Yes / Designer benzodiazepines are one of the primary new psychoactive substances (NPS) threats around the world, being found in large numbers in post-mortem, driving under the influence of drugs (DUID) and drug facilitated sexual assault (DFSA) cases. Even though when compared to many other NPS types, such as opioids and cathinones, there are relatively few designer benzodiazepines being monitored. Recently a new NPS benzodiazepine has been reported in Europe, the USA and Canada, desalkygidazepam, also known as bromonordiazepam. This substance is a metabolite of the pro-drug gidazepam, a drug licenced for use in Ukraine and Russia under the name Gidazepam IC®. In the paper we review what is currently known about the use, pharmacology and analytical detection of gidazepam, its metabolite desalkygidazepam, and their other possible metabolites.

The sheer number, continuous emergence, heterogeneity and wide chemical and structural diversity of New Psychoactive Substance (NPS) products are factors being exploited by illicit drug designers to obscure detection of these compounds. Despite the advances in analytical techniques currently used by forensic and toxicological scientists in order to enable the identification of NPS, the lack of a priori knowledge of sample content makes it very challenging to detect an 'unknown' substance. The work presented in this thesis serves as a proof-of-concept by combining similarity studies, Raman spectroscopy and chemometrics, underpinned by robust pre-processing methods for the identification of existing or newly emerging NPS. It demonstrates that the use of Raman spectroscopy, in conjunction with a 'representative' NPS Raman database and chemometric techniques, has the potential for rapidly and non-destructively classifying NPS according to their chemical scaffolds. The work also demonstrates the potential of indicating the purity in formulations typical of those purchased by end users of the product i.e. 'street-like' mixtures. Five models were developed, and three of these provided an insight into the identification and classification of NPS depending on their purity. These are: the 'NPS and non-NPS/benchtop' model, the 'NPS reference standards/handheld' model and the 'NPS and non-NPS/handheld' model. In the 'NPS and non-NPS/benchtop' model (laser λex = 785 nm), NPS internet samples were projected onto a PCA model derived from a Raman database comprising 'representative' NPSs and cutting agent/ adulterant reference standards. This proved the most successful in suggesting the likely chemical scaffolds for NPS present in samples bought from the internet. In the 'NPS reference standards/handheld' model (laser λex = 1064 nm), NPS reference standards were projected onto a PCA model derived from a Raman database comprising 'representative' NPSs. This was the most successful of the three models with respect to the accurate identification of pure NPS. This model suggested chemical scaffolds in 89% of samples compared to 76% obtained with the benchtop instrument, which generally had higher fluorescent backgrounds. In the 'NPS and non-NPS/handheld' model (laser λex = 1064 nm), NPS internet samples were projected onto a PCA model derived from a Raman database comprising 'representative' NPSs and cutting agent/ adulterant reference standards. This was the most successful in differentiating between NPS internet samples dependent on their purity. In all models, the main challenges for identification of NPS were spectra displaying high fluorescent backgrounds and low purity profiles. The 'first pass' matching identification of NPS internet samples on a handheld platform was improved to ~50% using a laser source of 1064 nm because of a reduction in fluorescence at this wavelength. We outline limitations in using a handheld platform that may have added to problems with appropriate identification of NPS in complex mixtures. However, the developed models enabled the appropriate selection of Raman signals crucial for identification of NPS via data reduction, and the extraction of important patterns from noisy and/or corrupt data. The models constitute a significant contribution in this field with respect to suggesting the likely chemical scaffold of an 'unknown' molecule. This insight may accelerate the screening of newly emerging NPS in complex matrices by assigning them to: a structurally similar known molecule (supercluster/ cluster); or a substance from the same EMCDDA/EDND class of known compounds. Critical challenges in instrumentation, chemometrics, and the complexity of samples have been identified and described. As a result, future work should focus on: optimising the pre-processing of Raman data collected with a handheld platform and a 1064 nm laser λex; and optimising the 'representative' database by including other properties and descriptors of existing NPS.

En raison de leur diffusion sauvage sur le e-commerce, leur soi-disant sécurité d’usage et l’alternative légale aux stupéfiants habituels qu’ils constituent, les nouvelles substances psychoactives (NPS) sont un phénomène mondial émergeant. Au-delà de différents défis dans nos sociétés (législation, prévention,... ), la capacité d'identifier les NPS dans des échantillons biologiques présente de nombreux challenges analytiques : ces nouvelles substances ne sont pas référencées dans les bibliothèques habituelles de spectrométrie de masse commerciales, leur métabolisme est inconnu (avec parfois des métabolites actifs), les doses actives sont parfois très faibles et par conséquent, les concentrations dans le sang ou l'urine sont également faibles. Dans ce contexte, notre laboratoire effectue régulièrement des analyses toxicologiques dans un contexte clinique, et pour les forces de l’ordre, dans des échantillons biologiques à l'aide de deux principaux types d’analyseurs : la chromatographie liquide couplée à la spectrométrie de masse tandem (CL-SM/SM) pour le criblage ciblé et la chromatographie liquide couplée à la spectrométrie de masse haute résolution (CL-SMHR) pour le criblage non ciblé. Cette dernière technique est basée sur la masse exacte (mais également, le profil isotopique et le temps de rétention) des composés de l’échantillon à partir de laquelle la formule chimique est déterminée et recherchée dans une base de données spectrales en utilisant un logiciel dédié. L’objectif de ma thèse est de caractériser des NPS et leurs métabolites (afin d’alimenter cette base de données) en utilisant une stratégie combinant des approches in vitro, in silico et in vivo. Il s’agit, en particulier, d’augmenter la sensibilité de détection de la prise de NPS en se focalisant sur les métabolites qui sont le plus souvent les produits majeurs d’élimination des NPS.A cet effet, une méthode in vitro destinée à produire les métabolites des NPS et utilisant des microsomes hépatiques humains a été mise en oeuvre. Les métabolites obtenus, comparés aux prédictions in silico, ont été enregistrés dans la base de données. Cette approche a été confrontée à l’analyses de comprimés et d’autres produits non biologiques contenant des NPS, mais également, à des données in vivo d’exposition aux NPS : cas d’intoxications, études expérimentales et études épidémiologiques prospectives et rétrospectives dans des populations ciblées, ou non…Au total, ce travail basé sur cette stratégie in vitro, in silico, in vivo m’a permis d’enrichir notre base de données de spectres de masse haute résolution (SMHR) pour le criblage non ciblé et également notre base de données de criblage ciblé (SM/SM). Notre méthode en haute résolution, qui s’est enrichie au cours de ces 3 années de thèse de 83 nouveaux NPS et 281 métabolites, constitue aujourd’hui un outil analytique efficient pour la détection d’une exposition aux NPS. / Owing to wild e-commerce diffusion, alleging safety and legal alternative to usual drugs of abuse arguments, the new psychoactive substances (NPS) are emerging phenomenon in the world. In our societies, through various consecutive challenges (legislation, prevention, …), the ability to identify NPS in biological samples exhibits numerous analytical pitfalls: new substances which are not referenced in the usual commercial mass spectrometric libraries, unknown metabolism (with sometimes active metabolites), sometimes very low active dosages and consecutively low concentrations in blood or urine. In this context, clinical and forensic toxicological analyses in biological samples are routinely performed in our laboratory using two main analytical devices: liquid chromatography-tandem mass spectrometry (LC-MS/MS) for targeted screening and liquid chromatography-high resolution mass spectrometry (LC-HRMS) for non-targeted screening. This last technique is based on the accurate mass (together with isotopic pattern and retention time) of sample components, from which the chemical formula is calculated and searched against a database of mass spectra using dedicated software. The aim of my thesis is to characterize NPS and metabolites (in order to increase the spectral database) using a strategy combining in vitro, in silico, and in vivo approaches. Therefore, the main goal is to increase the detection sensitivity of the NPS use by focusing on the metabolites that are most often the major products of NPS elimination. For this purpose, an in vitro method designed to produce NPS metabolites using human liver microsomes incubations was applied. Obtained metabolites, after confrontation with metabolites in silico predicted, were saved in database. This approach was subsequently confronted with analysis of tablets or other non-biological product containing NPS, but also, with in vivo observed data from NPS exposure: intoxication cases, experimental studies and prospective and retrospective epidemiological studies in targeted population or not … All in all, this work based on this in vitro, in silico and in vivo strategy allowed me to enhance our high resolution spectra database (HRMS) for non-targeted screening and also our spectra database for targeted screening (MS/MS). Today, our HRMS device, with a database that was increased with 83 new NPS and 281 metabolites for the duration of my thesis, is an efficient analytical tool for NPS use detection.

En raison de la diffusion incontrôlée sur le e-commerce, la sécurité et l’alternative légale aux stupéfiants habituels, les nouvelles substances psychoactives (NPS), d’apparition récente (2008), sont au cœur des phénomènes récents d’addiction et de décès mal expliqués. Au-delà des différents défis dans nos sociétés (prévention, législation), la capacité d’identifier les NPS dans des échantillons biologiques pour caractériser leur utilisation, présente de nombreux challenges analytiques. L’objectif principal de cette thèse a été de collecter des échantillons biologiques (sang, urine, cheveux) provenant de cas d’exposition à des NPS et d’y caractériser les substances présentes à l’aide de méthodes analytiques originales, dans le but d’enrichir les librairies de spectres de masse et d’améliorer, en conséquence, la détection de la consommation de NPS. En particulier, il s’agissait d’augmenter la fenêtre de détection de la prise de NPS en se focalisant sur les métabolites qui sont, le plus souvent, les produits majeurs d’élimination. Le développement analytique, par chromatographie liquide ultra haute performance couplée à la spectrométrie de masse en tandem (UHPLC-MS/MS), a demandé plusieurs mois d’optimisation afin d’obtenir une méthode robuste, exhaustive et sensible. Actuellement, la librairie de spectres MS comporte 114 NPS et est mise à jour régulièrement. A la suite de ce développement, ma thèse a porté sur l’étude de cas d’intoxication vus au service des urgences du CHU de Strasbourg, mais aussi en médecine légale, avec des situations de décès et d’identification de produits inconnus provenant de saisies (poudres et cristaux). Il a également été nécessaire de développer des outils analytiques complémentaires, tels que la caractérisation de métabolite(s) par étude sur microsomes hépatiques humains (HLMs), et l’utilisation de la spectroscopie par résonance magnétique nucléaire (RMN) afin d’identifier avec certitude certains composés et de déterminer leur degré de pureté. Les outils analytiques développés et la stratégie mise en place ont permis la rédaction de 18 publications, ainsi que l’agencement de nombreuses collaborations. / Due to the uncontrolled spread on the Internet and their legal alternative to usual drugs, the new psychoactive substances (NPS), recently appeared (2008), are at the center of recent phenomena of addiction and badly explained deaths. Beyond different challenges in our societies (prevention, legislation), the ability to identify NPS in biological samples, in order to characterize their use, presents many analytical challenges. The main objective of this thesis was to collect biological samples (blood, urine, hair) from cases of exposure to NPS and to characterize the substances present using original analytical methods, in order to enlarge the libraries of mass spectra and improve, as a result, the detection of NPS consumption. In particular, it was intended to increase the detection sensitivity of NPS intake by focusing on the metabolites that are often the major products of elimination. This analytical development, by ultra-high liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), required several months of optimization in order to obtain a robust, exhaustive and sensitive method. At present, the mass spectra database has 114 NPS and is regularly updated. Thereafter, ma thesis focused on the study of cases of intoxication observed in the emergency department of Strasbourg, but also in legal medicine with situations of deaths and identification of unknown products collected from seizures (powders and crystals). It has also been necessary to implement complementary analytical tools, such as the characterization of metabolites by human liver microsomes (HLMs), and the use of nuclear magnetic resonance (NMR) spectroscopy to accurately identify the compounds and establish their purity degrees. The analytical tools developed, and the strategy adopted, allowed the writing of 18 publications, as well as the setting up of numerous collaborations.