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.

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.

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.

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.

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.

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.

Risks and implications for health and the environment associated with products and waste containing nanomaterials: regulatory and management issues in the European framework.

SUMMARY: Nanomaterials can revolutionize everyday products, but there are still many unanswered questions about the risks they may pose to human health and the environment. Inconsistent definitions and metrological issues are still of concern for manufacturers, importers and distributors who are demanded to comply with strict regulations. It is also likely that the increasing number of nanomaterial-containing products available on the market will vary the chemical and physical properties of the waste produced, which is currently treated in traditional plants without any particular differentiation. Treatment efficiency for nanomaterials-containing waste should then be addressed and the risks of uncontrolled emissions considered. Finally, the risks associated with the use of nanomaterials-containing products may not be sufficiently characterized as current exposure estimation models are not designed for estimating exposure to nanomaterials and they are likely to be affected by large uncertainties. Hence, it would be important for national and international institutions to provide, as soon as possible, harmonized regulations covering all aspects of the life cycle of products and waste containing nanomaterials. This paper is proposed as a starting point for reflection on the main regulatory and management issues associated with products and waste containing nanomaterials, focusing mainly on the European framework.

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.

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.

Forensic-metrological considerations on assessment of compliance (or non-compliance) in forensic blood alcohol content determinations: A case study with software application.

Blood alcohol concentration is the most frequent analytical determination carried out in forensic toxicology laboratories worldwide. It is usually required to assess if an offence has been committed by comparing blood alcohol levels with specified legal limits, which can vary widely among countries. Due to possible serious legal consequences associated with non-compliant alcohol levels, measurement uncertainty should be carefully evaluated, along with other metrological aspects which can influence the final result. The whole procedure can be time-consuming and error-generating in routine practice, increasing the risks for unreliable assessments. A software application named Ethanol WorkBook (EtWB) was developed at the author's laboratory by using Visual Basic for Application language and MS Excel(®), with the aim of providing help to forensic analysts involved in blood alcohol determinations. The program can (i) calculate measurement uncertainties and decision limits with different methodologies; (ii) assess compliance to specification limits with a guard-band approach; (iii) manage quality control (QC) data and create control charts for QC samples; (iv) create control maps from real cases data archives; (v) provide laboratory reports with graphical outputs for elaborated data and (vi) create comprehensive searchable case archives. A typical example of drink driving case is presented and discussed to illustrate the importance of a metrological approach for reliable compliance assessment and to demonstrate software application in routine practice. The tool is made freely available to the scientific community at request.

Detection of 3-methylmethcathinone and its metabolites 3-methylephedrine and 3-methylnorephedrine in pubic hair samples by liquid chromatography-high resolution/high accuracy Orbitrap mass spectrometry.

Hair testing is considered to be one of the most efficient tool to investigate drug-related histories, particularly when the period of use needs to be tested back to many days or even months before sampling. High-resolution mass spectrometry represents today one of the most specific and sensitive analytical techniques to detect psychoactive substances in hair samples following single or multiple drug exposures. In this study pubic hair testing, by means of liquid chromatography-high resolution/high accuracy Orbitrap mass spectrometry, was employed to document the potential intake of five new psychoactive substances by a drug dealer. Pubic hair samples were decontaminated and pulverized with a ball mill, and, after the addition of the internal standard 3,4-methylenedioxypropylamphetamine, extracted with methanol:trifluoroacetic acid 9:1 at 45°C for one night. The obtained extracts were analyzed on a Thermo Fisher Scientific Accela 1250 liquid chromatography system coupled to a Thermo Fisher Scientific single-stage Exactive HCD mass spectrometry system. 3-methylmethcathinone (3-MMC) was found to be present at a concentration of 25.8ng/mg in the pubic hair sample, whereas the other four designer drugs were found to be absent. 3-methylephedrines and 3-methylnorephedrines, metabolites of 3-MMC, were identified in the same sample, thereby proving the 3-MMC intake by the drug dealer.

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.

Understanding the risks associated with the use of new psychoactive substances (NPS): high variability of active ingredients concentration, mislabelled preparations, multiple psychoactive substances in single products.

New psychoactive substances (NPS), are now a large group of substances of abuse not yet completely controlled by international drug conventions, which may pose a public health threat. Anxiety, paranoia, hallucinations, seizures, hyperthermia and cardiotoxicity are some of the common adverse effects associated with these compounds. In this paper, three case reports taken from the archive of processed cases of the authors' laboratory are presented and discussed to stress the risks of possible adverse consequences for NPS users: in particular, (i) the risk deriving from the difficulty of predicting the actual consumed dose, due to variability of active ingredients concentration in consumed products, (ii) the risk deriving from the difficulty of predicting the actual active ingredients present in consumed products, as opposed to those claimed by the manufacturer, and (iii) the risk deriving from the difficulty of predicting the actual pharmacological and toxicological effects related to the simultaneous consumption of different psychoactive ingredients contained in single products, whose interactions are mostly unknown. Each of them individually provide a source of concern for possible serious health related consequences. However, they should be considered in conjunction with each others, with the worldwide availability of NPS through the web and also with the incessantly growing business derived from the manipulation and synthesis of new substances. The resulting scenario is that of a cultural challenge which demands a global approach from different fields of knowledge.

Variability of blood alcohol content (BAC) determinations: the role of measurement uncertainty, significant figures, and decision rules for compliance assessment in the frame of a multiple BAC threshold law.

The measurement of blood-alcohol content (BAC) is a crucial analytical determination required to assess if an offence (e.g. driving under the influence of alcohol) has been committed. For various reasons, results of forensic alcohol analysis are often challenged by the defence. As a consequence, measurement uncertainty becomes a critical topic when assessing compliance with specification limits for forensic purposes. The aims of this study were: (1) to investigate major sources of variability for BAC determinations; (2) to estimate measurement uncertainty for routine BAC determinations; (3) to discuss the role of measurement uncertainty in compliance assessment; (4) to set decision rules for a multiple BAC threshold law, as provided in the Italian Highway Code; (5) to address the topic of the zero-alcohol limit from the forensic toxicology point of view; and (6) to discuss the role of significant figures and rounding errors on measurement uncertainty and compliance assessment. Measurement variability was investigated by the analysis of data collected from real cases and internal quality control. The contribution of both pre-analytical and analytical processes to measurement variability was considered. The resulting expanded measurement uncertainty was 8.0%. Decision rules for the multiple BAC threshold Italian law were set by adopting a guard-banding approach. 0.1 g/L was chosen as cut-off level to assess compliance with the zero-alcohol limit. The role of significant figures and rounding errors in compliance assessment was discussed by providing examples which stressed the importance of these topics for forensic purposes.

Variability of cannabis potency in the Venice area (Italy): a survey over the period 2010-2012.

Cannabis is the most widely used illicit substance globally, with an estimated annual prevalence in 2010 of 2.6-5.0% of the adult population. Concerns have been expressed about increases in the potency of cannabis products. A high tetrahydrocannabinol (THC) content can increase anxiety, depression, and psychotic symptoms, and can increase the risk of dependence and adverse effects on the respiratory and cardiovascular systems in regular users. The aim of this study was to report statistical data about the potency of cannabis products seized in the north-east of Italy, in a geographical area centred in Venice and extending for more than 10,000 km(2) with a population of more than two million, by investigating the variability observed in THC levels of about 4000 samples of cannabis products analyzed over the period 2010-2012. Overall median THC content showed an increasing trend over the study period from about 6.0% to 8.1% (6.2-8.9% for cannabis resin, 5.1-7.6% for herbal cannabis). The variation in the THC content of individual samples was very large, ranging from 0.3% to 31% for cannabis resin and from 0.1 to 19% for herbal cannabis. Median CBN:THC ratios showed a slightly decreasing trend over the study period, from 0.09 (2010) to 0.03 (2012), suggesting an increasing freshness of submitted materials. Median CBD:THC ratios also showed a decreasing trend over the study from about 0.52 (2010) to 0.18 (2012), likely due to the increase in submissions of materials from indoor and domestic cultivation with improved breeding methods.

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.

Determination of illicit drugs in seized materials: role of sampling and analysis in estimation of measurement uncertainty.

The determination of illicit active ingredients in seized materials, in order to assess penal or administrative offences, is routinely carried out in many forensic toxicology laboratories. This paper presents main features of the protocol adopted in the Authors' laboratory for the above investigations. In particular, sampling and analysis are considered as the same measurement process quantifying their combined contribution to overall measurement uncertainty. Aspects concerning representative sampling in the case of single and multiple items are discussed. The effects of material heterogeneity are considered by analyzing separately distinct primary samples taken from different parts of the sampling target. Possible errors due to particles dimension that could arise when sub-sampling are also considered. Analytical precision, bias and other matrix effects are studied in order to quantify the component of the overall measurement uncertainty associated to the analysis of prepared test samples. Typical scenarios arising when measurement results are used to assess compliance with specification limits are also discussed revealing the crucial role of measurement uncertainty.