A molecular receptor for mephedrone, an amphetamine-like NPS, was developed using host-guest chemistry and pharmacophoric design. The in-field detection of new psychoactive substances (NPS) is an area that has garnered considerable attention in the last few years. With the continuously expanding number of NPS on the market, traditional detection mechanisms lack the selectivity needed. In this project a new methodology has been developed for the design of host molecules for use in in-field detection, based on biomimetic design. To understand what a sensory molecular needs to be selective against, GC-MS and HPLC analysis were employed to identify and quantify thirteen aminoindane internet samples. It was found that the composition of internet samples varies greatly in terms of concentration of active ingredient, with a range of 17-95 % w/w of active ingredient identified. It was also found that caffeine was the most common cutting agent with a range of 27.7-30.2 % w/w identified. This highlights the need for both selectivity and sensitivity in detection mechanisms. Using the principles of biomimetic design, a methodology for the treatment of protein-ligand interactions was developed. Protein-ligand binding data collected from the Protein Databank was analysed for mephedrone related structures and common cutting agents, identified through aminoindane internet sample analysis and literature sources. From this work a three-point pharmacophoric model was developed, upon which two host molecules were considered, macrocyclic calixarenes and acyclic anthraquinones. Both contained the three binding interactions deduced from the pharmacophore design; two p-stacking interactions and one hydrogen bond acceptor. The final host molecule taken forward for testing was 1,8-dibenzylthiourea anthracene (Probe 1). The binding affinity of Probe 1 to mephedrone was tested using 1H-NMR. An estimated association constant of 104 M-1 was calculated, with a 1:1 binding stoichiometry. Along with ESI-MS and DFT calculations, it was found that mephedrone binds to Probe 1 in a concerted fashion with a three-point binding geometry, with two hydrogen bonds and one p-stacking interaction. A modest optical response using fluorescence spectroscopy was also observed between mephedrone and Probe 1 at high molar concentrations. A more pronounced response was observed upon addition of high molar concentrations of flephedrone. 1H-NMR showed that Probe 1 selectively bound mephedrone over methamphetamine as well as the four most common cutting agents identified from literature: lidocaine, caffeine, paracetamol and benzocaine, which have been shown to cause false positives in previous studies. Probe 1 showed significant selectivity for the β-ketoamine arrangement. This is supported by the systematic analysis of mephedrone, methamphetamine, mephedrone precursor and flephedrone. This is the first time this has been achieved using host-guest chemistry. A protocol was developed to successfully detect mephedrone via Probe 1 using NMR spectroscopy in a simulated street sample containing two of the most common cutting agents, benzocaine and caffeine. To further aid future design of small host molecules a methodology for the in silico analysis of small molecule host-guest binding using metadynamics was explored. Solvent interactions with the host and guest molecules were observed, highlighting the importance of solvent choice in binding studies. Metadynamics shows potential to be used in further work for improving the approach in which host molecules are designed in future.

In the new era of drug abuse, the proliferation of new psychoactive substances (NPS), commonly referred to as designer drugs or legal highs, has been a global concern. These substances are produced to circumvent current legislation for controlled substances with minor modifications in their chemical structure. Although many efforts have been made previously, the characterization of such substances are still challenging because of (1) the continual emergence of newly identified substances, (2) the lack of a universal screening test for NPS that are structurally similar to each other, and (3) the complex and time-consuming chromatographic techniques currently used. Therefore, it is necessary to develop novel analytical methods that can be readily adapted by forensic laboratories to overcome these challenges. In this dissertation, various analytical techniques have been evaluated for qualitative analysis of these emerging NPS. For rapid screening purposes, a commercial ion mobility spectrometry with a 63Ni ion source (63Ni-IMS) and also direct analysis in real time coupled to a quadrupole time-of-flight mass spectrometer (DART-QTOF-MS) were investigated first. The results showed that rapid detection by 63Ni-IMS and identification by DART-QTOF-MS can be achieved with sub-nanogram detection capability and high speed total analysis time less than two minutes. In recent developments of gas chromatography mass spectrometry (GC-MS), gas chromatography (GC) has been coupled to state-of-the-art mass spectrometers, including triple quadrupole (MS/MS) and quadrupole time-of-flight (QTOF). It was revealed that the application of GC-MS/MS and GC-QTOF facilitates the unambiguous identification of emerging NPS with a chemical ionization (CI) source. In addition, constitutional isomers of NPS were differentiated with the capabilities of product ion scan and multiple reaction monitoring (MRM) modes. Finally, the coupling of IMS with a mass spectrometer (IMS-MS) was investigated as an alternative confirmatory technique. With the development of an optimal solvent system in the electrospray ionization (ESI) process, the rapid analysis and identification of synthetic cathinone was successfully achieved less than five minutes. As a proof-of-concept, seized drugs samples provided by a local forensic laboratory were analyzed using these developed methods by various analytical techniques. The results from these seized samples are also presented in this evaluation.

Indiana University-Purdue University Indianapolis (IUPUI) / In this work we developed paper spray mass spectrometry methods to obtain lower detection limits for pharmaceuticals and drugs of abuse. The second chapter investigates blood fractionation membranes for their ability to obtain lysis free plasma from whole blood without changing the drug concentration relative to centrifugation. We presented a device capable of obtaining and analyzing plasma samples from whole blood and obtaining quantitative results similar to traditional methods. In the third chapter the properties of the paper substrate are investigated systematically for their impacts on ionization efficiency and recovery in combination with the solvent choice. The fourth and fifth chapters detail a simple method for lowering detection limits using a method called paper strip extraction. In this method biofluids are wicked through either sesame seed oil or solid phase extraction powder on a paper strip to concentrate and preserve (in the case of THC) analytes out of biofluids. The use of 3D printing for rapid prototyping and how it potentially impacts paper spray MS sensitivity is outlined in the final chapter.

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.

"Benzo Fury" compounds and derivatives are enactogens similar to 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA) in various aspects. These compounds are similar in structure to MDMA and MDA, as well as, elicit similar effects such as elevated mood, euphoria and hallucinations. This similarity in effect increases the potential for abuse as MDMA has become less prevalent in some regions as the use of these new psychoactive substances (NPSs) has increased. The benzofury compounds are used as legal alternatives to MDMA because of their marketing as “not for human consumption”. With the relative ease in obtaining NPSs via the Internet, it is possible that these drugs may soon be prevalent in the United States. The project’s goal was to separate, detect, and quantitate the benzofury compounds and derivatives as well as MDA and MDMA in one method of analysis using ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS). The project also examined which method of sample preparation is more effective for these compounds. Six benzofury compounds were researched: 5-(2-aminopropyl)benzofuran) (5-APB), 6-(2-aminopropyl)benzofuran) (6-APB), 5-(2-aminopropyl)-2,3-dihydrobenzofuran (5-APDB), 6-(2-aminopropyl)-2,3-dihydrobenzofuran (6-APDB), 1-(benzofuran-5-yl)-N-methylpropan-2-amine (5-MAPB) and 1-(benzofuran-6-yl)-N-methylpropan-2-amine (6-MAPB) as well as MDMA and MDA. These drugs were analyzed in blood. A liquid-liquid extraction (LLE) method and solid phase extraction (SPE) method were examined to determine which would be better for the separation, detection and quantitation of the benzofury compounds. For the development of the overall method, accuracy, precision, calibration curve, carryover, limit of detection, limit of quantitation, analyte stability, and recovery were examined. The accuracy of the methods examined was greater than +/- 20%. For most analytes, the precision within-run and between-run did not exceed 20%, regardless of the sample preparation method used. A weighting of 1/x was applied to the calibration curve regardless of sample preparation method utilized. The carryover was less than 2% with the SPE method having less carryover (0.02% to 0.50%) than the LLE method (0.05% to 1.56%). The limit of quantitation was determined to be greater than 10 ng/mL. While this was unexpected, the limit of detection calculations determined that this was correct. Using the LLE method in combination with the UFLC-MS/MS method developed, the limit of detection was determined to be at least 9.98 ng/mL. Compared to the LLE method, the SPE limit of detection was lower and calculated to be 3.75 ng/mL. The percent recovery was examined for each of the analytes. It was determined that the SPE was capable of recovering 80% or more of the benzofury compounds and derivatives regardless of the concentration level. The LLE was not as successful in the recovering the benzofury compounds, the best recovery occurred at the 200 ng/mL level with only 65% or less recovered. Analyte stability exhibited a general decrease with variation prior to day 7 and then remains relatively stable until day 14. It was anticipated that the quantitation of the drugs might be complicated due to the similarity in structure between the isomers as well as the similarity of structure between all of the compounds. While this may still be the case, the difficult separation resulted in a re-evaluation and alterations to the UFLC-MS/MS method to correct for these issues. With the change in the UFLC-MS/MS method, further method optimization is required to achieve the appropriate accuracy and limit of quantitation. It was found that the best combination of sample preparation and detection of the benzofury compounds and derivatives is to use SPE followed by an UFLC-MS/MS method.

Analýza obsahu webových diskuzních fór jako zdroj dat pro výzkum nových trendů v užívání psychoaktivních látek ve srovnání s daty získanými od jiných populací Mgr. Eva Drápalová ABSTRACT Background: One of the most significant new trends in substance abuse today is an unprecedented rate of new psychoactive substances (NPS) emergence on a global drug market. While collection of scientific data about the effects and risks of numerous NPS is a timely process, the substantial amount of information about the very same substances is being exchanged on drug discussion forums, creating a pool of data with a potential for research of the NPS trend. Aims: This dissertation aims to evaluate a potential of internet discussion forum content analysis methods by (i) describing and comparing the characteristics of discussion forum visitors to NPS users who do not visit such forums; and subsequently by comparing the internet forum content analysis results describing (ii) a prevalence of NPS use, (iii) effects and context of NPS use and (iv) discussed internet retailers selling NPS with data retrieved by other methods. Methods: Differences between two groups of internet survey respondents were analyzed using Pearson Chi-square and Student's t-test to address the first aim of this dissertation. Furthermore the results of both...

Yes / Purpose: The number of benzodiazepines appearing as new psychoactive substances (NPS) is continually increasing. Information about the pharmacological parameters of these compounds is required to fully understand their potential effects and harms. One parameter that has yet to be described is the blood-to-plasma ratio. Knowledge of the pharmacodynamics of designer benzodiazepines is also important, and the use of quantitative structure–activity relationship (QSAR) modelling provides a fast and inexpensive method of predicting binding affinity to the GABAA receptor. Methods: In this work, the blood-to-plasma ratios for six designer benzodiazepines (deschloroetizolam, diclazepam, etizolam, meclonazepam, phenazepam, and pyrazolam) were determined. A previously developed QSAR model was used to predict the binding affinity of nine designer benzodiazepines that have recently appeared. Results: Blood-to-plasma values ranged from 0.57 for phenazepam to 1.18 to pyrazolam. Four designer benzodiazepines appearing since 2017 (fluclotizolam, difludiazepam, flualprazolam, and clobromazolam) had predicted binding affinities to the GABAA receptor that were greater than previously predicted binding affinities for other designer benzodiazepines. Conclusions: This work highlights the diverse nature of the designer benzodiazepines and adds to our understanding of their pharmacology. The greater predicted binding affinities are a potential indication of the increasing potency of designer benzodiazepines appearing on the illicit drugs market. / Engineering and Physical Sciences Research Council. / Research Development Fund Publication Prize Award winner, Feb 2022.

As chamadas club drugs compreendem um vasto grupo de substâncias frequentemente utilizadas em bares, festas e raves, com a finalidade de intensificar o contato social e a estimulação sensorial. Englobam desde substâncias sintéticas comumente conhecidas, como a anfetamina, a metanfetamina, o MDMA, até moléculas de surgimento mais recente, denominadas novas substâncias psicoativas. Isoladas ou associadas a outras drogas, é possível que sejam causa de morte per se, ou que predisponham o usuário a envolver-se em situações potencialmente fatais, sendo necessário que os órgãos de Perícia Criminal (Institutos Médico Legais e Institutos de Criminalística) estejam aptos a detectar e quantificar essas substâncias em amostras biológicas. O presente trabalho teve como objetivo desenvolver um método analítico para identificação e quantificação de club drugs em sangue total, utilizando cromatografia líquida acoplada a espectrometria de massas com analisador híbrido quadrupolotempo de voo (LC-QTOF). Após o desenvolvimento do método, este foi validado utilizando as diretrizes do guia de validação do Scientific Working Group for Forensic Toxicology (SWGTOX), sendo analisados de linearidade, limite de detecção, limite de quantificação, efeito matriz, precisão intradia, precisão interdia, exatidão e integridade de diluição, além de recuperação e eficiência do processo. O método desenvolvido compreendeu a determinação de MDA, MDMA, 2C-B, DOB, cetamina, mCPP, cocaína e cocaetileno. Amostras provenientes de casos reais de morte não natural, oriundas do Instituto Médico Legal Aristoclides Teixeira de Goiânia - GO foram analisadas pelo método desenvolvido. 56 casos foram selecionados, em sua maioria com histórico de morte por projétil de arma de fogo e acidente de transito. Das 56 amostras analisadas, 28,5% (n=16) foram positivas para cocaína e/ou cocaetileno. As demais substâncias pesquisadas não foram encontradas nas amostras. / Club drugs are a large group of substances consumed in pubs, parties and raves, aiming to intensify social contact and sensorial stimulation. The term comprises largely known substances such as amphetamine, methamphetamine, 3,4-methylenodioxymethamphetamine (MDMA), as well as so-called new psychoactive substances, which are synthetic drugs recently developed or recently introduced in drug market. Club drugs can be taken alone, combined with each other or, most frequently, with alcohol or other commonly abused drugs such as cocaine. In any of these situations, club drugs can possibly be the cause of death or potentialize the involvement of the user with crime and potentially fatal behavior. Thus, official organisms in charge of criminal investigation must be capable of identifying and quantifying these substances in biological samples. The present work aimed the development of an analytical method to identify and quantify club drugs in whole blood, using liquid chromatography - mass spectrometry with hybrid analyzer quadrupole - time of flight (LC-QTOF). After analytical development, the method was validated according to do Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines, evaluating linearity, limit of detection, limit of quantification, matrix effect, precision, intermediate precision, bias and dilution integrity, besides recovery and process efficiency. The developed method comprised MDA, MDMA, 2C-B, DOB, ketamine, mCPP, cocaine and cocaethylene determination. Real samples related to non-natural deaths were collected at Institute of the Legal Medicine Aristoclides Teixeira, Goiânia, Goiás, Brazil, and analyzed by the developed method. 56 cases were selected, most of them related to fire gun injury and traffic events, 28,5% (n=16) of them being positive for cocaine and/or cocaethylene. None of the other drugs comprised in the analysis were detected in these samples.

As chamadas club drugs compreendem um vasto grupo de substâncias frequentemente utilizadas em bares, festas e raves, com a finalidade de intensificar o contato social e a estimulação sensorial. Englobam desde substâncias sintéticas comumente conhecidas, como a anfetamina, a metanfetamina, o MDMA, até moléculas de surgimento mais recente, denominadas novas substâncias psicoativas. Isoladas ou associadas a outras drogas, é possível que sejam causa de morte per se, ou que predisponham o usuário a envolver-se em situações potencialmente fatais, sendo necessário que os órgãos de Perícia Criminal (Institutos Médico Legais e Institutos de Criminalística) estejam aptos a detectar e quantificar essas substâncias em amostras biológicas. O presente trabalho teve como objetivo desenvolver um método analítico para identificação e quantificação de club drugs em sangue total, utilizando cromatografia líquida acoplada a espectrometria de massas com analisador híbrido quadrupolotempo de voo (LC-QTOF). Após o desenvolvimento do método, este foi validado utilizando as diretrizes do guia de validação do Scientific Working Group for Forensic Toxicology (SWGTOX), sendo analisados de linearidade, limite de detecção, limite de quantificação, efeito matriz, precisão intradia, precisão interdia, exatidão e integridade de diluição, além de recuperação e eficiência do processo. O método desenvolvido compreendeu a determinação de MDA, MDMA, 2C-B, DOB, cetamina, mCPP, cocaína e cocaetileno. Amostras provenientes de casos reais de morte não natural, oriundas do Instituto Médico Legal Aristoclides Teixeira de Goiânia - GO foram analisadas pelo método desenvolvido. 56 casos foram selecionados, em sua maioria com histórico de morte por projétil de arma de fogo e acidente de transito. Das 56 amostras analisadas, 28,5% (n=16) foram positivas para cocaína e/ou cocaetileno. As demais substâncias pesquisadas não foram encontradas nas amostras. / Club drugs are a large group of substances consumed in pubs, parties and raves, aiming to intensify social contact and sensorial stimulation. The term comprises largely known substances such as amphetamine, methamphetamine, 3,4-methylenodioxymethamphetamine (MDMA), as well as so-called new psychoactive substances, which are synthetic drugs recently developed or recently introduced in drug market. Club drugs can be taken alone, combined with each other or, most frequently, with alcohol or other commonly abused drugs such as cocaine. In any of these situations, club drugs can possibly be the cause of death or potentialize the involvement of the user with crime and potentially fatal behavior. Thus, official organisms in charge of criminal investigation must be capable of identifying and quantifying these substances in biological samples. The present work aimed the development of an analytical method to identify and quantify club drugs in whole blood, using liquid chromatography - mass spectrometry with hybrid analyzer quadrupole - time of flight (LC-QTOF). After analytical development, the method was validated according to do Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines, evaluating linearity, limit of detection, limit of quantification, matrix effect, precision, intermediate precision, bias and dilution integrity, besides recovery and process efficiency. The developed method comprised MDA, MDMA, 2C-B, DOB, ketamine, mCPP, cocaine and cocaethylene determination. Real samples related to non-natural deaths were collected at Institute of the Legal Medicine Aristoclides Teixeira, Goiânia, Goiás, Brazil, and analyzed by the developed method. 56 cases were selected, most of them related to fire gun injury and traffic events, 28,5% (n=16) of them being positive for cocaine and/or cocaethylene. None of the other drugs comprised in the analysis were detected in these samples.

Synthetic cathinones and related agents represent an international drug abuse problem, and at the same time an important class of clinically useful compounds. Structure-activity relationship studies are needed to elucidate molecular features underlying the pharmacology of these agents. Illicit methcathinone (i.e., MCAT), the prototype of the synthetic cathinone class, exists as a racemic mixture. Though the differences in potency and target selectivity between the positional and optical isomers of synthetic cathinones and related agents have been demonstrated to have important implications for abuse and therapeutic potential, the two MCAT isomers have never been directly compared at their molecular targets: the monoamine transporters (MATs). Additionally, previous studies have found that the carbonyl oxygen atom can be replaced with a methoxy group, but this results in two chiral centers (i.e., four possible optical isomers for synthesis and evaluation). Here, the individual isomers of MCAT, their racemate, and achiral MCAT analogs were prepared where necessary, and examined in vitro and in silico at the MATs. All agents were active as substrates, with a rank order of potency suggesting that α-position chirality, in either configuration, is favored but not required, with the S(-) configuration slightly preferred. Either chiral center removal approach resulted in a reduction in potency, suggesting both favorable interactions with the α-methyl, and limited bulk tolerance. To further investigate this possibility, docking studies were conducted using homology models of the MATs. Common binding modes were identified that were similar to the binding mode of S(+)amphetamine co-crystallized at drosophila DAT. Taken together, these studies supported our conclusions, as steric hindrance was observed in the α-methyl region of the proposed binding site for the R(+)MCAT isomer. Inclusion of the original synthetic cathinones among Schedule I controlled substances has driven the clandestine development of a second generation of agents, resulting in an array of new synthetic cathinones diverse in structure and effect.Pyrrolidinophenones are a major constituent of second-generation bath salts. Little is known about their structure-activity relationships. Here, we have synthesized and examined a series of aryl-substituted pyrrolidinophenone analogs, as well as an achiral pyrrolidinophenone analog, utilizing novel synthetic chemistry and an innovative cell-based epifluorescence Ca2+ imaging technique. Herein, we evaluated the neurochemical properties of these novel compounds at the dopamine transporter (DAT), considered to exert a major role in actions of drugs of abuse. For future structure-activity relationship studies, additional analogs of synthetic cathinone-related agents were produced using novel synthetic approaches, including analogs and isomers of known amphetamine drugs of abuse. Finally, though much has been learned about the role of the dopamine and serotonin transporters in the mechanisms of action of synthetic cathinones, the role of the norepinephrine transporter is poorly understood. Homology models of the human norephinephrine transporter were built and docking studies conducted to inform the study of MAT ligand selectivity, activity, and binding. In conclusion, these studies represent progress towards the establishment of comprehensive structure-activity relationships for synthetic cathinones and related agents. Particular emphasis was placed on the SAR of the phenylalkylamine α-carbon in the synthetic cathinone context, and the role of the norepinephrine transporter in their activity.