Periodic Reporting for period 1 - e-gainstRapeDrugs (Electrified Liquid-Liquid Interfaces Supported in 3D Printed Platforms for Rapid Detection of Benzodiazepine Date Rape Drugs)
Reporting period: 2023-04-01 to 2025-03-31
Objectives: The global objective of this work was to develop simple, cheap, and reliable (although still presumptive) sensing solutions for the benzodiazepines (BDZ, date rape drugs) detection. The innovative approach of this project is a resultant of a three main streams: (i) Use of 3D printing to develop sensing platforms, (ii) electroanalysis at the electrified liquid-liquid interface (eLLI) defining sensing and selectivity by molecular partitioning properties and (iii) comprehensive electroanalytical screening focused on BDZ and their potential co-existing interfering species.
Background: BDZ are psychoactive chemical substances renowned for their ability to induce sedation and aid sleep; thus, prescribed to treat a range of clinical conditions that include anxiety, insomnia, muscle spasms, epilepsy, and alcohol withdrawal symptoms. At present, about 38 BDZ drugs are authorized as psychotropic substances based on the United Nations Convention from 1971 with the Etizolam and Flualprazolam being the latest addition to the list from 2020.
Scientific concern and ambitious challenge: The 2021 report of the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) has listed about thirty new BDZ analogues into the EU early warning system and all of them were discovered within the past two decades (two of them attained legal status in 2020). Among the thirty substances, about 53% (16) were found on the EU illicit drug market within a period of four-year time (2016-2019). In 2019, a total number of 1334 BDZ abuse were recorded in the EU among this about 1240 (93%) cases were related to the designer BDZ substances listed in the EU Early Warning System. Owing to the multiple reports of drug-facilitated sexual assaults a renowned BDZ drug, flunitrazepam (Rohypnol) was revoked from the EU market in 2007. In 2018, about 7 out of 10 opioid-related deaths in Scotland were correlated to poly-drug use related to the combination of BDZ substances. In Poland, post-mortem toxicology examination data of 202 drug-related deaths in Warsaw (2015-2016) have documented the presence of BDZ in 5% of the cases. In the year 2020, the EMCDDA has reported concerning numbers of BDZ linked drug-facilitated sexual assaults and thefts. Reports mentioned the presence of clozapine, clonazolam, diclazepam, flubromazolam, and flualprazolam in the liquid samples collected from the spot of crime.
The BDZ-related problem is much more complicated as compared with other illicit drugs. New designer BDZ analogues can be relatively cheaper and readily available than the prescribed BDZ drugs. Thanks to the ease of synthetic procedures, more and more new designer BDZ compounds with high potency are emerging into the market. Given these concerns on the social security, personal health, and economic complexity of BDZ drugs abuse it is necessary to strengthen the on the spot screening protocols to test BDZ drug spiking. This project was aimed develop such tools to evade BDZ related social crimes and health hazards. Solutions presented in this project are fully applicable for the presumptive analysis of BDZ drugs on the sport and by non-expects with minimal trainings.
Background: BDZ are psychoactive chemical substances renowned for their ability to induce sedation and aid sleep; thus, prescribed to treat a range of clinical conditions that include anxiety, insomnia, muscle spasms, epilepsy, and alcohol withdrawal symptoms. At present, about 38 BDZ drugs are authorized as psychotropic substances based on the United Nations Convention from 1971 with the Etizolam and Flualprazolam being the latest addition to the list from 2020.
Scientific concern and ambitious challenge: The 2021 report of the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) has listed about thirty new BDZ analogues into the EU early warning system and all of them were discovered within the past two decades (two of them attained legal status in 2020). Among the thirty substances, about 53% (16) were found on the EU illicit drug market within a period of four-year time (2016-2019). In 2019, a total number of 1334 BDZ abuse were recorded in the EU among this about 1240 (93%) cases were related to the designer BDZ substances listed in the EU Early Warning System. Owing to the multiple reports of drug-facilitated sexual assaults a renowned BDZ drug, flunitrazepam (Rohypnol) was revoked from the EU market in 2007. In 2018, about 7 out of 10 opioid-related deaths in Scotland were correlated to poly-drug use related to the combination of BDZ substances. In Poland, post-mortem toxicology examination data of 202 drug-related deaths in Warsaw (2015-2016) have documented the presence of BDZ in 5% of the cases. In the year 2020, the EMCDDA has reported concerning numbers of BDZ linked drug-facilitated sexual assaults and thefts. Reports mentioned the presence of clozapine, clonazolam, diclazepam, flubromazolam, and flualprazolam in the liquid samples collected from the spot of crime.
The BDZ-related problem is much more complicated as compared with other illicit drugs. New designer BDZ analogues can be relatively cheaper and readily available than the prescribed BDZ drugs. Thanks to the ease of synthetic procedures, more and more new designer BDZ compounds with high potency are emerging into the market. Given these concerns on the social security, personal health, and economic complexity of BDZ drugs abuse it is necessary to strengthen the on the spot screening protocols to test BDZ drug spiking. This project was aimed develop such tools to evade BDZ related social crimes and health hazards. Solutions presented in this project are fully applicable for the presumptive analysis of BDZ drugs on the sport and by non-expects with minimal trainings.
The execution of e-gainstRapeDrugs project involves eight key steps namely,
(i) Formulation of electrified liquid-liquid interface (eLLI; chemical composition, electrochemical characterization, and optimization - pH, concentration, solvents, etc.) that was found to give superior sensing output focused on BDZ.
(ii) Electrochemical characterization of five different BDZ drugs at formulated eLLI (interfacial ion transfer behaviour of BDZ drugs, ion partition characteristics, effect of pH, and alcohol on BDZn+ ion transfer reactions, analytical parameters, etc.).
(iii) Studies focused on electrochemical behaviour of BDZ and intrinsic chemical reagents of soft and hard drinks at eLLI (explored electrochemical profile of various beverage samples at eLLI with ion transfer voltammetry).
(iv) Electrochemical characterization and analytical determination of BDZ drugs spiked in alcoholic and non-alcoholic beverages at eLLI was performed.
(v) Computer aided design of 3D models of electrochemical cells for 3D printing was developed using CAD tool. These models were sliced using proprietary slicer software, printed and studied as the sensing unit platforms.
(vi) Developed 3D printed microporous solid supports for eLLI through dynamic light printing (DLP) and fused deposition modelling (FDM) printers and surface characterization of 3D printed objects through optical microscope and scanning electron microscope.
(vii) Physiochemical and electrochemical studies aiming at characterization of the 3D printed microporous solid supports of eLLI through contact angle analysis, and cyclic voltammetry.
(viii) Explored electrochemical behaviour of BDZ drugs at eLLI supported with 3D printed micropores electrochemical cells (interfacial ion transfer properties, analytical parameters, analytical determination of BDZ spiked in beverage and bio samples)
(i) Formulation of electrified liquid-liquid interface (eLLI; chemical composition, electrochemical characterization, and optimization - pH, concentration, solvents, etc.) that was found to give superior sensing output focused on BDZ.
(ii) Electrochemical characterization of five different BDZ drugs at formulated eLLI (interfacial ion transfer behaviour of BDZ drugs, ion partition characteristics, effect of pH, and alcohol on BDZn+ ion transfer reactions, analytical parameters, etc.).
(iii) Studies focused on electrochemical behaviour of BDZ and intrinsic chemical reagents of soft and hard drinks at eLLI (explored electrochemical profile of various beverage samples at eLLI with ion transfer voltammetry).
(iv) Electrochemical characterization and analytical determination of BDZ drugs spiked in alcoholic and non-alcoholic beverages at eLLI was performed.
(v) Computer aided design of 3D models of electrochemical cells for 3D printing was developed using CAD tool. These models were sliced using proprietary slicer software, printed and studied as the sensing unit platforms.
(vi) Developed 3D printed microporous solid supports for eLLI through dynamic light printing (DLP) and fused deposition modelling (FDM) printers and surface characterization of 3D printed objects through optical microscope and scanning electron microscope.
(vii) Physiochemical and electrochemical studies aiming at characterization of the 3D printed microporous solid supports of eLLI through contact angle analysis, and cyclic voltammetry.
(viii) Explored electrochemical behaviour of BDZ drugs at eLLI supported with 3D printed micropores electrochemical cells (interfacial ion transfer properties, analytical parameters, analytical determination of BDZ spiked in beverage and bio samples)
The e-gainstRapeDrugs project produced sizable portion of interesting research findings that are crucial in moving the boundaries of eLLI-based electroanalytical chemistry outside, academic environment such as onsite drug screening and food drug-spiking screening.
# Fundamental knowledge on the interfacial properties of multiple hard and soft drinks at eLLI was created and has high potential for practical application (e.g. quality assessment of beverage samples, food spoilage, change in chemical compositions, food adulteration, etc.,).
# For the first time interfacial behaviour of five different benzodiazepines were studied at eLLI and we gathered a few important parameters (such as dissociation constants, concentration fractions diagrams, log P values, Galvani potential difference values, etc) that are used for predicting physicochemical and pharmacological behaviour of the drugs. These parameters are also during the design of the electrochemical sensors having a role of presumptive analytical devices.
# The eLLI based date rape drug sensors were successful in discriminating two different benzodiazepine drugs (sharing a similar chemical core) in a mixture; that is first of its kind to discriminate two benzodiazepine drugs based on their ionic properties (rather than its redox behaviour) under electroanalytical conditions. This presents a new piece of knowledge to the scientific community and a much wider audience in the public.
# We also developed micro-eLLI systems supported by 3D printing technology to minimise the sample size and improved analytical performance. Two different 3D printing techniques were testified showing the practicality of the 3D printing techniques in developing eLLI based date rape drug sensors. 3D printed sensing unit compartments lower down the production cost and improve the electroanalytical performance of the system (lower limits of detection due to reduced capacitive currents and higher sensitivities due to improved mass transfer).
# To the best of our knowledge, proper design of the pore along with the optimized printing parameters, for first time, allowed the formation of 3D-printed micropores of sub one hundred micrometres. This was achieved with low cost and widely available hardware based on FDM 3D printers. These pores were used to support eLLI based detection of benzodiazepine date rape drugs in spiked drinks and biosamples.
# Fundamental knowledge on the interfacial properties of multiple hard and soft drinks at eLLI was created and has high potential for practical application (e.g. quality assessment of beverage samples, food spoilage, change in chemical compositions, food adulteration, etc.,).
# For the first time interfacial behaviour of five different benzodiazepines were studied at eLLI and we gathered a few important parameters (such as dissociation constants, concentration fractions diagrams, log P values, Galvani potential difference values, etc) that are used for predicting physicochemical and pharmacological behaviour of the drugs. These parameters are also during the design of the electrochemical sensors having a role of presumptive analytical devices.
# The eLLI based date rape drug sensors were successful in discriminating two different benzodiazepine drugs (sharing a similar chemical core) in a mixture; that is first of its kind to discriminate two benzodiazepine drugs based on their ionic properties (rather than its redox behaviour) under electroanalytical conditions. This presents a new piece of knowledge to the scientific community and a much wider audience in the public.
# We also developed micro-eLLI systems supported by 3D printing technology to minimise the sample size and improved analytical performance. Two different 3D printing techniques were testified showing the practicality of the 3D printing techniques in developing eLLI based date rape drug sensors. 3D printed sensing unit compartments lower down the production cost and improve the electroanalytical performance of the system (lower limits of detection due to reduced capacitive currents and higher sensitivities due to improved mass transfer).
# To the best of our knowledge, proper design of the pore along with the optimized printing parameters, for first time, allowed the formation of 3D-printed micropores of sub one hundred micrometres. This was achieved with low cost and widely available hardware based on FDM 3D printers. These pores were used to support eLLI based detection of benzodiazepine date rape drugs in spiked drinks and biosamples.