OILVEQ: an Italian external quality control scheme for cannabinoids analysis in galenic preparations of cannabis oil.

OBJECTIVES: Italy legalized cannabis oil for specific medical conditions (neuropathic pain, refractory epilepsy and other established pathologies) in 2015, but mandates titration of principal cannabinoids before marketing each batch using iphenated techniques coupled with mass spectrometry. To assess reliability of laboratories from the Italian National Health Service in charge of titrating the batches, the Italian National Institute of Health set up an quality control program on determination of Δ9-tetrahydrocannabinol l (THC), cannabidiol (CBD), Δ9-tetrahydrocannabinolic acid A (THCA-A) and cannabidiolic acid (CBDA) in cannabis oil preparations. METHODS: Two rounds of exercises have been carried out since 2019, involving sixteen Italian laboratories. Five different cannabis oil samples (19-1A and 19-1B for the first round and 22-1A, 22-1B and 22-1C for the second one were prepared and 1 mL amount of each sample was sent to the laboratories. The quantitative performance of each laboratory was assessed calculating the z-score value, a statistical measurement for value's relationship to the mean of a group of values. RESULTS: In the first round, eight out of fourteen laboratories employed an LC-MS while the remaining six used GC-MS. Differently, in the second round, six out of eleven laboratories employed a GC-MS while the remaining five used LC-MS. In the first round, only 28.6 % laboratories achieved an acceptable performance (z-score±2), and all of them used LC-MS as analytical method. In the second round, none of the laboratories achieved an acceptable performance. Satisfactory results, based on z-scores, were generally low (0.0-75.0 %), with only one exception of 100 % for THCA-A determination in sample 22-1B. In the second round, three false negatives (two THC and one CBD by GC-MS determination) were reported while no false positives were described in the blank sample. The two rounds yielded a mean ERR% of 42 % approximately and a mean CV% around 70 % in GC-MS determination. When applying LC-MS determination, the two rounds yielded a mean ERR% of 36 % approximately and a mean CV% around 33 %. CONCLUSIONS: The obtained results underline the need for a clear and consistent protocol to be adopted by all laboratories intending to include the titration of oily cannabis-based products into their routinely analytical techniques. This emphasis on methodology standardization and participation to quality control schemes is essential for ensuring reliable and accurate measurements, ultimately enhancing the overall effectiveness and reliability of medical cannabis treatments.

NPS-EQA PART I: Four years' experience in external quality assessment program in Italy for classical and new psychoactive substances analysis in oral fluid.

In 2019, Italian National Institute of Health established an external quality assessment program (EQA) to evaluate the performance of oral fluid testing for classical and new psychoactive substances by laboratories participating in the National Early Warning System collaborative centres. This report presents the results of four rounds between 2019 and 2023. Eleven oral fluid specimens, including 3 blank samples, were prepared by adding different classes of and new psychoactive drugs at known concentrations to pre-screened drug-free oral fluid. False-negative and false-positive results were calculated for the qualitative data evaluation. The quantitative evaluation measured the imprecision and accuracy of the results, in terms of coefficient of variation (CV%) and percent error (ERR%), respectively, with respect to a mean value obtained by reference laboratories. Z-score values were then calculated. Over the years, there has been a significant improvement in false-negative results (from 42.7% in the first year to 19.4% in the last year), but not in false-positive results (from 33.3% in the first year to 22.2% in the last one). In addition to the classic drugs of abuse (e.g. cocaine, amphetamine, methadone), the substances found in false positive samples belonged to the class of synthetic cannabinoids (e.g 5-fluoro CUMYL-PINACA and 5-fluoro-EDMB-PICA), synthetic opioids (e.g butyrylfentanyl) and tryptamines (e.g. 5-methoxy-N-methyl-N-isopropyltryptamine). The four rounds yielded a mean ERR% of approximately 22.1% and a mean CV% of around 41.5%. The participating laboratories demonstrated variable performances in relation to the class of analysed psychoactive substances, as evidenced by the calculated Z-scores. Between 25% and 60% of the reported results in all rounds should be considered satisfactory. EQA is a crucial element of laboratory quality management systems. It promotes continuous improvement and maintains high standards in the field of forensic and clinical drug testing.

NPS-EQA PART II: Four years' experience in external quality assessment program in Italy for classical and new psychoactive substances analysis in hair.

In 2019, the Italian National Institute of Health established an external quality assessment (EQA) program to evaluate the performance of laboratories of collaborative centres participating in the National Early Warning System in hair testing for classical and new psychoactive substances (NPS). The results obtained in the four rounds (2019-2023) and the evolution in hair testing performance for classic drugs of abuse and new psychoactive substances are presented. A total of 11 hair specimens, including 3 blank samples, were prepared by adding different classes of classical and NPS at known concentrations to pre-screened drug-free hair. False negative and false positive results were calculated for the qualitative data evaluation. The quantitative evaluation included the imprecision (as % coefficient of variation, CV%) and the accuracy (as % error, ERR%) of the results with respect to a mean value obtained by reference laboratories and Z-score values were assessed. Over the years, an improvement in false negative results (from 52.4% in the first year to 34.3% in the last one) and false positive results (from 55.0% in the first year to 30.8.% in the last one) was observed. In the first round, the mean ERR% ranged from 6.2% to 112.8% due to NPS determination. However, in the subsequent three rounds, the mean ERR% ranged from 10.4% to 22.4%, The mean CV% in the four rounds was approximately 41.5% (ranging from 44.3% to 53.3%). Between 12.0% and 56.6% of the reported results in all rounds should be considered satisfactory. EQA programs help laboratories to identify and correct problems within their processes by highlighting errors and variations. This ensures that the results produced are accurate and reproducible.

Hexahydrocannabinol Pharmacology, Toxicology, and Analysis: The First Evidence for a Recent New Psychoactive Substance.

BACKGROUND: During the last two years, hexahydrocannabinol (HHC), the hydrogenated derivative of tetrahydrocannabinol has been freely sold by internet websites as a "legal" replacement to THC and cannabis in a range of highly attractive branded and unbranded products, some of which are sold as "legal highs". Potentially, there could be a large demand for HHC products by individuals in Europe and internationally. METHODS: Studies reporting HHC pharmacology, toxicology and analysis were identified from Pubmed and Scopus databases, and official international organizations' websites were considered. RESULTS: HHC showed the effects of the typical cannabinoid on the central nervous system, with lower potency than Δ9-THC. A few studies highlighted that 9(R)-HHC is more potent than 9(S)-HHC. This molecule showed an affinity for cannabinoid receptor CB1 both in vitro and in vivo, suggesting a possible therapeutic effect in several pathologies. However, the affinity for the CB1 receptor suggests a possible addiction potential, inducing the users to misuse it. Since actual intoxication cases have not yet been reported, the HHC harmful potential was not described, probably due to the lack of effective analytical methods to detect HHC in biological matrices. Conversely, different analytical assays were developed and validated to separate HHC epimers in natural and non-natural sources. CONCLUSION: Similarly to other NPS, the HHC represents a cheaper alternative to the controlled Δ9-THC. Its monitoring is a crucial challenge for toxicological and forensic purposes. To this concern, it is essential to further investigate HHC to support health providers in the identification of related intoxications.

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.

Ketamine: From Prescription Anaesthetic to a New Psychoactive Substance.

Discovered in the United States of America (USA) in the 1960s, ketamine was introduced as an anaesthetic drug to specifically replace phencyclidine. Briefly, the substance moved from the medical world to recreational users, since it was discovered that intense psychedelic experiences were obtained with dosages lower than those prescribed for anesthesia. At the end of the 90's, it was circulated in London nightclubs as a drug itself and as counterfeit 3,4-methylenedioxymethamphetamine tablets. In 1997, the Drug Enforcement Administration (DEA) alerted the United States (US) government about the increasing diffusion of ketamine in American 'clubs', and in 1999, the substance was added to Schedule III of drugs controlled by federal authorities. In 2002, ketamine epidemics moved to Europe, and the European Monitoring Centre for Drugs and Drug Addiction carried out a risk assessment monitoring of the phenomenon. An estimated ninety-nine percent of all global ketamine seizures occurred in Asia. Its growing popularity is due to the fact that this new psychoactive substance is cheaper than other stimulants, such as MDMA. Moreover, the amount used for recreational purposes does not cause respiratory depression and its legal use as a drug makes it widely available for a diversion towards illicit markets. Nevertheless, acute intoxication and several deaths have been related to exclusive ketamine use both in Europe and internationally. Since 2015, there has been an increasing rise in the illicit ketamine market, and currently, the drug is being used with unprecedented peaks and a consequent significant increase in seizures and clinical cases worldwide.

The complex and constantly evolving public health threat of new psychoactive substances in Italy: addressing the main functions of a national observatory of drugs.

At the end of the 90s in Europe, the new psychoactive substances (NPS) phenomenon was limited to a small number of molecules created to mimic the actions and psychoactive effects of licensed medicines and existing drugs that are controlled by the United Nations drug conventions and therefore traded as their "legal" replacements. NPS were mostly circulating in rave parties and electronic music festivals. The globalization, the evolution of e-commerce and the growing popularity of NPS, facilitated the development of a wide illegal market in constant expansion. The dynamic nature of this phenomenon has led to an evolution in the prevention and monitoring of NPS trafficking within the European Union. The European legislative system has been amended with the aim of creating a faster and more effective regulatory system to tackle NPS diffusion and ban their sale and circulation. At the end of 2008, in compliance with the European Council Decision 2005/387/JHA, the Anti-Drug Policies Department of the Presidency of the Council of Ministers activated the National Early Warning System to promote a rapid exchange of information on NPS between Italy and the EU.

New Psychoactive Substances: Evolution in the Exchange of Information and Innovative Legal Responses in the European Union.

At the end of 2019, the European Monitoring Centre for Drugs and Drug Addiction was monitoring around 790 new psychoactive substances, more than twice the total number of controlled substances under the United Nations Conventions. These substances, which are not subject to international drug controls, include a wide range of molecules, including the assortment of drugs such as synthetic cannabinoids, stimulants, opiates, and benzodiazepines. Most of them are sold as "legal" substitutes for illicit drugs, while others are intended for small groups willing to experiment with them in order to know their possible new effects. At the national level, various measures have been taken to control new substances and many European countries have responded with specific legislation in favor of consumer safety and by extending or adapting existing drug laws to incorporate the new psychoactive substances. Moreover, since 1997, an early warning system has been created in Europe for identifying and responding quickly to the risks of new psychoactive substances. In order to establish a quicker and more effective system to address the criminal activities associated with new dangerous psychoactive substances, the European legal framework has considerably changed over the years.

Evolution of Italian laws banning trafficking, use and abuse of psychotropic drugs.

The penalty system implemented by Italian law still represents a barrier against psychoactive drugs and drug addiction, especially at a time when the age of first consumption has considerably dropped. Presidential Decree n. 309 of October 9, 1990 entitled "Consolidation of the laws governing drugs and psychotropic substances, the prevention, treatment and rehabilitation of drug addicts", and referred to as Presidential Decree 309/90, is the reference text for the cultivation, production, trade and use of narcotics and other psychoactive substances in Italy. The Presidential Decree has its origins in the now-forgotten law of December 22, 1975, n. 685, amended by law 162/90, which provided a draft of the current Presidential Decree 309/90. The current text has been amended numerous times over the years.

Nails Analysis for Drugs Used in the Context of Chemsex: A Pilot Study.

Nail analysis can be performed as a substitute or complement to hair analysis for the retrospective determination of psychotropic drugs consumption in forensic contexts. The application of nail analysis in a "chemsex" context is reported herein. An ultra-performance liquid chromatography-tandem mass spectrometry method was developed to quantify the most common drugs of abuse, synthetic cannabinoids, synthetic cathinones and GHB in fingernails and toenails of individuals presumptively using these drugs in music and sex settings. Results were compared to the concentrations measured in hair. Nail and hair keratin matrices were digested with VMA-TM3® buffer reagent and underwent a solid phase extraction before chromatographic separation with reversed phase columns and a linear gradient elution with 5 mM ammonium formate and acetonitrile, for detecting classic drugs of abuse, or 0.1% formic acid and methanol, for detecting synthetic cathinones, synthetic cannabinoids, and GHB. Analytes were detected with a triple quadrupole mass spectrometer operated in multiple reaction monitoring mode after positive electrospray ionization. Nails of individuals practicing "chemsex" contained at least three different psychoactive drugs, and up to eight drugs simultaneously. Identified drugs included cocaine, benzoylecgonine, cocaethylene, delta-9-tetrahydrocannabinol, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, ketamine, norketamine, mephedrone, methylone, 4-methyletcathinone, methcathinone, γ-hydroxybutyric acid (GHB) and γ-butyrolactone. Most used drugs were MDMA and GHB followed by cocaine and ketamine. Drugs concentrations ranged from 0.1 to 690 ng/mg in fingernails. In the two individuals who also provided toenails, concentrations were always higher in fingernails than in toenails, while in two other individuals who donated also hair, concentrations in this latter matrix were either higher or lower than those measured in fingernails. This study demonstrated that nails may well represent an appropriate non-conventional biological matrix to provide additional information in forensic toxicology.

Simple and alpha,beta-unsaturated aldehydes: correct prediction of genotoxic activity through structure-activity relationship models.

Aldehydes are widespread environmental and industrial compounds, able to stimulate a range of adverse health effects (e.g., general toxicity, allergenic reactions, mutagenicity, and carcinogenicity). We have previously presented quantitative structure-activity relationships (QSARs) for the genotoxicity of simple and alpha,beta-unsaturated aliphatic aldehydes. In this study, we show that the QSAR models are able to correctly predict--based only on the knowledge of the chemical structure--the genotoxicity of other aldehydes, not considered in the development phase of the models. This adds confidence to the reliability of our QSAR models as tools for the theoretical assessment of the genotoxic hazard posed by aldehydes. The analysis of SOS Chromotest induction ability and the ease of formation of DNA adducts by the aldehydes provided further mechanistic insights.