Hair analysis: Assessment of homemade hair treatment effects on drug concentrations in the keratin matrix.

Hair is the matrix of choice for investigating a subject's drug history over time, usually with specific forensic applications (license renewal, workplace drug testing, toxicological evaluation), and it is generally considered difficult to be tampered with. Nevertheless, some treatments promising to lower drug concentrations in hair are described online as how to "pass" a drug test. We selected three of these practices, claiming to be effective in decreasing drug concentrations-Treatment 1: (A) baking soda, (B) salicylic acid, (C) bleach; Treatment 2: (A) bleaching and (B) dyeing; Treatment 3: (A) white vinegar, (B) salicylic acid moisturizer, (C) liquid cleanser, and (D) dyeing. Quantitative results were compared with those of untreated hair strands, used as reference. We evaluated the efficacy of the treatment on drugs of abuse and benzodiazepines. Treatment 1 proved to be the most effective, since drug concentrations in treated hair were significantly lower than in untreated ones, although methadone and tetrahydrocannabinol (THC) seemed to be less affected than cocaine and 6-monoacetylmorphine (MAM). The mean percentage values of treatment-induced decrease were up to 90% for cocaine, 81% for benzoylecgonine, 77% for morphine, 89% for MAM, 37% for methadone, 67% for ketamine, 80% for MDMA, 76% for methamphetamine, and 60% for THC, compared with the reference samples. There was no noticeable damage or discoloration of the keratin matrix, making it difficult for the technicians to determine if there was a treatment. This could be an issue for the application of cutoffs or when low concentrations of drugs are incorporated into the keratinic matrix.

Pharmaco-Toxicological Effects of Atypical Synthetic Cathinone Mephtetramine (MTTA) in Mice: Possible Reasons for Its Brief Appearance over NPSs Scene.

Over the last year, NPSs have been steadily on the rise in the illicit drug market. Among these, synthetic cathinones seem to become increasingly popular among young adults, mainly because of their ability to replicate the effects of traditional psychostimulant drugs, such as cocaine, MDMA and amphetamines. However, scarce data are available about the in vivo pharmaco-toxicology of these new substances. To this end, this study focused on evaluation of effects induced by repeated administration of mephtetramine (MTTA 0.1-30 mg/kg i.p.) in mice. This atypical cathinone highlighted a sensorial (inhibition of visual and acoustic reflexes) and transient physiological parameter (decrease in breath rate and temperature) change in mice. Regarding motor activity, both a dose-dependent increase (accelerod test) and biphasic effect (drag and mobility time test) have been shown. In addition, blood and urine samples have been analysed to enrich the experimental featuring of the present study with reference to evaluation of potential toxicity related to consumption of MTTA. The latter analysis has particularly revealed important changes in blood cells count and blood and urine physicochemical profile after repeated treatment with this atypical cathinone. Moreover, MTTA induced histological changes in heart, kidney and liver samples, emphasizing its potential toxicity.

Behavioral and Pharmacokinetics Studies of N-Methyl-2-Aminoindane (NM2AI) in Mice: An Aminoindane Briefly Used in the Illicit Drug Market.

Drug forums are considered as the main platform sources that have contributed to the increase in NPS popularity, especially for those not yet known to law enforcement and therefore not yet illegal. An example is the new synthetic stimulant NM2AI, which has a very short history of human use and abuse. Little is known regarding this compound, but some information from internet forums and the scientific literature indicates NM2AI as a structural derivate of MDAI, which is known for its entactogenic activity. Indeed, the purpose of this study is to evaluate, for the first time, the in vivo acute effect induced by the intraperitoneal injection of NM2AI (1-10-30-100 mg/kg) in mice. We demonstrate the sensory (by visual placing and object tests) and physiological (core temperature measurement) function variations, nociceptor (by tail pinch test) and strength (grip test) alterations, and sensorimotor (time on rod and mobility) decrease. Moreover, we verify the mild hallucinogenic effect of NM2AI (by startle/prepulse inhibition test). Lastly, we perform a pharmacokinetic study on mice blood samples, highlighting that the main active metabolite of NM2AI is 2-aminoindane (2AI). Taken together, our data confirm the suspected entactogenic activity of NM2AI; however, these in vivo effects appear atypical and less intense with respect to those induced by the classic stimulants, in surprising analogy with what is reported by networked users.

Drug-impaired driving and traffic collisions: Study on a cross section of the Italian population.

The present study focuses on the association between road accidents and the presence of drugs of abuse markers in the biological fluids of the drivers. Biological fluids collected from 1236 drivers involved in road accidents (54 fatal and 1182 non-fatal crashes) in the Rome area were analyzed for alcohol and psychotropic drugs, as required by judicial authorities. The substance most frequently detected was alcohol (in 19% of non-fatal and 32% of fatal crashes), followed by cannabinoids (12% of non-fatal crashes) and cocaine (9% of non-fatal and 20% of fatal crashes). The results obtained for cocaine and cannabinoids in blood and urine were compared. We observed the absence or low concentrations of the active drug in blood (cocaine was often below 5 ng/ml and THC below 1 ng/ml), whereas urinary concentrations of metabolites were generally high (benzoylecgonine 250-above 5000 ng/ml, THCCOOH 15-270 ng/ml). The risk of being involved in a road accident if cocaine or cannabis markers were present in the urine specimens was evaluated compared to a control population. The odds ratios calculated, being 8.13 for cannabis and 5.32 for cocaine, suggest a strong association between the presence of these drugs in the urine of drivers and traffic accidents, regardless of their presence in blood samples. The present data suggest that the chance of being involved in a road accident is higher than in the control population even if the subject is no longer "under the influence" of cannabis or cocaine at the time of the accident.

A forensic procedure based on GC-MS, HPLC-HRMS and IBA to analyse products containing sildenafil or the doping agent oxandrolone.

The market of falsified or sub-standard medical products is a global scale phenomenon. This issue affects a wide range of medications, including life-saving medical products. In high-income countries the most falsified products are those defined "lifestyle", which include foremost anabolic steroids and phosphodiesterase 5 inhibitors. The spread of these products in the last years has been possible also because of their online purchase, since they can be bought anonymously and without any medical supervision or prescription. Their use can pose a serious threat for public health, especially because often are manufactured without adherence to quality standards. This leads to final products containing active ingredients different from those declared, at the wrong or unknown dose and contaminated with metals, synthesis by-products and other chemical substances. In this work, we present results on characterisation of illegal pharmaceutical products and doping agents by combining different techniques: chromatography coupled to mass spectrometry for organic analysis and accelerator-based nuclear analytical techniques, such as ion beam analysis (IBA), for elemental analysis. Three IBA techniques, namely PIXE (particle induced X-ray emission), PIGE (particle induced gamma-ray emission) and EBS (elastic backscattering spectrometry) were used in external beam mode to provide an elemental characterisation of the as-is material, placed simply in front of the proton beam, thus avoiding the need of preparing them with pre-analytical steps and greatly enhancing the measurement throughput. Several elements (F, Mg, Al, Si, P, S, Cl, K, Ca, Ti, V, Mn, Fe, Co, Ni, Cu, Zn, Br and Sr) were identified in the analysed products. External beam IBA measurements provided the quantitative elemental characterisation of the illegal pharmaceutical products and doping agents under study, complementary to the organic analysis results by chromatography and mass spectrometry thus allowing a rapid (a few minutes) and non-destructive direct assessment of the material for forensic purposes. For the first time IBA results from doping products are reported and further analysis by IBA involving two different accelerator laboratories (one in Italy and one in Brazil) allowed the comparison of results obtained on the same pharmaceutical product. Starting from the results obtained in our study, the actualisation of new research plans should be evaluated, which could lay the foundation for a classification system of illegal pharmaceutical products, doping products. and other substances, based on chromatography, mass spectrometry and IBA measurements; this could allow drawing inferences about the common characteristics of these substances, e.g. provenience of bulk materials, site of production etc. With this purpose, results obtained from two samples of the same pharmaceutical product by IBA in two different accelerator laboratories (one in Italy and one in Brazil) are compared.

Method development for the identification of methoxpropamine, 2-fluoro-deschloroketamine and deschloroketamine and their main metabolites in blood and hair and forensic application.

The constant increase of new psychoactive substances, often available on the illicit drug market as 'research chemicals', poses a concern for public health and a significant analytical and legislative challenge. Β-keto-arylcyclohexamines represent a class of dissociative anesthetics recently introduced on the market of New Psychoactive Substances (NPS). There is still a lack of information about the pharmacological activity of many of such substances, usually depending on the potential chemical modifications introduced to circumvent the law. Furthermore, their intake may not be fully intentional, since consumers do not always have knowledge of the content of online purchases. The present study describes the characterization by liquid chromatography-high resolution mass spectrometry (LC-HRMS), using a benchtop Orbitrap instrument, of the novel ketamine analogues methoxpropamine, 2-fluoro-deschloroketamine and deschloroketamine, found in the post-mortem blood and hair samples from a forensic case of suicide by fall from height, and of some of their metabolites. This allowed the development of analytical methods for the determination of both the ß-keto-arylcyclohexamines and the metabolites in LC-HRMS and in LC-MS/MS, providing a starting point for studying their toxicokinetics.

Metabolism study and toxicological determination of mephtetramine in biological samples by liquid chromatography coupled with high-resolution mass spectrometry.

The emerging market of new psychoactive substances (NPSs) is a global-scale phenomenon, and their identification in biological samples is challenging because of the lack of information about their metabolism and pharmacokinetic. In this study, we performed in silico metabolic pathway prediction and in vivo metabolism experiments, in order to identify the main metabolites of mephtetramine (MTTA), an NPS found in seizures since 2013. MetaSite™ software was used for in silico metabolism predictions and subsequently the presence of metabolites in the blood, urine, and hair of mice after MTTA administration was verified. The biological samples were analyzed by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) using a benchtop Orbitrap instrument. This confirmed the concordance between software prediction and experimental results in biological samples. The metabolites were identified by their accurate masses and fragmentation patterns. LC-HRMS analysis identified the dehydrogenated and demethylated-dehydrogenated metabolites, together with unmodified MTTA in the blood samples. Besides unmodified MTTA, 10 main metabolites were detected in urine. In hair samples, only demethyl MTTA was detected along with MTTA. The combination of Metasite™ prediction and in vivo experiment was a powerful tool for studying MTTA metabolism. This approach enabled the development of the analytical method for the detection of MTTA and its main metabolites in biological samples. The development of analytical methods for the identification of new drugs and their main metabolites is extremely useful for the detection of NPS in biological specimens. Indeed, high throughput methods are precious to uncover the actual extent of use of NPS and their toxicity.

Metabolism Study of N-Methyl 2-Aminoindane (NM2AI) and Determination of Metabolites in Biological Samples by LC-HRMS.

Since the widespread diffusion of new psychoactive substances, forensic laboratories are often required to identify new drugs and their metabolites for which information or reference standards are lacking. We performed a study on N-methyl-2-aminoindane (NM2AI) metabolism in silico and in vivo, in order to identify the main metabolites to be screened in the different biological samples. We performed the in silico metabolism prediction of NM2AI using MetaSiteTM software and subsequently verified the presence of metabolites in the blood, urine and hair of mice after NM2AI administration. The samples were analyzed by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) with a benchtop Orbitrap Exactive mass detector. This allowed the evaluation of the agreement between software prediction and experimental results in biological samples. LC-HRMS analysis identified seven main metabolites in the urine. They were identified, by their accurate masses and fragmentation patterns, as 2-aminoindane (2AI), two hydroxy-2AI and four hydroxy-NM2AI; one of the hydroxy-NM2AI and one of the hydroxy-2AI underwent also to conjugation. NM2AI and 2AI were also detected by LC-HRMS in the hair and blood. Based on these findings, we developed an LC-HRMS method for the screening of NM2AI and metabolites in urine, blood and hair samples. This can be of primary effectiveness to uncover the abuse of NM2AI and related possible intoxications.

An overview on performance and image enhancing drugs (PIEDs) confiscated in Italy in the period 2017-2019.

CONTEXT: The illegal market of counterfeit and falsified medicines and supplements containing unlabeled pharmaceuticals is expanding worldwide. They are usually referred to by the term "performance and image enhancing drugs" (PIEDs) and are mainly steroids, stimulants, hormones, and drugs for erectile dysfunction. PIEDs are easily accessible through the online or black markets. We analyzed over 400 such medicines confiscated in Italy in the period 2017-2019, to determine their composition. METHODS: Confiscated products were analyzed by gas chromatography/mass spectrometry and liquid chromatography/high-resolution mass spectrometry, in order to ascertain their composition and to evaluate the correspondence between what was declared on the label and the actual content, or to identify unknown products. RESULTS: The most commonly found substance was anabolic steroids, found in 64% of products, with 11% containing hormone modulators, 6% stimulants, 6% sexual enhancers (mainly sildenafil) and other drugs, including thyroid hormones, melanin stimulators, and vitamins. These substances were often in mixtures. The products were often mislabeled, containing contaminants in addition to the drug declared, or consisted of a drug completely different from the one reported on the label. Fifteen percent of products had a qualitative composition completely different from that declared, while 10% of products showed cross-contamination with other drugs, mainly testosterone esters, probably due to the presence of residues of other drugs in the production line. In addition, 11% of products were not labeled, so their purported composition was unknown. DISCUSSION: PIEDs pose a threat to public health. The main risks are related to the intrinsic toxicity of the substances found, especially when taken without a therapeutic indication. Another issue is related to the mislabeling of the fake medicines, and the poor-quality standard of counterfeit product preparation, with additional risks of the presence of other toxic ingredients or microbial contamination. CONCLUSIONS: The use of counterfeit products is a public health concern, as it constitutes a high risk for consumer health. It is mainly caused by the uncontrolled use of steroids, stimulants, sexual enhancers, and other medicaments, without medical indication or supervision, with variable and unknown compositions and doses, as well as other contaminants as a result of the absence of good manufacturing practices.

LC-HRMS characterization of the skin pigmentation and sexual enhancers melanotan II and bremelanotide sold on the black market of performance and image enhancing drugs.

The spread of performance and image enhancing drugs (PIEDs) often requires forensic toxicology laboratories to identify unknown compounds without reference standards. We characterized the PIEDs melanotan II and bremelanotide, not legally marketed, in eight unknown samples confiscated by police together with anabolic steroids, hormone modulators, sexual enhancers and stimulants, intended for the black market of bodybuilders, using liquid chromatography-high resolution/high accuracy Orbitrap mass spectrometry (LC-HRMS). The characterization was carried out by the accurate mass measurements of MH+ ionic species, the study of their isotopic patterns and the associated relative isotopic abundance (RIA) values, as well as the accurate mass measurements of collision-induced product ions obtained in fragmentation experiments. LC-HRMS confirmed itself as a powerful analytical tool to elucidate the elemental composition and structural characteristics of unknown compounds.

Application of ultrasound-assisted liquid-liquid microextraction coupled with gas chromatography and mass spectrometry for the rapid determination of synthetic cannabinoids and metabolites in biological samples.

The constant emergence of new psychoactive substances is a challenge to clinical and forensic toxicologists who need to constantly update analytical techniques to detect them. A large portion of these substances are synthetic cannabinoids. The aim of this study was to develop a rapid and simple method for the determination of synthetic cannabinoids and their metabolites in urine and blood using gas chromatography-mass spectrometry. The method involves an ultrasound-assisted dispersive liquid-liquid microextraction that implies a rapid procedure, giving excellent extraction efficiencies with minimal use of toxic solvents. This is followed by silylation and analysis with gas chromatography-mass spectrometry. The chromatographic method allows for the separation and identification of 29 selected synthetic cannabinoids and some metabolites. The method was validated on urine and blood samples with the ability to detect and quantify all analytes with satisfactory limits of detection (from 1 to 5 ng/mL), limits of quantification (5 ng/mL), and selectivity and linearity (in the range of 5-200 ng/mL). The developed assay is highly applicable to laboratories with limited instrumental availability, due to the use of efficient and low-cost sample preparation and instrumental equipment. The latter may contribute to enhance the detection of new psychoactive substances in clinical and forensic toxicology laboratories.

A Probable Fatal Case of Oleander (Nerium oleander) Poisoning on a Cattle Farm: A New Method of Detection and Quantification of the Oleandrin Toxin in Rumen.

Oleander (Nerium oleander) is an ornamental plant common in tropical and sub-tropical regions that is becoming increasingly widespread, even in temperate regions. Oleander poisoning may occur in animals and humans. The main active components contained in the plant are cardiac glycosides belonging to the class of cardenolides that are toxic to many species, from human to insects. This work describes a case of oleander poisoning that occurred on a small cattle farm and resulted in the fatality of all six resident animals. Furthermore, the investigation of the poisonous agent is described, with particular focus on the characterization of the oleandrin toxin that was recovered from the forage and rumen contents. The innovation of this study is the first description of the detection and quantification of the oleandrin toxin by liquid chromatography-high resolution mass spectrometry (LC-HRMS) in rumen.

HPLC-MS/MS combined with membrane-protected molecularly imprinted polymer micro-solid-phase extraction for synthetic cathinones monitoring in urine.

Synthetic cathinones are a type of drug belonging to group of new psychoactive substances (NPSs). The illicit market for these substances is characterized by the continuous introduction to the market of new analogs to evade legislation and to avoid detection. New screening and confirmation assays are therefore needed, mainly in forensic/clinical samples. In the current development, a porous membrane-protected, micro-solid-phase extraction (µ-SPE) has been developed for the assessment of several cathinones in urine. The µ-SPE device consisted of a cone-shaped polypropylene (PP) porous membrane containing the adsorbent (molecularly imprinted polymers, MIPs, synthesized for the first time for this class of drugs). MIPs were prepared using ethylone and 3-methylmethcathinone (3-MMC) as templates, ethylene glycol dimethacrylate (EGDMA) as a functional monomer, divinylbenzene (DVB) as a cross-linker, and 2,2´-azobisisobutyronitrile (AIBN) as an initiator. The prepared ethylone-based MIP and 3-MMC-based MIP have been fully characterized and evaluated as new selective adsorbents for µ-SPE. Cathinones separation/determination was performed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Optimum loading conditions (pH 5.0, loading for 4.0 minutes under orbital-horizontal shaking at 200 rpm) and elution conditions [2.0 mL of 75:20:5 heptane/2-propanol/ammonium hydroxide and ultrasounds assistance (37 kHz, 325 W) for 4.0 minutes] were found for ethylone-based MIP. Validation (intra-day and inter-day precision and analytical recovery) showed RSD values lower than 9 and 10% for intra-day and inter-day precision, and within the 88%-101% range for intra-day and inter-day analytical recovery.

Instrumental neutron activation analysis (INAA) and liquid chromatography (LC) coupled to high resolution mass spectrometry (HRMS) characterisation of sildenafil based products seized on the Italian illegal market.

The commerce of illegal and counterfeit medicinal products on internet is a serious criminal problem. Drugs for erectile dysfunction such as phosphodiesterase type 5 inhibitor are the most commonly counterfeited medicines in Europe. The search of possible toxic chemical substances in seized products is needed. Moreover, the profiling of the material can be the source of relevant forensic information. For the first time a combined approach based on liquid chromatography (LC) coupled to high resolution mass spectrometry (HRMS) and instrumental neutron activation analysis (INAA) is proposed and tested, allowing characterisation of both authentic and illegal pharmaceuticals containing sildenafil seized in Italy. LC-HRMS allowed the detection and identification of unknown impurities not reported on labels in illegal products and the quantitation of the sildenafil. INAA showed to be suitable to provide both qualitative and quantitative information for forensic purposes on 23 elements, allowing discrimination between legal and illegal products.

Development of a micro-solid-phase extraction molecularly imprinted polymer technique for synthetic cannabinoids assessment in urine followed by liquid chromatography-tandem mass spectrometry.

Several molecularly imprinted polymers (MIPs) have been synthesized for the first time using various synthetic cannabinoids (JWH007, JWH015 and JWH098) as template molecules. Ethylene dimethacrylate (EDMA) was used as a functional monomer for all cases. Similarly, divinylbenzene (DVB) and 2,2'-azobisisobutyronitrile (AIBN) were used as cross-linker and initiator, respectively. The prepared MIPs have been fully characterized and evaluated as new selective adsorbents for micro-solid phase extraction (µ-SPE) of synthetic cannabinoids in urine. The developed MIP-µ-SPE devices consisted of a polypropylene (PP) porous membrane containing the adsorbent (novel porous membrane protected micro-solid phase extraction based on a cone-shaped device) for operating in batch mode, which allowed a fast and integrated extraction-cleanup procedure. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used for quantifying the analytes after MIP-µ-SPE. The best performances were obtained for MIPs prepared from JWH015 as a template. Optimum loading conditions were found to be urine pH of 5.0 and adsorption time of 8.0 min under mechanical (orbital-horizontal) stirring at 100 rpm. The composition of the eluting solution consisted of 75:20:5 heptane/2-propanol/ammonium hydroxide. The elution was assisted by ultrasounds (37 kHz, 325 W) for 8.0 min. In addition, studies regarding selectivity have also been addressed for several drugs of abuse under optimized loading/adsorption conditions. Validation of the method showed good precision and analytical recovery by intra-day and inter-day assays (RSD values lower than 7 and 10% for intra-day and inter-day precision, and within the 83-100% range for intra-day and inter-day analytical recovery).

Rapid and simple procedure for the determination of cathinones, amphetamine-like stimulants and other new psychoactive substances in blood and urine by GC-MS.

In the last few years an increasing number of new psychoactive substances (NPS), with different chemical structures (of which 37% are stimulants), have been released into the illicit drug market. Their detection and identification in biological samples is hence of great concern. The aim of this work was to develop a high-throughput and rapid method for the determination of different classes of stimulants (amphetamine-type stimulants, cathinones, phenethylamines and ketamine analogues) from blood and urine samples using GC-MS. The proposed method allows the almost simultaneous derivatization and extraction of analytes from biological samples in a very short time, by using hexyl chloroformate as derivatization agent. The extraction of analytes was performed by Dispersive Liquid Liquid Microextraction (DLLME), a very rapid, cheap and efficient extraction technique that employs microliter amounts of organic solvents. The chromatographic method allowed for the separation of 26 stimulants including positional isomers (3-MMC and 4-MMC). The method was validated on urine and blood samples with the ability to detect and quantify all analytes with satisfactory limits of detection (LODs) ranging between 1 and 10ng/mL, limits of quantification (LOQs) between 2 and 50ng/mL, selectivity and linearity (5-1000ng/mL). The method was then applied to real samples from forensic cases, demonstrating its suitability for the screening of a wide number of stimulants in biological specimens.

A snapshot on NPS in Italy: Distribution of drugs in seized materials analysed in an Italian forensic laboratory in the period 2013-2015.

The diffusion of New Psychoactive Substances (NPS) in the illicit drug market is a worldwide problem. The aim of the study is to describe the qualitative distribution of drugs of abuse in seized materials confiscated in the Italian territory over the last two years. Between 2013 and 2015 162 seizures of substances purchased through the Internet and confiscated by police authorities were analyzed: 35 seizures (22%) were crystals of 3-methylmethcathinone (3-MMC). Although 3-MMC is subject to the relevant legislation in Italy, it is not controlled in other countries such as the Netherlands, from which the shipments originated. 33 seizures (20%) were crystals of 4-methylethcathinone (4-MEC), 19 seizures (12%) were powders containing methylenedioxypyrovalerone (MDPV). N,N-diallyl-5-methoxytryptamine (5-MeO-DALT) was identified in 5 powders, whereas ethylphenidate in six and pyrrolidinophenones in fourteen seized powders: 6 α-PVP (alpha-pyrrolidinovalerophenone), 6 α-PHP (alpha-pyrrolidinohexiophenone) and 1 α-PVT (alpha-pyrrolidinopentiothiophenone). Other substances identified were cathinones such as pentedrone, methylone, buthylone, ethylone, methedrone, 3-CMC (3-chloromethcathinone), 3,4-dimethylmethcathinone (3,4-DMMC), flephedrone (4-fluoromethcathinone or 4-FMC), 2-FMC and 3-FMC (2- and 3-fluoromethcathinone), MPPP (4-methyl-alpha-pyrrolidinopropiophenone), bk-2C-B (2-amino-1-(4-bromo-2,5-dimethoxyphenyl)ethan-1-one). Other compounds were NM2AI (N-methyl-2-aminoindane), MPA (1-(thiophen-2-yl)-2-methylaminopropane), MTTA (mephtetramine), 4-APB and 6-APB (4- and 6- (2-aminopropyl)benzofuran), 2-fluoromethamphetamine, 1mCPP (1-meta-chlorophenylpiperazine) and diphenidine, detected for the first time in Europe. Only three seizures contained synthetic cannabinoids, consisting of herbal blends soaked in N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide (AKB48), or a mixture of 5-F-AKB48 and BB-22 (1-(cyclohexylmethyl)-8-quinolinyl ester-1H-indole-3-carboxylic acid). In some mixtures of drugs - such as granules - 4-MEC and pentedrone were detected, also with traces of diphenidine on one occasion. In other cases 5-MeO-DALT, ethylphenidate and caffeine were mixed together. In one batch, the mixture was flephedrone and methoxethamine, whereas in another one the sample contained methylone, ethylone, methedrone, 4-fluoroamphetamine, 5-MeO-DALT and 5MeO-MiPT (N-methyl-N-isopropyl-5-methoxytryptamine). In 9 seizures, tablets shipped together with NPS were also found to contain sildenafil. The analyses performed on these seizures showed the presence of a wide number of NPS within the Italian boundaries coming from abroad, therefore this study confirms the threat for the public health, especially when the content of NPS being sold is not reported on the label or misleading.