Applying machine learning to international drug monitoring: classifying cannabis resin collected in Europe using cannabinoid concentrations.

In Europe, concentrations of ∆9-tetrahydrocannabinol (THC) in cannabis resin (also known as hash) have risen markedly in the past decade, potentially increasing risks of mental health disorders. Current approaches to international drug monitoring cannot distinguish between different types of cannabis resin which may have contrasting health effects due to THC and cannabidiol (CBD) content. Here, we compared concentrations of THC and CBD in different types of cannabis resin collected in Europe (either Moroccan-type, or Dutch-type). We then tested the ability of machine learning algorithms to classify the type of cannabis resin (either Moroccan-type, or Dutch-type) using routinely collected monitoring data on THC and CBD. Finally, we applied the optimal algorithm to new samples collected in countries where the type of cannabis resin was unknown, the UK and Denmark. Results showed that overall, Dutch-type samples had higher THC (Hedges' g = 2.39) and lower CBD (Hedges' g = 0.81) than Moroccan-type samples. A Support Vector Machine algorithm achieved classification accuracy exceeding 95%, with little variation in this estimate, good interpretability, and plausibility. It made contrasting predictions about the type of cannabis resin collected in the UK (94% Moroccan-type; 6% Dutch-type) and Denmark (36% Moroccan-type; 64% Dutch-type). In conclusion, we provide proof-of-concept evidence for the potential of machine learning to inform international drug monitoring. Our findings should not be interpreted as objective confirmatory evidence but suggest that Dutch-type cannabis resin has higher THC concentrations than Moroccan-type cannabis resin, which may contribute to variation in drug markets and health outcomes for people who use cannabis in Europe.

Characterization of the designer drug deschloroketamine (2-methylamino-2-phenylcyclohexanone) by gas chromatography/mass spectrometry, liquid chromatography/high-resolution mass spectrometry, multistage mass spectrometry, and nuclear magnetic resonance.

RATIONALE: Clinical and forensic toxicology laboratories are challenged every day by the analytical aspects of the new psychoactive substances phenomenon. In this study we describe the analytical characterization of a new ketamine derivative, deschloroketamine (2-methylamino-2-phenylcyclohexanone), contained in seized powders. METHODS: The analytical techniques employed include gas chromatography/mass spectrometry (GC/MS), liquid chromatography/electrospray ionization coupled with Orbitrap high-resolution/MS (LC/ESI-HRMS), multistage MS (ESI-MS(n)), and NMR. The LC/ESI-HRMS analyses consisted of accurate mass measurements of MH(+) ions in full-scan mode; comparison of experimental and calculated MH(+) isotopic patterns; and examination of the isotopic fine structure (IFS) of the M + 1, M + 2, M + 3 isotopic peaks relative to the monoisotopic M + 0 peak. The collision-induced product ions of the MH(+) ions were studied by both HRMS and MS(n). (1)H and (13)C NMR measurements were carried out to confirm the chemical structure of the analyte. RESULTS: The EI mass spectra obtained by GC/MS analysis showed the presence of molecular ions at m/z 203, and main fragment ions at m/z 175, 174, 160, 147, 146, and 132. The application of LC/ESI-HRMS allowed us to obtain: the accurate mass of deschloroketamine MH(+) ions with a mass accuracy of 1.47 ppm; fully superimposable experimental and calculated MH(+) isotopic patterns, with a relative isotopic abundance value of 3.69 %; and the IFS of the M + 1, M + 2, M + 3 isotopic peaks completely in accordance with theoretical values. Examination of the product ions of MH(+), as well as the study of both (1)H and (13)C NMR spectra, enabled the full characterization of the molecular structure of deschloroketamine. CONCLUSIONS: The combination of the employed analytical techniques allowed the characterization of the seized psychoactive substance, in spite of the lack of a reference standard. Deschloroketamine is a ketamine analogue considered to be more potent and longer lasting than ketamine, and this paper is probably the first to report on its analytical characterization.

Characterization of the designer drug bk-2C-B (2-amino-1-(bromo-dimethoxyphenyl)ethan-1-one) by gas chromatography/mass spectrometry without and with derivatization with 2,2,2-trichloroethyl chloroformate, liquid chromatography/high-resolution mass spectrometry, and nuclear magnetic resonance.

RATIONALE: We describe the analytical characterization of the designer drug bk-2C-B, a cathinone derivative, contained in a seized tablet, in the absence of an analytical standard. METHODS: The analytical techniques employed include gas chromatography/mass spectrometry (GC/MS), without and with derivatization with 2,2,2-trichloroethyl chloroformate, liquid chromatography/high-resolution-MS (LC/HRMS) with an Orbitrap® analyzer, and nuclear magnetic resonance (NMR). LC/HRMS measurements consisted of accurate mass measurements of MH(+) ionic species under full scan conditions; comparison of experimental and calculated MH(+) isotopic patterns; examination of the isotopic fine structure (IFS) of the M+1, M+2, M+3 isotopic peaks relative to the monoisotopic M+0 peak; study of MH(+) collision-induced dissociation (CID) product ions obtained in fragmentation experiments. RESULTS: GC/MS analysis gave highly informative EI mass spectra, particularly after the derivatization of bk-2C-B with 2,2,2-trichloroethyl chloroformate. The application of LC/HRMS, allowing for accurate mass measurements at 100,000 resolving power, greatly enhanced analytical capabilities in structural characterization of this new designer drug. HRMS allowed us to obtain the accurate mass measurements of bk-2C-B MH(+) ionic species, with a mass accuracy of 2.19 ppm; fully superimposable experimental and calculated MH(+) isotopic patterns, with RIA1 and RIA2 values <4%; the IFS of the M+1, M+2, M+3 isotopic peaks relative to the monoisotopic M+0 peak completely in accordance with theoretical values. These findings enabled us to obtain the elemental composition formula of the seized drug. Furthermore, characteristic MH(+) CID product ions enabled the characterization of the bk-2C-B molecular structure. The presence of (79)Br and (81)Br isotopes in the substance molecule produced a characteristic isotopic pattern in most MS spectra. Lastly, NMR spectra allowed us to obtain useful information about the position of substituents in the designer drug. CONCLUSIONS: The combination of all the analytical techniques employed allowed the characterization of the seized psychoactive substance, in spite of the lack of a reference standard.

Determination of cannabinoids in 50 µL whole blood samples by online extraction using turbulent flow chromatography and LC-HRAM-Orbitrap-MS: Application on driving under the influence of drugs cases.

The analysis of cannabinoids in whole blood is usually done by traditional mass spectrometry (MS) techniques, after offline cleanup or derivatization steps which can be lengthy, laborious, and expensive. We present a simple, fast, highly specific, and sensitive method for the determination of Δ9 -tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), 11-hydroxy-Δ9 -tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Δ9 -tetrahydrocannabinol (THC-COOH) in 50 µL whole blood samples. After the addition of deuterated internal standards (IS) and a simple protein precipitation step, an online extraction of sample supernatants using turbulent flow chromatography (TurboFlow-Thermo Scientific) was carried out. Analytes were separated on a C18 analytical column and detected by LC-HRAM-Orbitrap-MS using a Thermo Scientific Q Exactive Focus MS system. MS detection was performed in polarity switching and selected ion monitoring (SIM) modes using five specific acquisition windows, at a resolution of 70,000 (FWHM). Total run time was about 10 min including preanalytical steps. Method validation was carried out by determining limit of detection (LOD), lower limit of quantitation (LLOQ), linearity range, analytical accuracy, intra-assay and interassay precision, carry-over, matrix effect, extraction recovery, and selectivity, for all analytes. Measurement uncertainties were also evaluated, and a decision rule was set with confidence for forensic purposes. The method may become suitable for clinical and forensic toxicology applications, taking advantage of the small matrix volume required, the simple and cost-effective sample preparation procedure, and the fast analytical run time. Performances were monitored over a long-term period and tested on 7620 driving under the influence of drugs (DUID) samples, including 641 positive samples.

Drugs WorkBook (DWB): a tool for the analysis of illicit drugs in seized materials.

Accurate and reliable analytical measurements are essential when data are to be used to assist the Court in deciding whether or not a drug offence has been committed and therefore about either the innocence or guilt of the accused. The Italian law on drugs demands that compliance with specification limits be assessed on the basis of the actual content of controlled substance contained in seized materials. As a consequence, the role of measurement uncertainty, significant figures and rounding errors becomes critical. In order to assist analysts of forensic toxicology laboratories with illicit drug-related cases, a software tool named Drugs WorkBook (DWB) has been developed. The tool is useful for the quantification of illicit drugs in seized materials along with their measurement uncertainties, the assessment of compliance to specification limits, the printing of comprehensive laboratory reports and the organization of case archives. Other quality control topics, such as control charts, are included. The tool's databases can be edited by the user and maintained up to date. The tool is made freely available to the scientific community.

Predicting heroin potency from the analysis of paraphernalia: A tool for overdose prevention projects.

In recent years, fatal and non-fatal heroin-related overdoses have increased in northeastern Italy, and the change in potency of heroin available at street level has been identified as a prominent factor associated with acute toxicity. Two very different products, high-potency and low-potency heroin were becoming available on the street, and no clear morphological characteristics could be used to easily distinguish them. A theoretical model for predicting heroin potency from rapid analysis of cigarette filters was developed as part of an overdose prevention project. The model was derived from the analysis of real heroin samples and exploits the common presence of caffeine in heroin as an adulterant. It was tested on laboratory prepared filters, real filters used to prepare heroin injections, and other paraphernalia. The model showed strong predictive ability and was used to implement a rapid alert system to inform drug users and healthcare institutions about the potency of heroin or other psychoactive substances circulating in the area. Cigarette filters were used as standard material, but other paraphernalia were successfully tested. The developed model is a dynamic tool whose parameters can be updated according to the market characteristics, so it can be useful for laboratories involved in drug analysis and similar prevention programs.

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.

Analysis of the alcohol biomarker phosphatidylethanol by NACE with on-line ESI-MS.

Phosphatidylethanol (Peth), a group of aberrant phospholipids formed in cell membranes in the presence of ethanol, has been recently proposed as biomarker of chronic alcohol abuse. The aim of this study was to develop a new analytical method, based on NACE online coupled with a mass spectrometer for the analysis of Peth in blood. For this purpose an ion-trap mass spectrometer equipped with an orthogonal ESI source operating in negative ion mode was used. An alkaline solution of ammonium acetate 5 mM (pH 9) in water/methanol (MeOH) (80:20 v/v) was delivered as coaxial sheath liquid. All experiments were performed using an uncoated fused-silica capillary (90 cm x 75 microm id). The effects of variable percentages of ACN, MeOH, 2-propanol, dichloromethane, along with variable concentrations of ammonium acetate were investigated for the separation of Peth. Collectively, a separation medium composed of ACN (45% v/v), 2-propanol (20% v/v), dichloromethane (20% v/v), MeOH (10% v/v), water (5% v/v), and ammonium acetate (25 mM) was chosen. The estimated LOD was 0.1 microM, while LOQ was 0.4 microM. Within-run (intra-day) and between-run (inter-day) precision was always lower than 15%. The method proved to be robust and reliable. The MS detector allowed the simultaneous identification of several Peth homologues, and the use of an internal standard (phosphatidylbutanol) with similar electrophoretic properties of that of Peth increased quantitation effectiveness.

A novel and an effective analytical approach for the LC-MS determination of ethyl glucuronide and ethyl sulfate in urine.

An alternative liquid chromatography-mass spectrometry (LC-MS) method based on no discharge (ND) atmospheric pressure chemical ionization (APCI) was developed for the simultaneous determination of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in urine in negative ion conditions. Abundant [M-H](-) species of EtG and EtS were obtained, allowing to reach limits of quantification (0.1 microg/ml for both analytes), accuracy, and precision comparable to those proposed in the literature. Additionally, the LC-ND-APCI-MS method proved to be reliable, requiring little maintenance even when high throughput analyses (i.e., 6,000 samples per year) were required.

Neonatal hair analysis by liquid chromatography-high-resolution mass spectrometry to reveal gestational exposure to venlafaxine.

Venlafaxine (VEN) is a second generation antidepressant drug, belonging to the class of selective serotonine and norepinephrine reuptake inhibitors, widely used in the treatment of depression and anxiety disorders. Though its pharmacological profile is considered safe, treatment with VEN can cause several nervous, gastrointestinal, cardiovascular and genitourinary adverse effects. Therapeutic drug monitoring for VEN could be useful in specific situations, including exposure to the drug during pregnancy. A liquid chromatography-high-resolution mass spectrometry method was developed and validated for the assay of VEN in 2.5-mg hair samples from 2 newborn identical twin sisters. The analyte was extracted by a rapid, simultaneous pulverization and extraction step, allowing analysis when tiny amounts of hair are available, such as in the case of a newborn. Gradient elution on an Atlantis T3 column was performed using nordiazepam-d5 as an internal standard. Positive ion electrospray ionization and high-resolution full scan determination were performed in an Orbitrap mass spectrometer. The method was linear range in the range 0.2-25 ng/mg, and had a quantification limit of 0.2 ng/mg, a relative standard deviation in the range 0.7%-1.4% (intra-assay) and 2.9%-5.9% (interassay), and was accurate (as % relative error) in the range -9% to + 2%, using a hair sample size as low as 2.5 mg. The utilization of high-resolution/high accuracy mass spectrometry in full-scan mode allowed both the quantitative determination of VEN in the hair of the 2 newborns and the straightforward identification of 4 VEN metabolites, namely O-desmethylvenlafaxine, N-desmethylvenlafaxine, N,N-didesmethylvenlafaxine, and N,O-didesmethylvenlafaxine, by means of retrospective screening, thus unequivocally documenting in utero exposure.

Validation of a fast screening method for the detection of cocaine in hair by MALDI-MS.

The sensitivity and specificity of a novel method of screening for cocaine in hair, based on matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry (MS), have been evaluated. The method entails a rapid extraction procedure consisting of shaking 2.5 mg pulverised hair at high frequency in the presence of an acidic solution (160 microL of water, 20 microL of acetonitrile and 20 microL of 1 M trifluoroacetic acid) and a stainless-steel bullet. Following centrifugation, the supernatant is dried under a nitrogen stream, and the residue is reconstituted in 10 microL of methanol/trifluoroacetic acid (7:3; v/v). One microlitre of the extract is deposed on a MALDI sample holder previously scrubbed with graphite; an alpha-cyano-4-hydroxycinnamic acid (matrix) solution is electrosprayed over the dried sample surface to achieve a uniform distribution of matrix crystals. The identification of cocaine is obtained by post-source decay experiments performed on its MH(+) ion (m/z 304), with a limit of detection of 0.1 ng/mg of cocaine. A total of 304 hair samples were analysed in parallel by MALDI-MS and a reference gas chromatography-MS method. The obtained results demonstrate specificity and sensitivity of 100% for MALDI-MS. Evidence of cocaine presence was easily obtained even when hair samples exhibiting particularly low cocaine levels (<0.5 ng/mg) were analysed.

Cannabis potency in North-East Italy: A ten-year study (2010-2019).

This paper presents data about potency of herbal and resin cannabis products seized during 2010-2019 in north-east Italy. More than 12,000 cannabis samples were analyzed and concentrations of THC, CBD and CBN were collected. The results of our study provided clear evidence for an increase in the potency of cannabis products across the study period, which is consistent with other studies. Globally, the median THC concentrations increased from about 6%-11%, but differences were found between herbal and resin materials. THC potency in resin materials increased more consistently across the study period with a dramatic raise during 2018-2019, with median THC contents around 17%. CBD concentrations were found to decrease constantly over the study period, especially in herbal materials, which had a mean CBD concentration of 0.3%. In particular, about 75% of the analyzed herbal samples had a CBD concentration which was less than 3% of the corresponding THC concentration. In contrast, more than 50% of the analyzed resin materials had a CBD concentration which was about 30% of the corresponding THC concentration. This is consistent with the increase in prevalence of high-potency seedless female herbal products observed in the same period and indicates that herbal and resin materials were produced from different varieties of cannabis plants. However, resin materials derived from high THC/low CBD cannabis plants were recently found. Different routes (e.g. northern Europe) or different modalities of distribution were assumed for these products. CBN concentrations were also considered and found to be very low and consistent across the study period indicating reliability of THC values used in statistical analysis. In conclusion, this study provided an accurate picture of cannabis products seized over a decade over a definite geographical area which can be extremely helpful for comparative purposes and for national and international statistical analyses on cannabis products.

Cannabis knowledge and implications for health: Considerations regarding the legalization of non-medical cannabis.

Cannabis contains over a hundred of different cannabinoids, of which Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are the most well studied. The use of high-potency cannabis, containing high concentrations of THC and low concentrations of CBD, has been linked to adverse health outcomes, particularly for adolescents and young adults. Recently, an increase in cannabis potency has been observed in jurisdictions that legalized the sale of cannabis for non-medical purposes. Moreover, an increase of cannabis use and cannabis-related emergency treatment have also been observed in these jurisdictions. At the same time, risk perception regarding cannabis use has decreased in these populations. Trivializing language and an increased appearance of commercial cannabis in the public space may lead to a generalized underestimation of the risks of cannabis use. New regulation models principally focus on the creation of a legal cannabis market economy, the diversion of profits from illegal markets, and the reduction of costs associated with prohibition. However, an approach that specifically focuses on the rights to the health and safety of the individual should be considered in order to reduce the risks associated with cannabis legalization. Such an approach should promote and protect individual and social health and safety, establish a strict quality control of legal cannabis products regulated according to THC and CBD content, and eliminate all sorts of incentives to use, thus providing a more consistent, sustainable, and ethical framework for the legalization of non-medical cannabis use.

Gas Chromatography-Fourier Transform Infrared Spectroscopy for Unambiguous Determination of Illicit Drugs: A Proof of Concept.

The increasing number of synthetic molecules constantly introduced into the illicit drug market poses a great demand in terms of separation and identification power of the analytical tools. Therefore, forensic laboratories are challenged to develop multiple analytical techniques, allowing for the reliable analysis of illicit drugs. This goal is accomplished by means of spectroscopy measurements, usually after a separation step, consisting of liquid (LC) or gas (GC) chromatography. Within the wide range of hyphenated techniques, the coupling of GC to Fourier Transform Infrared Spectroscopy (FTIR) provides a powerful identification tool, also allowing discriminating between isobars and isomers. In this research, the effectiveness of GC-FTIR is demonstrated, in achieving structure elucidation of 1-pentyl-3-(1-naphthoyl)indole, commonly known as JWH-018, a synthetic cannabinoid identified as component of illegal "incense blends." Moreover, solid deposition FTIR enabled for boosting the sensitivity of the technique, over conventional flow (light pipe) cells, scaling down the limit of identification to the ng scale. Calibration curves for JWH-018 standard were obtained in the 20-1,000 ng range, and the limit of detection and limit of quantification were assessed as equal to 4.3 and 14.3 ng, respectively. Finally, the proposed methodology has been adopted for the identification of active principles in a real "street" sample seized by the law enforcement, consisting of an herbal matrix containing four different synthetic cannabinoids belonging to the JWH class. The correct identification of such compounds, with a high degree of chemical similarity, demonstrated the usefulness of the proposed approach for reliable analysis of complex mixtures of illicit drugs, as viable alternative to widespread mass spectrometry-based approaches.

Analytical Characterization of 3-MeO-PCP and 3-MMC in Seized Products and Biosamples: The Role of LC-HRAM-Orbitrap-MS and Solid Deposition GC-FTIR.

Among the phencyclidine (PCP) and synthetic cathinone analogs present on the street market, 3-methoxyphencyclidine (3-MeO-PCP) is one of the most popular dissociative hallucinogen drugs, while 3-methylmethcathinone (3-MMC) is a commonly encountered psychostimulant. Numerous 3-MeO-PCP- and 3-MMC-related intoxication cases have been reported worldwide. Identification of the positional isomers of MeO-PCP and MMC families are particularly challenging for clinical and forensic laboratories; this is mostly due to their difficult chromatographic separation (particularly when using liquid chromatography-LC) and similar mass spectrometric behaviors. 3-MeO-PCP and 3-MMC were identified in two powders, detained by two subjects and seized by the police, by different analytical techniques, including liquid chromatography-high-resolution accurate-mass Orbitrap mass spectrometry (LC-HRAM-Orbitrap-MS), and solid deposition gas chromatography-Fourier transform infrared spectroscopy (sd-GC-FTIR). LC-HRAM-Orbitrap-MS allowed us to assign the elemental formulae C18H27NO (MeO-PCP) and C11H15NO (MMC) through accurate mass measurement of the two MH+ ions, and the comparison of experimental and calculated MH+ isotopic patterns. However, MH+ collision-induced product ions spectra were not conclusive in discriminating between the positional isomers [(3-MeO-PCP vs. 4-MeO-PCP) and (3-MMC vs. 4-MMC and 2-MMC)]. Likewise, sd-GC-FTIR easily allowed us to differentiate between the MeO-PCP and MMC positional isomers unambiguously, confirming the presence of 3-MeO-PCP and 3-MMC, due to the high-quality match factor of the experimental FTIR spectra against the target FTIR spectra of MeO-PCP and MMC isomers in a dedicated library. 3-MeO-PCP (in contrast to 3-MMC) was also detected in blood and urine samples of both subjects and analyzed in the context of routine forensic casework by LC-HRAM-Orbitrap-MS following a simple deproteinization step. In addition, this untargeted approach allowed us to detect dozens of phase I and phase II 3-MeO-PCP metabolites in all biological specimens. Analysis of the extracted samples by sd-GC-FTIR revealed the presence of 3-MeO-PCP, thus confirming the intake of such specific methoxy-PCP isomer in both cases. These results highlight the effectiveness of LC-HRAM-Orbitrap-MS and sd-GC-FTIR data in attaining full structural characterization of the psychoactive drugs, even in absence of reference standards, in both non-biological and biological specimens.

A fast screening MALDI method for the detection of cocaine and its metabolites in hair.

Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry was used for the rapid detection of cocaine, benzoylecgonine and cocaethylene in hair. Different MALDI sample preparation procedures have been tested and the employment of a multi-layer 'graphite-sample-electrosprayed alpha-cyano-4-hydroxycinnamic acid (HCCA)' yielded the best results for standard solutions of the target analytes. The same approach was subsequently applied to hair samples that were known to contain cocaine, benzoylecgonine and cocaethylene, as determined by a classical GC-MS method. It was however necessary to extract hair samples by incubating them in methanol/trifluoroacetic acid for a short time (15 min) at 45 degrees C; 1 microl of the obtained supernatant was deposed on a metal surface treated with graphite, and HCCA was electrosprayed on it. This procedure successfully suppressed matrix peaks and was effective in detecting all the target analytes as their protonated species. The results obtained give further confirmation of the effectiveness of the MALDI for detecting drugs and their metabolites in complex biological matrices. The method can be useful as a fast screening procedure to detect the presence of cocaine and metabolites in hair samples.

Understanding and managing the new psychoactive substances phenomenon: a holistic approach.

The new psychoactive substances (NPS) phenomenon has emerged as a global threat that challenges public health and institutions. There are important qualitative differences between the NPS and traditional drugs phenomena. We discuss these differences and explore the complex structure of the NPS phenomenon. We analyse the entire phenomenon with a global, holistic approach. We present an original framework to help policy makers, healthcare practitioners, and community workers understand the NPS phenomenon's structure and to plan comprehensive policy responses and prevention strategies. We discuss fundamental characteristics, driving forces, routes of information, and social and individual health risks of the phenomenon. We conclude that a holistic approach integrating all aspects of the framework is essential for addressing this emerging threat. We give practical examples of interventions likely to be effective.

The role of time and storage conditions on the composition of hashish and marijuana samples: A four-year study.

The aim of this study was to investigate the role of time and different real-life storage conditions on the composition of different varieties of cannabis products (hashish and marijuana). Six high-potency cannabis products constituted by herbal and resin materials containing different initial concentrations of delta 9-Tetrahydrocannabinol (THC) were employed for this study. Four representative samples were collected from each study material and were maintained for a prolonged time (four years) under different controlled storage conditions: (A) light (24 h) and room temperature (22°C); (B) darkness (24 h) and room temperature; (C) darkness and refrigeration (4 °C); (D) darkness and freezing (-20 °C). The concentration of the three main cannabinoids, i.e. THC, Cannabinol (CBN, produced from the degradation of THC), and Cannabidiol (CBD), were measured by GC-FID around every 100 days along the four-year study. Significant changes in the THC (degradation) and CBN (formation) content were detected under storage conditions A and B, and almost 100% of THC was degraded after four years. A mono-exponential function was able to well fit both THC degradation and CBN formation, suggesting that these processes occur with a first order kinetics. Data treatment indicated that the storage temperature and light exposure had two different effects on the conversion of THC to CBN: temperature changed only the speed, light changed both the speed and the stoichiometry of this conversion. Models were proposed which allow to predict the storage time, if unknown, and the initial content of THC (i.e. the concentration of THC at the starting storage time), from the measurement of THC and CBN content at any time under storage condition A. Values predicted are more uncertain at larger storage times and have an accuracy of around 5-10%. These models were also tested on data reported in the literature, and can represent a starting point for further improvements. Prediction models may be helpful for forensic purposes, if the initial concentration of THC or the approximate age of a degraded material need to be estimated, or to plan the storage of delicate samples which need to be re-examined over time.

Inter-laboratory proficiency results of blood alcohol determinations at clinical and forensic laboratories in Italy.

BACKGROUND: Inter-laboratory proficiency schemes are widely used to control the performance of clinical and forensic toxicology laboratories. In 2016 the Laboratory of Environmental Hygiene and Forensic Toxicology - Venice (Italy) initiated an inter-laboratory proficiency test of blood-alcohol analysis. The number of participating laboratories gradually increased from 26 to 36. Furthermore, a few clinical laboratories were included if gas chromatographic (GC) methods were used for blood alcohol analysis. PROCEDURE: Whole blood was obtained from the Blood Transfusion Centre of the Venice Hospital and a mixture of sodium fluoride and potassium oxalate was added as a preservative and anticoagulant, respectively. Aliquots of the blood were spiked with certified pure ethanol to obtain target blood-alcohol concentrations (BACs) ranging from 0 to 5.0g/L. Two blood samples (4mL each) were included in each shipment to the participating laboratories. The laboratories were asked to provide information about number of replicate BAC determinations they made, the types of ethanol reference standards used, and inherent measurement uncertainty. The aim of the testing was to obtain a mean consensus value for the target BAC and to assess inter-laboratory imprecision. All procedures for registration and submission of results were done on-line. A confidential report and statistical evaluations were returned to the participants one week later. ANALYTICAL METHODS: All participants used head-space GC (HS-GC) for the analysis of ethanol in blood. More than 85% of participants used HS-GC with flame-ionization detection, whereas the others used mass spectrometry (MS) as a detector. More than 40% of the participating laboratories kept the blood samples frozen (-20°C) prior to analysis, whereas the others used refrigeration (+4°C). The preliminary validation tests showed that there were no statistically significant differences between BAC in frozen or refrigerated samples for a period of 20 days. RESULTS AND CONCLUSION: The statistical evaluation of results was done using an iterative procedure known as Algorithm A (ISO 13528:2015, C.3.1). This provides robust estimates for mean and standard deviation between laboratories and these were used as consensus values. More than 85% of participants provided satisfactory results (z-score <1) and 94% of laboratories were within z-score <2, based on five control samples. When a blood sample without any alcohol (blank) was sent for analysis, laboratories reported this as zero, 0.00g/L, below limit of detection (LOD) or not detected. Some type of consensus should be reached for reporting blank samples.