions for each dextromethorpan, levomethorphan, dextrorphan and dextromethorphan-d3 and two transitions for each levorphanol, levorphanol-d3 and dextrorphan-d3. Application of this method to human post-mortem blood samples confirmed cases of severe overdosing with dextromethorphan, levomethorphan, and less commonly with both.

Development of enantioselective high-performance liquid chromatography-tandem mass spectrometry method for quantitative determination of methylone and some of its metabolites in oral fluid.

In the present study an enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the first time for quantitative determination of the recreational drug of abuse methylone and its major metabolites in oral fluid. The simultaneous chemo- and enantioseparation of methylone and its major metabolites was performed on a polysaccharide-based chiral column based on amylose tris(5-chloro-3-methylphenylcarbamate) as chiral selector (Lux i-Amylose-3) with methanol containing 0.4 % (v/v) aqueous ammonium hydroxide as mobile phase. The time required for enantioselective analysis of methylone and its 2 major metabolites was 15 min. This method was fully validated following the Organization of Scientific Area Committees (OSAC) for Forensic Science guidelines. This method was applied for the enantioselective determination of methylone and its metabolites in oral fluid and enantioselectivity in metabolism and pharmacokinetic of the parent compound and metabolites was observed. While the first enantiomer of methylone was found at higher concentration, both metabolites shown greater concentration for the second enantiomer. The results revealed that MET undergoes an enantioselective biotransformation to its metabolites HMMC and MDC, with S-(-)-MET more rapidly metabolized and eliminated from the body.

Usefulness of Oral Fluid for Measurement of Methylone and Its Metabolites: Correlation with Plasma Drug Concentrations and the Effect of Oral Fluid pH.

The aim of this study was to investigate methylone and its metabolites concentration in oral fluid following controlled increasing doses, focusing on the effect of oral fluid pH. Samples were obtained from a clinical trial where twelve healthy volunteers participated after ingestion of 50, 100, 150 and 200 mg of methylone. Concentration of methylone and its metabolites 4-hydroxy-3-methoxy-N-methylcathinone (HMMC) and 3,4-methylenedioxycathinone in oral fluid were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pharmacokinetic parameters were estimated, and the oral fluid-to-plasma ratio (OF/P) at each time interval was calculated and correlated with the oral fluid pH using data from our previous study in plasma. Methylone was detected at all time intervals after each dose; MDC and HMMC were not detectable after the lowest dose. Oral fluid concentrations of methylone ranged between 88.3-503.8, 85.5-5002.3, 182.8-13,201.8 and 214.6-22,684.6 ng/mL following 50, 100, 150 and 200 mg doses, respectively, peaked between 1.5 and 2.0 h, and were followed by a progressive decrease. Oral fluid pH was demonstrated to be affected by methylone administration. Oral fluid is a valid alternative to plasma for methylone determination for clinical and toxicological studies, allowing for a simple, easy and non-invasive sample collection.

New Psychoactive Substances and receding COVID-19 pandemic: really going back to "normal"?

To the Editor, The ongoing rise of New Psychoactive Substances (NPS), i.e. psychotropic molecules devised and synthesized to replicate the effects of traditional drugs of abuse in order to circumvent banned substances schedules, has been posing a challenge of enormous magnitude to substance detection systems and law enforcement worldwide. Still, it would be remiss to ignore the role played by the unprecedented public health emergency relating to the COVID-19 pandemic in the exacerbation of the NPS crisis. The diversion of resources has in fact hindered conventional approaches to drug monitoring, surveillance, control, and public health responses. The dangerous path ahead in our struggle against NPS abuse is best exemplified by the rather recent emergence of isotonitazene, an analogue of a benzimidazole class of analgesic compounds, powerful synthetic opioid and full mu-opioid receptor agonist belonging to the 2-benzylbenzimidazole group of compounds, which comprises the structurally different clonitazene, metonitazene and etonitazene (1). Isonitazene has reportedly been detected on European markets in at least five different forms and could even supplant fentanyl derivatives (2). Currently available data on isonitazene-related abuse and fatalities seem to be emblematic of the volatile, elusive nature of NPS: deaths in which isotonitazene was involved in fact presented substantial differences from casualties arising from synthetic opioids abuse. Case reports have highlighted how flualprazolam was detected in most fatalities associated with isotonitazene whereas flualprazolam was involved in only 8% of other synthetic opioid overdose deaths (3). Rather than rising background use, such a finding seems to suggest likely co-use or co-distribution of flualprazolam and isotonitazene. The key element of polysubstance involvement is rife in synthetic opioid overdose deaths. That being said, significantly more substances were implicated in isotonitazene-related deaths than fatalities linked to other synthetic opioid overdose (4, 5). Such dynamics and mortality patterns further stress the urgency of expanding health services for those suffering from opioid addiction disorders. Fine-tuned and standardized detection mechanisms relying on specialized assays based on sensitive instrumentation are essential for the timely and accurate characterization of such novel synthetic opioids (6-8). Isotonitazene in fact cannot be detected by common fentanyl testing strips (9). Hence, the essential nature of clinical and toxicological cannot be overstated, if we are to effectively deal with the public health risks arising from new substances or classes, along with the healthcare and social costs thereof (10). As new substances appear on illicit markets and are detected, their distinctive traits can only be identified by user experience, in the early stages (11-13). Nonetheless, the pandemic scenario has brought about a profound alteration of substance abuse patterns, and opened up new avenues of supply and demand for which our surveillance/detection systems may not be fully prepared or well-suited. As the pandemic appears to recede and hopefully turn into an endemic context based on coexistence with the SARS-CoV-2 and its less harmful variants, it would be a mistake to take for granted that drug abuse/trafficking dynamics will also get back to where they were before the pandemic. Putting in place policies aimed at monitoring web-based platforms and social media can potentially constitute a valuable tool in terms of keeping in check emerging substances, given how during the COVID-19 pandemic many interactions between traffickers and buyers have moved online (14). After all, social media have been playing an increasingly relevant role as interacting platforms, which users and drug dealers can take advantage of in order to discuss drug prices, substance purity, distinctive traits of the "high" (i.e. desired drug effects) they are seeking, ways of taking the substances, dosages, and characteristics of any new NPS becoming available on such back-alley marketing channels (15). Softwares designed and specifically programmed to sift through and analyze all detectable online information in that regard may prove valuable to figure out evolving dynamics of trafficking, purchases and use. Probing social media users has proven effective tool for public health concerns, e.g. drug checking services which have been harnessed due to their harm reduction potential in places estimated to be at risk, with large crowds gathering (concerts, clubs and the like). Nonetheless, research efforts need to be directed towards the new realm of criminality, the "Dark Web", in which all sorts of illegal exchanges and interactions are known to take place. A 2020 study has highlighted the appalling risks for drug users who choose to pursue that option in order to buy drugs (16). Three dealers were selected on a specific "Dark Web" marketplace, and NPS were ordered through such a channel. All these exchanges were thoroughly documented, and an analysis was undertaken of all the substances thus bought, totaling nine samples, by NMR, HRMS, LC-UV, and two also by x-ray diffraction. It was ultimately concluded that four out of five substances bought had been labeled with NPS names that did not match the actual substance, and two out of three samples of substances sold as new (i.e. unscheduled) NPS were instead found to be already documented substances, mislabeled and peddled under false pretenses. Drug dealers were therefore either deceiving their clients or were unaware as to the actual substances which they were selling. In light of such extremely worrisome findings, it is not hard to understand the implications and the major public health risks that such new trends of trafficking and abuse may entail. It is therefore incumbent upon the scientific community and law enforcement agencies to adapt and strive to meet the new challenges brought by the new criminal ecosystems in terms of drug enforcement, first and foremost the impervious environment known as "Dark Web" relying on untraceable cryptocurrencies for illegal transactions.