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- Cannabis and Its Secondary Metabolites: Their Use as Therapeutic Drugs, Toxicological Aspects, and Analytical DeterminationPublication . Gonçalves, Joana; Rosado, Tiago; Soares, Sofia; Simão, Ana; Caramelo, Débora Almeida; Luís, Ângelo; Fernández, Nicolás; Barroso, Mário; Gallardo, Eugenia; Duarte, Ana PaulaAlthough the medicinal properties of Cannabis species have been known for centuries, the interest on its main active secondary metabolites as therapeutic alternatives for several pathologies has grown in recent years. This potential use has been a revolution worldwide concerning public health, production, use and sale of cannabis, and has led inclusively to legislation changes in some countries. The scientific advances and concerns of the scientific community have allowed a better understanding of cannabis derivatives as pharmacological options in several conditions, such as appetite stimulation, pain treatment, skin pathologies, anticonvulsant therapy, neurodegenerative diseases, and infectious diseases. However, there is some controversy regarding the legal and ethical implications of their use and routes of administration, also concerning the adverse health consequences and deaths attributed to marijuana consumption, and these represent some of the complexities associated with the use of these compounds as therapeutic drugs. This review comprehends the main secondary metabolites of Cannabis, approaching their therapeutic potential and applications, as well as their potential risks, in order to differentiate the consumption as recreational drugs. There will be also a focus on the analytical methodologies for their analysis, in order to aid health professionals and toxicologists in cases where these compounds are present.
- In Vitro Study of the Bioavailability and Bioaccessibility of the Main Compounds Present in Ayahuasca BeveragesPublication . Gonçalves, Joana; Castilho, Miguel; Rosado, Tiago; Luís, Ângelo; Restolho, José; Fernández, Nicolás; Gallardo, Eugenia; Duarte, Ana PaulaAyahuasca is a psychoactive beverage that contains the psychoactive compound N,N-dimethyltryptamine and β-carboline alkaloids. This study aims at determining in vitro the bioavailability and bioaccessibility of the main compounds present in decoctions of four individual plants, in a commercial mixture and in four mixtures of two individual plants used in the preparation of Ayahuasca. The samples were subjected to an in vitro digestion process, and the Caco-2 cell line was used as an absorption model. The integrity and permeability of the cell monolayer were evaluated, as well as the cytotoxicity of the extracts. After digestion and cell incubation, the compounds absorbed by the cell monolayer were quantified by high-performance liquid chromatography coupled to a diode array detector. The results showed that compounds such as N,N-dimethyltryptamine, Harmine, Harmaline, Harmol, Harmalol and Tetrahydroharmine were released from the matrix during the in vitro digestion process, becoming bioaccessible. Similarly, some of these compounds, after being incubated with the cell monolayer, were absorbed, becoming bioavailable. The extracts did not show cytotoxicity after cell incubation, and the integrity and permeability of the cell monolayer were not compromised.
- Julbernardia paniculata and Pterocarpus angolensis: From Ethnobotanical Surveys to Phytochemical Characterization and Bioactivities EvaluationPublication . Santos, Eugénia Solange; Luís, Ângelo; Gonçalves, Joana; Rosado, Tiago; Pereira, L.; Gallardo, Eugenia; Duarte, A. P.Julbernardia paniculata and Pterocarpus angolensis are two plant species with important application in African traditional medicine, particularly in Angola, in the treatment of several diseases. However, scientific studies concerning these species are scarce. The goal of this work was to know better which medicinal approaches are used by the Huíla population in Angola by means of ethnobotanical surveys. Furthermore, extracts of both plants were phytochemically characterized. Antioxidant, anti-inflammatory, wound-healing activities, and potential cytotoxicity were also studied. With this study it was possible to verify that 67% of the individuals that use medicinal plants are women, and their main therapeutic uses are the treatment of problems of the digestive system and skin disorders. Barks of J. paniculata and leaves of P. angolensis are the most often used plant parts. Through high-performance liquid chromatography coupled to diode-array detector (HPLC-DAD) and GC-MS it was possible to characterize the chemical composition of the two species, which are rich in phenolic compounds, terpenes, terpenoids, sesquiterpenoids and fatty acids. Both plants showed to possess antioxidant, anti-inflammatory proprieties, and wound-healing activity. To the best of our knowledge, this is the most comprehensive study of these two species and the first ethnobotanical and ethnopharmacological study of medicinal plants from this region of Angola.
- Assessment of the Bioaccessibility and Bioavailability of the Phenolic Compounds of Prunus avium L. by in Vitro Digestion and Cell ModelPublication . Gonçalves, Joana; Ramos, Rodrigo; Luís, Ângelo; Rocha, Sandra; Rosado, Tiago; Gallardo, Eugenia; Duarte, Ana PaulaThe fruit of Prunus avium L., commonly known as sweet cherry, is an excellent source of phytochemicals, namely, phenolic compounds. This study aims to determine in vitro the bioavailability and the bioactivity of phenolic compounds present in cherries. Caco-2 cells were used, and the permeability and integrity of the monolayer formed were investigated. After incubation, the phenolic compounds that permeabilized the cell monolayer were quantified using a high-performance liquid chromatography–diode array detector, and the antioxidant activity was evaluated by the 1,1-diphenyl-2-picrylhydrazyl method. The results demonstrated that the phenolic compounds of sweet cherries after undergoing a simulated digestion were absorbed by the cellular barrier, becoming bioavailable. Contrary to what was found after incubation with the in vitro digested extract, the integrity of the cell monolayer was altered and its permeability increased upon incubation with the sweet cherry phenolic extract. Regarding the antioxidant activity, it was verified that this decreased after the absorption by the cellular monolayer. This study suggests that digestion is an indispensable process for absorption because without it the phenolic content of the food matrix does not become bioaccessible.
- Novel synthetic opioids - toxicological aspects and analysisPublication . Tabarra, Inês Pires; Soares, Sofia; Rosado, Tiago; Gonçalves, Joana; Luís, Ângelo; Malaca, Sara; Barroso, Mário; Keller, Thomas; Restolho, José; Gallardo, EugeniaOver the past few years, there has been an emerging number of new psychoactive drugs. These drugs are frequently mentioned as "legal highs", "herbal highs", "bath salts" and "research chemicals". They are mostly sold and advertised on online forums and on the dark web. The emerging new psychoactive substances are designed to mimic the effects of psychoactive groups, which are often abused drugs. Novel synthetic opioids are a new trend in this context and represent an alarming threat to public health. Given the wide number of fatalities related to these compounds reported within the last few years, it is an important task to accurately identify these compounds in biologic matrices in order to administer an effective treatment and reverse the respiratory depression caused by opioid related substances. Clinicians dealing with fentanyl intoxication cases should consider that it could, in fact, be a fentanyl analogue. For this reason, it is a helpful recommendation to include synthetic opioids in the routine toxicological screening procedures, including analysis in alternative matrices, if available, to investigate poly-drug use and possible tolerance to opioids. To address this public health problem, better international collaboration, effective legislation, effective investigation, control of suspicious "research chemicals" online forums and continuous community alertness are required. This article aims to review diverse reported fatalities associated with new synthetic opioids describing them in terms of pharmacology, metabolism, posology, available forms, as well as their toxic effects, highlighting the sample procedures and analytical techniques available for their detection and quantification in biological matrices.
- Synthetic cannabinoids in biological specimens: a review of current analytical methods and sample preparation techniquesPublication . Rosado, Tiago; Gonçalves, Joana; Luís, Ângelo; Malaca, Sara; Soares, Sofia; Vieira, Duarte Nuno; Barroso, Mário; Gallardo, EugeniaSynthetic cannabinoids are a new class of chemical drugs capable of modifying human behavior. These products do not contain cannabis, but produce similar effects after consumption. The fact that they are easily accessed, and are many times considered to be harmless, justifies their widespread use among young people. This fact, together with the difficulty in their detection by routine drug tests, makes it extremely important to develop new procedures able to detect and monitor their consumption. The aim of this work is to perform a critical review regarding the human biological samples that can be used for the determination of synthetic cannabinoids, paying special attention to analytical methods and sample preparation techniques. The reviewed articles deal with the determination of synthetic cannabinoids in the context of forensic and toxicological analysis.
- Miniaturized approaches for sample preparation in hair testing for drugs of abuse and their application in clinical and forensic scenariosPublication . Rosado, Tiago Alexandre Pires; Gallardo Alba, Maria Eugénia; Barroso, Mário Jorge Dinis; Vieira, Duarte Nuno PessoaHair is nowadays one of the most important alternative matrices that have attracted attention for the analysis of various drugs. The fact that it can be collected under supervision, the lower probability of tampering with, and the greater stability are cited as major advantages. As with conventional matrices, the preparation of hair samples is an important step for clean-up and pre-concentration of analytes, which significantly affects the reliability and accuracy of the analysis. The use of miniaturized pre-concentration techniques, driven by the concept of “green chemistry”, has minimized the waste usually associated to classical techniques, and microextraction techniques are known for using lower solvent volumes and for saving time; however, their applicability to hair samples is still poorly explored. The aim of this dissertation is to discuss the status of miniaturized clean-up approaches for hair samples. In addition, the advantages of microextraction by packed sorbent (MEPS) in different analytical fields are addressed, and the practical applicability of this technique in hair samples is demonstrated by three different works using gas chromatography coupled to tandem mass spectrometry (GC-MS/MS): determination of selected opioids; determination methadone and EDDP; determination of cocaine and metabolites. These novel methods were optimized and validated according to internationally accepted guidelines. Regarding the current status of the application of miniaturized approaches to hair samples, an increased research has been observed in both solid-phase (SPME) and liquid phase microextraction (LPME), with SPME showing higher representativeness. In this last approach, dispersive micro-solid phase extraction (D-μ-SPE) emerges as the most used in the last 5 years, if we do not include the different variants of fibre microextraction, namely the direct immersion (DI-SPME), headspace (HS-SPME) or in tube (IT-SPME) approaches. Moreover, D-μ-SPE was the one that showed more innovations in terms of solid sorbent material, driven by developments with carbon nanotubes, graphene, graphene oxide (GO), and the use of modified magnetic nanoparticles (MNPs) and ion-imprinted polymers (IIPs). As for LPME, the use of hollow fibre (HF)-LPME has been extensively explored for hair samples, showing great versatility for target analytes. Improvements were also observed by functionalization with GO and by the use of ionic liquids (IL). However, when all variants of dispersive liquid-liquid microextraction (DLLME) are considered, this is undoubtedly the most researched approach. The inclusion of methods involving solidification of a floating organic drop (DLLME-SFO), supramolecular solvents (SM-DLLME), and temperature-controlled ionic liquids (TIL-DLLME) were considered very beneficial due to their lower toxicity. Of all the microextraction techniques applied to hair samples, MEPS seems to be little explored. In fact, before this project was initialized , there was only one paper that had applied MEPS to pre-concentrate analytes from hair samples. Nonetheless, MEPS has been successfully used to extract a variety of compounds from different matrices, and its applicability has been demonstrated in a number of areas, including therapeutic monitoring, forensic toxicology, and food and environmental analysis. This technique is considered as a miniaturization of the classical solid phase extraction (SPE) and was developed in 2004 by Abdel-Rehim with the aim of reducing the volumes of both the sample and the solvents. It also became very attractive as it allows the reuse of the sorbent material and offers an automated procedure by easy coupling to chromatographic systems. In MEPS, the sorbent is reduced (1-4 mg) and is located in a micro-syringe instead of a cartridge. In turn, the sample flows bidirectionally through the sorbent (aspirations), improving the efficiency of the process due to the increased interaction with the sorbent. Considering the potential of MEPS in the context of sample preparation, we decided to test its usefulness for hair samples in three applications. In the first work, we present an analytical method which was developed and validated for the determination of tramadol (TRM), codeine (COD), morphine (MOR), 6- acetylcodeine (6-AC), 6-monoacetylmorphine (6-MAM) and fentanyl (FNT) using gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). Using an M1 sorbent (4 mg; 80% C8 and 20% SCX), the procedure included the following steps: (i) conditioning (3 x 250 μL of methanol and 3 x 250 μL of 2% formic acid); (ii) sample load (15 x 150 μL); (iii) washing (150 μL of 3.36% formic acid); and (iv) elution (8 x 100 μL of 2.36% ammonium hydroxide in methanol). Linearity was obtained for all compounds between the lower limit of quantification (LLOQ) and 5 ng/mg, with determination coefficients higher than 0.99. The obtained LLOQs were 0.01 ng/mg for TRM, COD and 6-AC and 0.025 ng/mg for MOR, 6-MAM and FNT. The recoveries ranged from 74 to 90% (TRM), 51 to 59% (COD), 22 to 36% (MOR), 69 to 99% (6-AC), 53 to 61% (6-MAM) and 75 to 86% (FNT). The method proved to be precise and accurate with coefficients of variation typically below 15% and relative errors within a range of ± 15%, respectively. In the second work, a procedure was developed for the rapid concentration of methadone and its main metabolite (EDDP). The miniaturized approach was coupled to GC-MS/MS. MEPS was performed with an M1 (4 mg; 80% C8 and 20% SCX) sorbent conditioned with three cycles of methanol (250 μL) and three cycles of 2% formic acid (250 μL). Subsequently, the sample was loaded through nine cycles of 150 μL followed by a washing step that involved three cycles of 50 μL with 3.36% formic acid. For the elution of the compounds, six cycles of 100 μL with 2.36% ammonium hydroxide in methanol were used. The method was linear from 0.01 to 5 ng/mg for both compounds, with determination coefficients greater than 0.99. The recoveries ranged from 73 to 109% for methadone and from 84 to 110% for EDDP. Finally, precision and accuracy were in accordance with the international guidelines for analytical method validation. In the third work, a MEPS technique, with M1 (4 mg; 80% C8 and 20% SCX) sorbent, was developed for the pre-concentration of cocaine (COC), benzoylecgonine (BEG), ecgonine methyl ester (EME), norcocaine (NCOC), cocaethylene (COET) and anhydroecgonine methyl ester (AEME). The determination of the compounds was carried out using GC-MS/MS. The final procedure consisted of the following steps: (i) conditioning (250 μL of methanol and 250 μL of deionized water); (ii) sample load (21 x 150 μL); (iii) washing (50 μL of deionized water and 50 μL of acetate buffer pH 4); and (iv) elution (3 x 100 μL of 2% ammonium hydroxide in methanol). The obtained recoveries were considered acceptable for most compounds, namely 44-64% for COC, 63-73% for COET, 21-28% for BEG and 36-44% for NCOC. Lower recoveries were obtained for AEME (4-6%) and EME (1-3%). The method was linear between the LLOQs (0.010 ng/mg for COC and COET, 0.025 ng/mg for EME, BEG and NCOC and 0.150 ng/mg for AEME) and 5 ng/mg. In turn, the method was considered precise and accurate with coefficients of variation below 15%, and with an average relative error within ± 15% for all compounds, except for LLOQ (20%). With the successful application of MEPS it has been demonstrated that this miniaturized technique is an excellent alternative for toxicological analysis in hair samples. MEPS has the advantage of reducing solvent use, and the sorbent may be reused (> 100 extractions), which can be economically attractive to laboratories. Although there has been considerable academic interest in miniaturized clean-up approaches over the past five years, few implementations have been observed in routine laboratories. With the relevance and applicability herein described, we expect this panorama to change in the near future.
- Comparative study of sample preparation procedures to determine the main compounds in ayahuasca beverages by QuEChERS and high‐performance liquid chromatography analysisPublication . Gonçalves, Joana; Rosado, Tiago; Barroso, Mário; Restolho, José; Fernández, Nicolás; Luís, Ângelo; Gallardo, Eugenia; Duarte, Ana PaulaIntroduction Ayahuasca is a psychoactive drink originally consumed by indigenous people of the Amazon. The lack of regulation of this drink leads to uncontrolled consumption, and it is often consumed in religious contexts. Objective The aim of this work is to compare three miniaturised extraction techniques for extracting the main ayahuasca compounds from beverages. Methodology Three sample pretreatment techniques were evaluated (dispersive liquid–liquid microextraction [DLLME], microextraction by packed sorbent [MEPS] and QuEChERS [Quick, Easy, Cheap, Effective, Rugged and Safe]) for the simultaneous extraction of N,N-dimethyltryptamine (DMT), tetrahydroharmine (THH), harmine, harmaline, harmol and harmalol from ayahuasca beverage samples. Then, the most promising technique (QuEChERS) was chosen to pre-concentrate the analytes, subsequently detected by high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD). Results The procedure was optimised, with the final conditions being 500 μL of extractor solvent, 85 mg of primary secondary amine (PSA) and 4 s of vortexing. The analytical method was validated, showing to be linear between 0.16 and 10 μg/mL for β-carbolines and between 0.016 and 1 μg/mL for DMT, with coefficients of determination (R2) between 0.9968 and 0.9993. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.16 μg/mL for all compounds, except for DMT (0.016 μg/mL) and extraction efficiencies varied between 60.2% and 88.0%. Conclusion The analytical methodology proved to be accurate and precise, with good linearity, LODs and LLOQs. This method has been fully validated and successfully applied to ayahuasca beverage samples.
- Low-Cost Carriers Socio-Economic Impact in Tourism Development: The Case of Faro’s Airport HinterlandPublication . Rosado, Tiago; Baltazar, Maria; Silva, JorgeDue to airline market deregulation in Europe LCC’s (Low-Cost Carriers) depicts a fast growth in the last decade and it’s expected that this growth continues in the next years. Also, thisEuropean airline market change has affected the way many airports operate and it’s likely that thischange impacts not only airports performance and efficiency, but also its hinterland. Tourism development is one of the main beneficiaries of this new paradigm. Airport hinterland definition is very broad.Traditionally hinterland is measured by several kilometres’ radiuscentred on the airport or a certain travel time from one pointto the airport. However, this definition may be considered too simplistic because there are other indicators that can determine such influence area.Therefore, current literature prefers to do it in combination with certain pre-defined criteria: airport impact or effectiveness assessment, or a tourism destination perspective. This paper presents a study on airport hinterland socio-economic activity, with emphasis on tourism development due to LCC operations. Thestudy analyses socio-economic indicators from 2006 to 2012, a period whichrepresents the full operation entry and evolution of LCC’s in the Portuguese south airport of Faro. Results are aligned with the expectations created by literature review as well by the empirical preliminary analysis from the case study, showing a possible correlation between LCC movements and some hinterland indicators with direct impact on the tourism sector.
- Mitragyna speciosa: Clinical, Toxicological Aspects and Analysis in Biological and Non-Biological SamplesPublication . Meireles, Vânia Sofia de Oliveira; Rosado, Tiago; Barroso, Mário; Soares, Sofia; Gonçalves, Joana; Luís, Ângelo; Caramelo, Débora Almeida; Simão, Ana Y; Fernández, Nicolás; Duarte, Ana Paula; Gallardo, EugeniaThe abuse of psychotropic substances is a well-known phenomenon, and many of them are usually associated with ancestral traditions and home remedies. This is the case of Mitragyna speciosa (kratom), a tropical tree used to improve work performance and to withstand great heat. According to several published studies, the main reasons for kratom consumption involve improving sexual performance and endurance, but also social and recreational uses for the feeling of happiness and euphoria; it is also used for medical purposes as a pain reliever, and in the treatment of diarrhea, fever, diabetes, and hypertension. However, this plant has gained more popularity amongst young people over the last years. Since it is available on the internet for purchase, its use is now widely as a drug of abuse, namely as a new psychoactive substance, being a cheaper alternative to opioids that does not require medical prescription in most countries. According to internet surveys by the European Monitoring Centre for Drugs and Drug Addiction in 2008 and 2011, kratom was one of the most widely supplied new psychoactive substances. The composition of kratom is complex; in fact, more than 40 different alkaloids have been identified in Mitragyna speciosa so far, the major constituent being mitragynine, which is exclusive to this plant. Besides mitragynine, alkaloids such as corynantheidine and 7-hydroxamitragynine also present pharmacological effects, a feature that may be attributed to the remaining constituents as well. The main goal of this review is not only to understand the origin, chemistry, consumption, and analytical methodologies for analysis and mechanism of action, but also the use of secondary metabolites of kratom as therapeutic drugs and the assessment of potential risks associated with its consumption, in order to aid health professionals, toxicologists, and police authorities in cases where this plant is present.