Full Characterisation of Heroin Samples Using Infrared Spectroscopy and Multivariate Calibration.

The analysis of heroin samples, before use in the protected environment of user centra, could be a supplementary service in the context of harm reduction. Infrared spectroscopy hyphenated with multivariate calibration could be a valuable asset in this context, and therefore 125 heroin samples were collected directly from users and analysed with classical chromatographic techniques. Further, Mid-Infrared spectra were collected for all samples, to be used in Partial Least Squares (PLS) modelling, in order to obtain qualitative and quantitative models based on real live samples. The approach showed that it was possible to identify and quantify heroin in the samples based on the collected spectral data and PLS modelling. These models were able to identify heroin correctly for 96% of the samples of the external test set with precision, specificity and sensitivity values of 100.0, 75.0 and 95.5%, respectively. For regression, a root mean squared error of prediction (RMSEP) of 0.04 was obtained, pointing at good predictive properties. Furthermore, during mass spectrometric screening, 10 different adulterants and impurities were encountered. Using the spectral data to model the presence of each of these resulted in performant models for seven of them. All models showed promising correct-classification rates (between 92 and 96%) and good values for sensitivity, specificity and precision. For codeine and morphine, the models were not satisfactory, probably due to the low concentration of these impurities as a consequence of acetylation. For methacetin, the approach failed.

Evaluation of an electrochemical sensor and comparison with spectroscopic approaches as used today in practice for harm reduction in a festival setting-A case study: Analysis of 3,4-methylenedioxymethamphetamine samples.

More and more countries and organisations emphasise the value of harm reduction measures in the context of illicit drug use and abuse. One of these measures is drug checking, a preventive action that can represent a quick win by tailored consultation on the risks of substance use upon analytical screening of a submitted sample. Unlike drop-in centres that operate within a fixed setting, enabling drug checking in a harm reduction context at events requires portable, easy to use analytical approaches, operated by personnel with limited knowledge of analytical chemistry. In this case study, four different approaches were compared for the characterisation of 3,4-methylenedioxymethamphetamine samples and this in the way the approaches would be applied today in an event context. The four approaches are mid-infrared (MIR), near-infrared, and Raman spectroscopy, which are today used in drug checking context in Belgium, as well as an electrochemical sensor approach initially developed in the context of law enforcement at ports. The MIR and the electrochemical approach came out best, with the latter allowing for a direct straightforward analysis of the percentage 3,4-methylenedioxymethamphetamine (as base equivalent) in the samples. However, MIR has the advantage that, in a broader drug checking context, it allows to screen for several molecules and so is able to identify unexpected active components or at least the group to which such components belong. The latter is also an important advantage in the context of the growing emergence of new psychotropic substances.

Substandard and falsified ivermectin tablets obtained for self-medication during the COVID-19 pandemic as a source of potential harm.

In 2019, a global viral pandemic, due to the SARS-CoV-2 virus, broke out. Soon after, the search for a vaccine and/or antiviral medicine began. One of the candidate antiviral medicines tested was ivermectin. Although several health authorities warned the public against the use of this medicine outside clinical trials, the drug was widely used at the end of 2020 and in 2021. Simultaneously, several reports started to emerge demonstrating serious adverse effects after self-medicating with ivermectin. It stands to reason that the self-administration of substandard or falsified (SF) medicines bearing harmful quality deficiencies have contributed to this phenomenon. In order to have a better view on the nature of these harmful quality deficiencies, SF ivermectin samples, intercepted in large quantities by the Belgian regulatory agencies during the period 2021-2022, were analyzed in our official medicines control laboratory. None of the samples (n = 19) were compliant to the quality criteria applicable to medicinal products. These SF products either suffered from a systematic underdosing of the active pharmaceutical ingredient or were severely contaminated with bacteria, two of which were contaminated with known pathogens that cause gastrointestinal illness upon oral intake. In addition to the direct risks of self-medicating with such a product, the improper usage and dosage of ivermectin medication might also facilitate ivermectin tolerance or resistance in parasites. This may have detrimental consequences on a global scale, certainly as the number of newly developed active pharmaceutical ingredients that can safely be used to combat parasites is rather scarce.

Discrepancies between validated GC-FID and UHPLC-DAD methods for the analysis of Δ-9-THC and CBD in dried hemp flowers.

Herbal products for smoking containing cannabidiol (CBD) are available as "low-tetrahydrocannabinol cannabis products" in most EU countries. In Belgium, Δ9-tetrahydrocannabinol (THC) content of these products must be less than 0.2% w/w, which is also the limit for agricultural hemp. For agricultural hemp, the official and only valid method for European regulators is gas-chromatography coupled to flame ionization detector (GC-FID). There is no such method, for smoking products. Many of these herbal for smoking products are analyzed as part of their quality control and have certificate of analysis. During surveillance by official labs, discrepancies were seen between the official results and the certificate of analysis. In this study, a GC-FID method based on the European method and an ultra-high-performance liquid chromatography coupled to diode array detection (UHPLC-DAD) method were validated and applied for samples analysis in order to investigate these discrepancies. The GC-FID method shows better results for the validation parameters; notably, it has ß-expectation tolerance limits within 10% with a ß value of 95% while the validated UHPLC-DAD method has ß-expectation tolerance limits within 15% with a ß value of 90%. Furthermore, the other parameters evaluated are generally better with the GC-FID method. The statistic t test shows that the difference between both methods was significantly different for total-THC, but not significantly different for the total-CBD. The authors state that, as for agricultural hemp, the GC-FID method is to be preferred for the analysis of THC and CBD in products for smoking.

CBD oils on the Belgian market: A validated MRM GC-MS/MS method for routine quality control using QuEChERS sample clean up.

Quality control of CBD oils on the Belgium market showed that the CBD content not always corresponds to the label claim. There is a pressing need to develop new analytical methods specifically developed to the assay of such oily samples. Analytical issues are, however, encountered for routine analyses due to the matrix complexity, high cost of cannabinoid standards and low Δ9-THC concentrations. An oily matrix could cause technical damages to analytical instruments and reduce the lifetime of the chromatographic columns. This paper proposes a procedure combining a sample cleanup by QuEChERS, removing the oily matrix, followed by a validated MRM GC-MS/MS method for the routine analysis of CBD oil samples. Eighteen CBD samples were selected on the Belgium market for analysis. This method allows the quantification of CBD, the legality check for the Δ9-THC content by a CBN standard and the screening of seven other cannabinoids namely CBN, CBDV, CBT, CBC, Δ8-THC, THCV and CBG. The method was validated at three concentration levels (0.5-1-2% (w/v)) for CBD and (0.05-0.1-0.2% (w/v)) for CBN. The detection limits for CBT, CBD, CBC, Δ8-THC, CBN and for the other cannabinoids of interest, were 10 and 14 ng/mL respectively. The accuracy profile values for CBD and CBN showed that the ß-expectation tolerance intervals did not exceed the acceptance limits of ± 20%, meaning that 90% of future measurements will be included within this error range.

Analytical characterization of "etonitazepyne," a new pyrrolidinyl-containing 2-benzylbenzimidazole opioid sold online.

This paper reports on the identification and full chemical characterization of the substance colloquially called "etonitazepyne" or "N-pyrrolidino etonitazene" (2-(4-ethoxybenzyl)-5-nitro-1-(2-(pyrrolidin-1-yl)ethyl)-1H-benzo[d]imidazole), a potent NPS opioid of the 5-nitrobenzimidazole class. Identification of etonitazepyne was performed, on a sample purchased during routine monitoring of the drug market, using GC-MS, HRAM LC-MS/MS, 1 H NMR, and FTIR. The chromatographic data, together with the FTIR data, indicated the presence of a highly pure compound and already indicated a benzimidazole structure. The specific benzimidazole regio-isomer was confirmed using 1 H NMR spectroscopy, resulting in the unambiguous identification of etonitazepyne.

Impact of the Revised European Tobacco Product Directive on the Quality of E-cigarette Refill Liquids in Belgium.

INTRODUCTION: Since its introduction, the e-cigarette has become a commonly used consumer product. In this study, we investigate whether regulatory changes had an impact on the quality of refill liquids (e-liquids) available on the Belgian market through analysis of their chemical composition. Hence, the nicotine concentration accuracy was investigated in samples before, during and after the implementation of the revised Tobacco Product Directive (TPD) as an indicator of good manufacturing practices. This is, however, not enough to assure the quality. Therefore, extra criteria were also assessed based on TPD requirements. METHODS: By using in-house validated methods, a total of 246 e-liquids purchased prior (2013-2015), during (2016) and after (2017-2018) the implementation of the TPD revisions, were analyzed for the presence of nicotine, nicotine-related impurities, volatile organic compounds (VOCs), caffeine and taurine, and the flavors diacetyl and acetylpropionyl. RESULTS: Although not all manufacturers managed to produce and label their products accurately, nicotine labeling discrepancies have decreased over time. Moreover, also the number of e-liquids, containing high-risk VOCs (10% in 2016 vs. none of the samples in 2017-2018), caffeine (16% in 2017 vs. 5% in 2018), and diacetyl and acetylpropionyl (50% in 2017 vs. 27% in 2018 of sweet-flavored samples) diminished over time. CONCLUSION: Our results demonstrate that the overall quality of the e-liquids has improved after the implementation of the revised TPD. However, the results also show that periodic quality control might be required to ensure further compliance to the TPD. IMPLICATIONS: This study clearly demonstrates that the implementation of the revised TPD has improved the quality of the e-liquids on the Belgian market. However, there are still e-liquids that are not in agreement with the TPD due to nicotine concentration label discrepancies, presence of e-liquid impurities and controversial flavors diacetyl and acetylpropionyl or the additive caffeine.

An infrared spectroscopic approach to characterise white powders, easily applicable in the context of drug checking, drug prevention and on-site analysis.

More and more events, such as the summer music festivals, are considering the possibilities for implementing on-site testing of psychoactive drugs in the context of prevention and harm reduction. Although the on-site identification is already implemented by plenty of drug checking services, the required rapid quantitative dosing of the composition of illicit substances is still a missing aspect for a successful harm reduction strategy at events. In this paper, an approach is presented to identify white powders as amphetamine, cocaine, ketamine or others and to estimate the purity of the amphetamine, cocaine and ketamine samples using spectroscopic techniques hyphenated with partial least squares (PLS) modelling. For identification purposes, it was observed that mid-infrared spectroscopy hyphenated with PLS-discriminant analysis allowed the distinction between amphetamine, cocaine, ketamine and other samples and this with a correct classification rate of 93.1% for an external test set. For quantitative estimation, near-infrared spectroscopy was more performant and allowed the estimation of the dosage/purity of the amphetamine, cocaine and ketamine samples with an error of more or less 10% w/w. An easily applicable, practical and cost-effective approach for on-site characterisation of the majority of the psychoactive samples encountered in Belgian nightlife settings based on IR spectroscopy was proposed.

Identification of flavouring substances of genotoxic concern present in e-cigarette refills.

E-cigarettes have become very popular, a trend that has been stimulated by the wide variety of available e-liquid flavours. Considering the large number of e-liquid flavours (>7000), there is an urgent need to establish a screening strategy to prioritize the flavouring substances of highest concern for human health. In the present study, a prioritization strategy combining analytical screening, in silico tools and literature data was developed to identify potentially genotoxic e-liquid flavourings. Based on the analysis of 129 e-liquids collected on the Belgian market, 60 flavourings with positive in silico predictions for genotoxicity were identified. By using literature data, genotoxicity was excluded for 33 of them whereas for 5, i.e. estragole, safrole, 2-furylmethylketon, 2,5-dimethyl-4-hydroxyl-3(2H)-furanone and transhexanal, there was a clear concern for in vivo genotoxicity. A selection of 4 out of the remaining 22 flavourings was tested in two in vitro genotoxicity assays. Three out of the four tested flavourings induced gene mutations and chromosome damage in vitro, whereas equivocal results were obtained for the fourth compound. Thus, although there is a legislative framework which excludes the use of CMR compounds in e-liquids, flavourings of genotoxic concern are present and might pose a health risk for e-cigarette users.

Awareness of users and motivational factors for using new psychoactive substances in Belgium.

BACKGROUND: Few data on motivations for using new psychoactive substances (NPS) are available. However, the cost, the legal status, and their accessibility through channels like internet contributed to the popularity of NPS. The objective of this article are first to gain a deeper understanding of the culture surrounding NPS in Belgium and second to define the awareness of the users concerning the content of the NPS they are consuming. METHODS: Snowball sampling and partners in the drug demand reduction field were used as a gateway in order to reach a heterogeneous study population. In total, 45 users were recruited and in-depth interviews were conducted. The personal experiences of NPS users and their needs for support along the continuum of care were explored through an interview guideline, while subjects were given the opportunity to deposit a NPS sample for forensic analysis in a recognized laboratory. RESULTS: A diversity of profiles was found among NPS users but also a wide diversity in the motives to consume NPS: personal reasons such as pleasure, mind exploration, being connected to others, or out of curiosity, but also external reasons such as price, accessibility or the specific effects procured by certain NPS. The results showed as well that a majority of NPS users seem to be aware of the substances they are using. CONCLUSION: Understanding the motivations of use is of importance to determine which type of NPS targeted interventions are adapted to different profiles of users.

Discrimination of legal and illegal Cannabis spp. according to European legislation using near infrared spectroscopy and chemometrics.

Aerial parts containing cannabidiol can be purchased in a legal way but cannabis used as recreational drug is illegal in most European countries. Δ9-tetrahydrocannabinol is one of the main cannabinoids responsible for the psychotropic effect. European Union countries and Switzerland authorize a concentration of THC of 0.2 % and 1.0 % w/w, respectively, for smoking products and industrial hemp. Public health inspectors and law enforcement officers need to check the legality of samples. Therefore there is a need for innovative approaches, allowing quality control of these products in an easy way and preferably on site. In many countries, cultivation of industrial hemp is permitted if the THC content does not exceed 0.2 % w/w. A portable equipment could be a useful measuring tool for farmers to check for the THC content at regular time. In this work, 189 samples were analysed with a benchtop and a handheld NIR device in order to create two classification methods according to European and Swiss laws. All samples were also analysed by GC-FID to determine their THC concentration. Supervised analysis was applied in order to establish the best model. For the first classification, the accuracy was 91% for the test set with the benchtop data and 93 % for the test set with the handheld data. For the second classification, the accuracies were respectively 91 % and 95 %. The obtained models, hyphenating spectroscopic techniques and chemometrics, enable to discriminate legal and illegal cannabis samples according to European and Swiss laws.

Impurity profiling of the most frequently encountered falsified polypeptide drugs on the Belgian market.

Advances in biotechnology and the chemical synthesis of peptides have made biopharmaceuticals and synthetic peptide drugs viable pharmaceutical compounds today and an important source for tomorrow's drugs and therapies. Unfortunately, also falsifications and counterfeit versions of these powerful and promising drugs are offered illegally via the internet. Since these falsified preparations are produced outside the legally required quality systems, end-users have no guarantee regarding the efficacy and safety of these products. Although falsified samples of biotherapeutics were already analysed, looking at a specific aspect of their quality or identity, no systematic studies have been performed regarding the presence of different impurities or possible contaminations. Therefore, in order to obtain a better understanding of the potential health risks related to the usage of falsified polypeptide drugs we performed a systematic screening of the ten most frequently encountered falsified peptide drugs on the Belgian market acquired from three different suspected illegal internet pharmacies. The screening incorporated the analysis of the active pharmaceutical ingredient (API), API-related impurities, small molecule contaminants (defined as organic small molecules not belonging to the other categories), elemental impurities and residual solvents. This comprehensive study showed that these type of falsified drugs not only have a high variation in amount of drugs per unit and a low purity (ranging between 5% and 75% for cysteine containing peptides), but also contained the known toxic class one elemental impurities arsenic (As) and lead (Pb). One sample was contaminated with Pb while multiple samples were found with concentrations up to ten times the ICH toxicity limit for parenteral drugs. Subsequent speciation of As confirmed the elevated concentrations for As and demonstrated that all As was present in the more toxic inorganic form. Together with the (sometimes) high amount of peptide impurities and the inherent dangers associated with the use of unauthorized peptide drugs (such as doping peptides or preclinical drugs) this study confirms the reported potential health risks patients/users take when resorting to falsified peptide drugs. Moreover, the presence of the carcinogen As and the known accumulation in human tissues of Pb raises questions about potential sub-acute to chronic toxicity due to the long term administration of these falsified peptide drugs.

Development and validation of a HS/GC-MS method for the simultaneous analysis of diacetyl and acetylpropionyl in electronic cigarette refills.

The use of e-cigarettes as alternative for tobacco cigarettes has become increasingly popular, even though their safety has not yet been scientifically established. One of the frequently raised concerns is the potential toxicity of certain flavours present in the e-liquids, such as diacetyl and acetylpropionyl. It is therefore important to be able to identify and quantify both compounds. Numerous analytical methods have been published for determining e-liquid compositions, but concerns exist with respect to the lack of analytical evaluation. Hence in this study, a new HS/GC-MS-based method was developed for the screening and quantification of diacetyl and acetylpropionyl in e-liquids. This method was fully validated using the 'total error' approach. The LOQ of the analytical method was 5ppm for diacetyl and acetylpropionyl. The obtained accuracy profiles show that the ß-expectation tolerance intervals did not exceed the acceptance limits of±10%, meaning that 95% of future measurements will be included in the [-10%, 10%] bias limits. As proof of applicability, the validated method was successfully applied on a small set of e-liquid samples, indicating that this methodology could be used for routine quality control analyses of e-liquids.

Assessment of human exposure to benzene through foods from the Belgian market.

Benzene is a volatile organic compound known to be carcinogenic to humans (Group 1) and may be present in food. In the present study, 455 food samples from the Belgian market were analyzed for benzene contents and some possible sources of its occurrence in the foodstuffs were evaluated. Benzene was found above the level of detection in 58% of analyzed samples with the highest contents found in processed foods such as smoked and canned fish, and foods which contained these as ingredients (up to 76.21 µg kg(-1)). Unprocessed foods such as raw meat, fish, and eggs contained much lower concentrations of benzene. Using the benzene concentrations in food, a quantitative dietary exposure assessment of benzene intake was conducted on a national representative sample of the Belgian population over 15 years of age. The mean benzene intake for all foods was 0.020 µg kg bw d(-1) according to a probabilistic analysis. These values are below the minimum risk level for oral chronic exposure to benzene (0.5 µg kg bw d(-1)).

Factors influencing benzene formation from the decarboxylation of benzoate in liquid model systems.

Benzene may occur in foods due to the oxidative decarboxylation of benzoate in the presence of hydroxyl radicals. This study investigated factors influencing benzene formation in liquid model systems. The type of buffer, other sources of hydroxyl radical formation in food (photo oxidation of riboflavin and lipid oxidation), transition metal ion concentrations, and the inhibitory effect of antioxidants were tested in benzoate containing model systems. Regarding the hydroxyl radical sources tested, the highest benzene formation was observed in light exposed model systems containing ascorbic acid, Cu(2+), and riboflavin in Na-citrate buffer (1250 ± 131 µg kg(-1)). In practice, it seems that the combination ascorbic acid/transition metal ion remains the biggest contributor to benzene formation in food. However, the concentration of Cu(2+) influences significantly benzene formation in such a system with highest benzene yields observed for Cu(2+) 50 µM (1400 µg kg(-1)). The presence of antioxidants with metal chelation or reduction properties could prevent completely benzene formation.

Determination of benzene in different food matrices by distillation and isotope dilution HS-GC/MS.

Benzene is classified by the IARC as carcinogenic to humans. Several sources may contribute for the occurrence of benzene in foods, such as, environmental contamination and the reaction of benzoate salts with ascorbic acid (naturally present or added as food additives). Matrix effect on benzene recovery (e.g. in fatty foods) and artefactual benzene formation from benzoate during analysis in the presence of ascorbate are some of the challenges presented when determining benzene in a wide range of foodstuffs. Design of experiment (DOE) was used to determine the most important variables in benzene recovery from headspace GC/MS. Based on the results of the DOE, a versatile method for the extraction of benzene from all kind of food commodities was developed. The method which consisted of distillation and isotope dilution HS-GC/MS was in-house validated. Artefactual benzene was prevented by addition of a borate buffer solution (pH 11) under distillation conditions. The method presented in this study allows the use of a matrix-independent calibration with detection limits below the legal limit established by the European Council for benzene in drinking water (1 microg L(-1)).