Identification and Analytical Characterization of a Novel Synthetic Cannabinoid-Type Substance in Herbal Material in Europe.

The rapid diffusion of new psychoactive substances (NPS) presents unprecedented challenges to both customs authorities and analytical laboratories involved in their detection and characterization. In this study an analytical approach to the identification and structural elucidation of a novel synthetic cannabimimetic, quinolin-8-yl-3-[(4,4-difluoropiperidin-1-yl) sulfonyl]-4-methylbenzoate (2F-QMPSB), detected in seized herbal material, is detailed. An acid precursor 4-methyl-3-(4,4-difluoro-1-piperidinylsulfonyl) benzoic acid (2F-MPSBA), has also been identified in the same seized material. After extraction from the herbal material the synthetic cannabimimetic, also referred to as synthetic cannabinoid receptor agonists or "synthetic cannabinoids", was characterized using gas chromatography-mass spectrometry (GC-MS), 1H, 13C, 19F and 15N nuclear magnetic resonance (NMR) and high-resolution tandem mass spectrometry (HR-MS/MS) combined with chromatographic separation. A cheminformatics platform was used to manage and interpret the analytical data from these techniques.

Identification of 1-Butyl-Lysergic Acid Diethylamide (1B-LSD) in Seized Blotter Paper Using an Integrated Workflow of Analytical Techniques and Chemo-Informatics.

The rapid dispersion of new psychoactive substances (NPS) presents challenges to customs services and analytical laboratories, which are involved in their detection and characterization. When the seized material is limited in quantity or of a complex nature, or when the target substance is present in very small amounts, the need to use advanced analytical techniques, efficient workflows and chemo-informatics tools is essential for the complete identification and elucidation of these substances. The current work describes the application of such a workflow in the analysis of a single blotter paper, seized by Swedish customs, that led to the identification of a lysergic acid diethylamide (LSD) derivative, 1-butyl-lysergic acid diethylamide (1B-LSD). Such blotter paper generally contains an amount in the range of 30-100 ug. This substance, which is closely related to 1-propionyl-lysergic acid diethylamide (1P-LSD), seems to have only recently reached the drug street market. Its identification was made possible by comprehensively combining gas chromatography with mass spectrometry detection (GC-MS), liquid chromatography coupled with high-resolution tandem MS (LC-HR-MS/MS), Orbitrap-MS and both 1D and 2D nuclear-magnetic-resonance (NMR) spectroscopy. All the obtained data have been managed, assessed, processed and evaluated using a chemo-informatics platform to produce the effective chemical and structural identification of 1B-LSD in the seized material.

Airway metabolic anomalies in adolescents with bronchopulmonary dysplasia: new insights from the metabolomic approach.

OBJECTIVES: To assess a group of adolescents with bronchopulmonary dysplasia (BPD) from a biochemical-metabolic standpoint, applying the metabolomic approach to studying their exhaled breath condensate (EBC). STUDY DESIGN: Twenty adolescents with BPD (mean age 14.8 years) and 15 healthy controls (mean age 15.2 years) were recruited for EBC collection, exhaled nitric oxide measurement, and spirometry. The EBC samples were analyzed using a metabolomic approach based on mass spectrometry. The obtained spectra were analyzed using multivariate statistical analysis tools. RESULTS: A reliable Orthogonal Projections to Latent Structures-Discriminant Analysis model showed a clear discrimination between cases of BPD and healthy controls (R(2) = 0.95 and Q(2) = 0.92). The search for putative biomarkers identified an altered complex lipid profile in the adolescents with BPD. CONCLUSIONS: The metabolomic analysis of EBC distinguishes cases of BPD from healthy individuals, suggesting that the lung of survivors of BPD is characterized by long-term metabolic abnormalities. The search for putative biomarkers indicated a possible role of an altered surfactant composition, which may persist far beyond infancy.

Policy recommendations and cost implications for a more sustainable framework for European human biomonitoring surveys.

The potential of Human Biomonitoring (HBM) in exposure characterisation and risk assessment is well established in the scientific HBM community and regulatory arena by many publications. The European Environment and Health Strategy as well as the Environment and Health Action Plan 2004-2010 of the European Commission recognised the value of HBM and the relevance and importance of coordination of HBM programmes in Europe. Based on existing and planned HBM projects and programmes of work and capabilities in Europe the Seventh Framework Programme (FP 7) funded COPHES (COnsortium to Perform Human Biomonitoring on a European Scale) to advance and improve comparability of HBM data across Europe. The pilot study protocol was tested in 17 European countries in the DEMOCOPHES feasibility study (DEMOnstration of a study to COordinate and Perform Human biomonitoring on a European Scale) cofunded (50%) under the LIFE+ programme of the European Commission. The potential of HBM in supporting and evaluating policy making (including e.g. REACH) and in awareness raising on environmental health, should significantly advance the process towards a fully operational, continuous, sustainable and scientifically based EU HBM programme. From a number of stakeholder activities during the past 10 years and the national engagement, a framework for sustainable HBM structure in Europe is recommended involving national institutions within environment, health and food as well as European institutions such as ECHA, EEA, and EFSA. An economic frame with shared cost implications for national and European institutions is suggested benefitting from the capacity building set up by COPHES/DEMOCOPHES.

A new cannabinoid receptor 1 selective agonist evading the 2021 "China ban": ADB-FUBIATA.

Following the class-wide ban of synthetic cannabinoid receptor agonists (SCRAs) in China, SCRAs carrying new core and linker structures, aimed at circumventing the recent Chinese generic legislation, have appeared on the recreational drug market. A very recent example is (S)-2-(2-(1-(4-fluorobenzyl)-1H-indol-3-yl)acetamido)-3,3-dimethylbutanamide (ADB-FUBIATA), which is structurally closely related to the potent SCRA ADB-FUBICA, but carries an additional methylene in the linker region of the molecule. ADB-FUBIATA has recently been identified in seized materials in China, Russia, the United States, and also Belgium; however, its pharmacological characteristics were unknown. The aim of this study was to evaluate the intrinsic cannabinoid receptor (hCB1 and hCB2 ) activation potential of this previously unknown substance via two distinct yet similar in vitro ß-arrestin2 recruitment assays, based on the NanoLuc Binary Technology®. At CB1 , a potency of 635 nM (EC50 ) was found, with an efficacy (Emax ) of 141% relative to the reference compound CP55,940. On the other hand, ADB-FUBIATA had almost no activity at CB2 , indicative of a clear CB1 selectivity. Interestingly, this activation pattern differs markedly from that observed for ADB-FUBICA, which was previously found to be potent and efficacious at both cannabinoid receptors. Additionally, the bioassays were applied to a seized powder containing ADB-FUBIATA, as analytically confirmed by high-performance liquid chromatography coupled to diode-array detection (HLPC-DAD), gas chromatography coupled to mass spectrometry (GC-MS), liquid chromatography couple to time-of-flight mass spectrometry (LC-QTOF-MS), Fourier transform infrared spectroscopy (FTIR), and nuclear magnetic resonance (NMR). The EC50 and Emax values obtained for this powder were very similar to those of the ADB-FUBIATA analytical standard, suggesting a high purity of the powder, although analytical techniques did reveal that the sample was not entirely pure.

Cannabinoid receptor activation potential of the next generation, generic ban evading OXIZID synthetic cannabinoid receptor agonists.

In recent years, several nations have implemented various measures to control the surge of new synthetic cannabinoid receptor agonists (SCRAs) entering the recreational drug market. In July 2021, China put into effect a new generic legislation, banning SCRAs containing one of seven general core scaffolds. However, this has driven manufacturers towards the synthesis of SCRAs with alternative core structures, exemplified by the recent emergence of "OXIZID SCRAs." Here, using in vitro ß-arrestin2 recruitment assays, we report on the CB1 and CB2 potency and efficacy of five members of this new class of SCRAs: BZO-HEXOXIZID, BZO-POXIZID, 5-fluoro BZO-POXIZID, BZO-4en-POXIZID, and BZO-CHMOXIZID. All compounds behaved as full agonists at CB1 and partial agonists at CB2 . Potencies ranged from 84.6 to 721 nM at CB1 and 2.21 to 25.9 nM at CB2 . Shortening the n-hexyl tail to a pentyl tail enhanced activity at both receptors. Fluorination of this pentyl analog did not yield a higher receptor activation potential, whereas an unsaturated tail resulted in decreased potency and efficacy at CB1 . The cyclohexyl methyl analog BZO-CHMOXIZID was the most potent compound at both receptors, with EC50 values of 84.6 and 2.21 nM at CB1 and CB2 , respectively. Evaluation of the activity of a seized powder containing BZO-4en-POXIZID suggested a high purity, in line with high-performance liquid chromatography coupled to diode-array detection (HPLC-DAD), gas chromatography coupled to mass spectrometry (GC-MS), liquid chromatography coupled to time-of-flight mass spectrometry (LC-QTOF-MS), and Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) analysis. Furthermore, all tested compounds showed a preference for CB2 , except for BZO-POXIZID. Overall, these findings inform public health officials, law enforcement agencies, and clinicians on these newly emerging SCRAs.

Synthesis of Citrate-Stabilized Silver Nanoparticles Modified by Thermal and pH Preconditioned Tannic Acid.

Silver nanoparticles (AgNPs) may be synthesized by many different methods, with those based on the thermal reduction of silver salts by citric acid or citric acid/tannic acid being amongst the most commonly used. These methods, although widely used and technically simple, can produce particles in which the size, polydispersivity and morphology can vary greatly. In this work nearly mono-dispersed spherical AgNPs have been synthesized via a one-step reduction method by using sodium citrate and varying quantities of Tannic Acid (TA), which was thermally conditioned prior to use in the growth process. It was found that the final size can be further tailored by controlling the amount of TA and the thermal conditioning of the TA at 60 °C at different time points, which changes the size and polydispersivity of AgNPs. To better understand the origin of this effect, optical spectroscopic analysis and 1H NMR of the TA following mild thermal conditioning of the solution have been done. Comparison of thermally conditioned TA and TA exposed to basic pH shows that similar chemical modifications occur and consequently produce similar effects on growth when used in the synthesis of AgNPs. It is proposed that thermal preconditioning of the TA introduces either chemical or structural changes, which decrease the final particle size under a given total silver content.

Isolation, Characterization and Structural Elucidation of Polybutylene Terephthalate Cyclic Oligomers and Purity Assessment Using a ¹H qNMR Method.

The use of polybutylene terephthalate (PBT) as a food contact material is increasing over the last years. Typical contaminations in the final PBT product include its cyclic oligomers, which are allowed as additives in food contact plastics according to Regulation (EU) No. 10/2011. Their investigation is currently limited by the lack of analytical standards and physical-chemical information. Therefore, four PBT cyclic oligomers have been isolated and purified from a PBT raw material with an automated preparative HPLC-DAD system. Comprehensive characterization of the compounds was performed using Ultra-High Performance Liquid Chromatography (UHPLC) with high resolution time-of-flight mass spectrometry, Fourier-Transform Infrared spectroscopy (FTIR), Differential Scanning Calorimetry (DSC) and Nuclear Magnetic Resonance (NMR) spectroscopy. The purity of each oligomer was assessed using a ¹H qNMR method and ranged from 96.1% to 97.0% for PBT tetramer and trimer respectively. The availability of pure and well characterized PBT cyclic oligomer standards will facilitate future studies of release from plastic food packaging materials.

Collaboration of the Joint Research Centre and European Customs Laboratories for the Identification of New Psychoactive Substances.

BACKGROUND: The emergence of psychoactive designer drugs has significantly increased over the last few years. Customs officials are responsible for the control of products entering the European Union (EU) market. This control applies to chemicals in general, pharmaceutical products and medicines. Numerous products imported from non-EU countries, often declared as 'bath salts' or 'fertilizers', contain new psychoactive substance (NPS). REVIEW: These are not necessarily controlled under international law, but may be subject to monitoring in agreement with EU legislation. This situation imposes substantial challenges, for example, for the maintenance of spectral libraries used for their detection by designated laboratories. The chemical identification of new substances, with the use of powerful instrumentation, and the time needed for detailed analysis and interpretation of the results, demands considerable commitment. The EU Joint Research Centre endeavors to provide scientific support to EU Customs laboratories to facilitate rapid identification and characterisation of seized samples. In addition to analysing known NPS, several new chemical entities have also been identified. Frequently, these belong to NPS classes already notified to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) by the European Early- Warning System (EWS). CONCLUSION: The aim of this paper is to discuss the implementation of workflow mechanisms that are in place in order to facilitate the monitoring, communication and management of analytical data. The rapid dissemination of this information between control authorities strives to help protect EU citizens against the health risks posed by harmful substances.

Classification of gilthead sea bream (Sparus aurata) from 1H NMR lipid profiling combined with principal component and linear discriminant analysis.

The combination of (1)H NMR fingerprinting of lipids from gilthead sea bream (Sparus aurata) with nonsupervised and supervised multivariate analysis was applied to differentiate wild and farmed fish and to classify farmed specimen according to their areas of production belonging to the Mediterranean basin. Principal component analysis (PCA) applied on processed (1)H NMR profiles made a clear distinction between wild and farmed samples. Linear discriminant analysis (LDA) allowed classification of samples according to the geographic origin, as well as for the wild and farmed status using both PCA scores and NMR data as variables. Variable selection for LDA was achieved with forward selection (stepwise) with a predefined 5% error level. The methods allowed the classification of 100% of the samples according to their wild and farmed status and 85-97% to geographic origin. Probabilistic neural network (PNN) analyses provided complementary means for the successful discrimination among classes investigated.

Antibacterial agent discovery using thymidylate synthase biolibrary screening.

Thymidylate synthase (TS, ThyA) catalyzes the reductive methylation of 2'-deoxyuridine 5'-monophosphate to 2'-deoxythymidine 5'-monophosphate, an essential precursor for DNA synthesis. A specific inhibition of this enzyme induces bacterial cell death. As a second round lead optimization design, new 1,2-naphthalein derivatives have been synthesized and tested against a TS-based biolibrary, including human thymidylate synthase (hTS). Docking studies have been performed to rationalize the experimentally observed affinity profiles of 1,2-naphthalein compounds toward Lactobacillus casei TS and hTS. The best TS inhibitors have been tested against a number of clinical isolates of Gram-positive-resistant bacterial strains. Compound 3,3-bis(3,5-dibromo-4-hydroxyphenyl)-1H,3H-naphtho[1,2-c]furan-1-one (5) showed significant antibacterial activity, no in vitro toxicity, and dose-response effects against Staphylococcus epidermidis (MIC=0.5-2.5 microg/mL) clinical isolate strains, which are resistant to at least 17 of the best known antibacterial agents, including vancomycin. So far this compound can be regarded as a leading antibacterial agent.

Concentration and mean degree of polymerization of Rubus ellagitannins evaluated by optimized acid methanolysis.

Ellagitannins are a major class of phenolics largely responsible for the astringent and antioxidant properties of raspberries and blackberries. The Rubus ellagitannins constitute a complex mixture of monomeric and oligomeric tannins. Rubus oligomeric ellagitannins contain, beside the well-known ellagic acid and gallic acid moieties, the sanguisorboyl linking ester group. When exposed to acids or bases, ester bonds are hydrolyzed and the hexahydroxydiphenic acid spontaneously cyclizes into ellagic acid. This study describes a new, rapid procedure for the acid hydrolysis of Rubus ellagitannins in methanol, which results in maximal yield and enables the quantification of all the major reaction products. Additionally, the method provides the rationale for estimating the mean degree of polymerization of Rubus ellagitannins.

Systematic analytical characterization of new psychoactive substances: A case study.

New psychoactive substances (NPS) are synthesized compounds that are not usually covered by European and/or international laws. With a slight alteration in the chemical structure of existing illegal substances registered in the European Union (EU), these NPS circumvent existing controls and are thus referred to as "legal highs". They are becoming increasingly available and can easily be purchased through both the internet and other means (smart shops). Thus, it is essential that the identification of NPS keeps up with this rapidly evolving market. In this case study, the Belgian Customs authorities apprehended a parcel, originating from China, containing two samples, declared as being "white pigments". For routine identification, the Belgian Customs Laboratory first analysed both samples by gas-chromatography mass-spectrometry and Fourier-Transform Infrared spectroscopy. The information obtained by these techniques is essential and can give an indication of the chemical structure of an unknown substance but not the complete identification of its structure. To bridge this gap, scientific and technical support is ensured by the Joint Research Centre (JRC) to the European Commission Directorate General for Taxation and Customs Unions (DG TAXUD) and the Customs Laboratory European Network (CLEN) through an Administrative Arrangement for fast recognition of NPS and identification of unknown chemicals. The samples were sent to the JRC for a complete characterization using advanced techniques and chemoinformatic tools. The aim of this study was also to encourage the development of a science-based policy driven approach on NPS. These samples were fully characterized and identified as 5F-AMB and PX-3 using (1)H and (13)C nuclear magnetic resonance (NMR), high-resolution tandem mass-spectrometry (HR-MS/MS) and Raman spectroscopy. A chemoinformatic platform was used to manage, unify analytical data from multiple techniques and instruments, and combine it with chemical and structural information.

Stable isotope characterization of the ortho-oxygenated phenylpropanoids: coumarin and melilotol.

The natural abundance 2H NMR spectra of extractive coumarin 10 and of its dihydroderivative melilotol 11 produced by baker's yeast reduction has been compared with synthetic materials. Diagnostic for the differentiation of 10 are the (D/H)beta values, which are in the 128.1-133.6 ppm interval for the natural compounds but 258.5 and 189.8 ppm for the synthetic materials. Such a dramatic difference is also found for methyl cinnamate 12, which shows (D/H)beta values of 127.2 and 515.8 ppm, respectively. In extractive 10, the ratio (D/H)4para/(D/H)6ortho = 1.24 is similar to that observed in structurally related salicin and methyl salicylate. Coumarin 10 is transformed in salicyl alcohol 9, providing diacetate 14, showing in the natural series the trend (D/H)3meta > (D/H)4para > (D/H)5meta approximately (D/H)6ortho. A similar trend is shown also by the synthetic 10. A clear distinction between extractive and synthetic 10 is obtained through delta18O determinations on 10 and on chroman 13. The bulk delta18O values in the extractive series of 10 are 20.3, 23.6, and 22.6 per thousand, while those of the aromatic oxygen are 2.3, 0.5, and -0.5 per thousand. In the synthetic sample, the values are 12.6 and 5.6 per thousand, respectively. As a final product, the reduction of 10 leads to the dihydroderivative 11. Both the baker's yeast reduction and the catalytic hydrogenation lead to a marked decrease of the deuterium content of 11, which is stronger for the beta-position than for the alpha-position.

Characterization of European wine glycerol: stable carbon isotope approach.

Glycerol of about 170 European wines was analyzed using gas chromatography-combustion-isotope ratio mass spectrometry technique. (13)C/(12)C isotopic ratio measurements were performed to characterize glycerol's delta(13)C values of genuine wine samples from European Union wine-producing countries. Glycerol was also successfully dosed using an internal reference, 1,5-pentanediol.

Site-specific 13C content by quantitative isotopic 13C nuclear magnetic resonance spectrometry: a pilot inter-laboratory study.

Isotopic (13)C NMR spectrometry, which is able to measure intra-molecular (13)C composition, is of emerging demand because of the new information provided by the (13)C site-specific content of a given molecule. A systematic evaluation of instrumental behaviour is of importance to envisage isotopic (13)C NMR as a routine tool. This paper describes the first collaborative study of intra-molecular (13)C composition by NMR. The main goals of the ring test were to establish intra- and inter-variability of the spectrometer response. Eight instruments with different configuration were retained for the exercise on the basis of a qualification test. Reproducibility at the natural abundance of isotopic (13)C NMR was then assessed on vanillin from three different origins associated with specific δ (13)Ci profiles. The standard deviation was, on average, between 0.9 and 1.2‰ for intra-variability. The highest standard deviation for inter-variability was 2.1‰. This is significantly higher than the internal precision but could be considered good in respect of a first ring test on a new analytical method. The standard deviation of δ (13)Ci in vanillin was not homogeneous over the eight carbons, with no trend either for the carbon position or for the configuration of the spectrometer. However, since the repeatability for each instrument was satisfactory, correction factors for each carbon in vanillin could be calculated to harmonize the results.

Application of multielectrode array (MEA) chips for the evaluation of mixtures neurotoxicity.

Cortical neurons grown on multielectrode array (MEA) chips have been shown to be a valuable alternative method to study electrophysiological properties of the central nervous system neurons and to perform functional toxicological screening. Here we studied the effects of binary mixtures on neuronal networks cultured on MEAs. We have considered compounds with similar and different mode-of-action (MoA) to characterize and assess their combined effects. Individual and binary mixture dose-response curves based on spontaneous neuronal activity have been generated and the IC(50) has been considered as the end-point for neurotoxicity assessment. The two classical approaches of mixtures toxicity studies: concentration addition (CA) and independent action (IA) have been applied to compare calculated and experimental results. Nuclear magnetic resonance (NMR) spectroscopy has been employed to confirm no chemical reaction or complexation between mixtures components. The results suggest that both CA and IA are able to predict the toxicity of the mixture and that the combination of in vitro test methods with theoretical dose-response models has a strong potential as an alternative tool for the prediction of mixtures neurotoxicity.

Virgin olive oil authentication by multivariate analyses of 1H NMR fingerprints and delta13C and delta2H data.

(1)H NMR fingerprints of virgin olive oils (VOOs) from the Mediterranean basin (three harvests) were analyzed by principal component analysis, linear discriminant analysis (LDA), and partial least-squares discriminant analysis (PLS-DA) to determine their geographical origin at the national, regional, or PDO level. Further delta(13)C and delta(2)H measurements were performed by isotope ratio mass spectrometry (IRMS). LDA and PLS-DA achieved consistent results for the characterization of PDO Riviera Ligure VOOs. PLS-DA afforded the best model: for the Liguria class, 92% of the oils were correctly classified in the modeling step, and 88% of the oils were properly predicted in the external validation; for the non-Liguria class, 90 and 86% of hits were obtained, respectively. A stable and robust PLS-DA model was obtained to authenticate VOOs from Sicily: the recognition abilities were 98% for Sicilian oils and 89% for non-Sicilian ones, and the prediction abilities were 93 and 86%, respectively. More than 85% of the oils of both categories were properly predicted in the external validation. Greek and non-Greek VOOs were properly classified by PLS-DA: >90% of the samples were correctly predicted in the cross-validation and external validation. Stable isotopes provided complementary geographical information to the (1)H NMR fingerprints of the VOOs.

Authentication of Trappist beers by LC-MS fingerprints and multivariate data analysis.

The aim of this study was to asses the applicability of LC-MS profiling to authenticate a selected Trappist beer as part of a program on traceability funded by the European Commission. A total of 232 beers were fingerprinted and classified through multivariate data analysis. The selected beer was clearly distinguished from beers of different brands, while only 3 samples (3.5% of the test set) were wrongly classified when compared with other types of beer of the same Trappist brewery. The fingerprints were further analyzed to extract the most discriminating variables, which proved to be sufficient for classification, even using a simplified unsupervised model. This reduced fingerprint allowed us to study the influence of batch-to-batch variability on the classification model. Our results can easily be applied to different matrices and they confirmed the effectiveness of LC-MS profiling in combination with multivariate data analysis for the characterization of food products.