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- Analytical and in vitro cytotoxicity studies of synthetic cannabinoids found in seized ‘herbal incenses’Publication . Alves, Vera Lúcia Gouveia; Câmara, José Sousa; Teixeira, Helena Maria de Sousa Ferreira eInitially developed as therapeutic agents, synthetic cannabinoids rapidly appeared on drug market as recreational drugs with over 200 substances notified in Europe. Marketed as ‘synthetic marijuana’, they are usually sold in herbal mixtures, as smoking products. Cheaper, accessible, and with stronger effects compared to cannabis, have led to a dramatic increase in its consumption. New compounds emerge constantly, and the side effects and toxicity of these products have not yet been properly studied, being a challenge for laboratories. In our study, nineteen seized herbal products provided by the criminal police were analysed by gas chromatography – mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) techniques. Ten synthetic cannabinoids (JWH-018, JWH-073, JWH-122, JWH-210, MAM-2201, APINACA, XLR-11, UR-144, CP47,497-C8 and 3-epi-CP47,497-C8), and three adulterants (oleamide, vitamin E and vitamin E acetate) were identified. Subsequently, the determination of eight cannabinoids (MAM-2201, JWH-073, JWH-018, JWH-122, JWH-210, UR-144, XLR-11 and APINACA) in oral fluid samples using microextraction by packed sorbent (MEPS) and μSPEed techniques combined with ultra-high performance liquid chromatography (UHPLC) analysis was developed. Both methodologies were validated, presenting good recovery values (above 70%) and low detection limits (9.5 – 13.2 and 4.4 – 10.3 ng/mL, respectively) and applied to two real samples. Finally, the cytotoxicity of eight seized herbal extracts and damiana extract was evaluated in A549 lung cell line. A significant concentration-dependent decrease in cell viability was observed after 48h and 72h exposure. Damiana extract and JWH-018 (single compound of one of the samples) significantly induced more reactive species formation and a significant decline in ATP synthesis was observed in damiana and in sample with XLR-11 and APINACA compounds. In summary, the applied methodologies revealed as promising approaches. Inconsistency in composition was verified among the analysed samples, and in vitro results highlighted the potential harmful effects of these synthetic drugs.
- Beer volatile fingerprinting at different brewing stepsPublication . Alves, Vera; Gonçalves, João; Figueira, José A.; Ornelas, Laura P.; Branco, Ricardo N.; Câmara, José S.; Pereira, Jorge A. M.Volatile fingerprints of a lager beer were carried out throughout five brewing steps to characterize the changes encompassing this process. Overall, 60 volatile organic metabolites (VOMs) were identified by headspace solid phase microextraction followed by gas chromatography mass spectrometry (HS-SPME/GC–MS). Specific profiles were observed at different brewing steps - aldehydes and furans dominate in wort, whereas the aliphatic esters and alcohols predominate in the following steps. Such variations can be assigned to specific VOMs, as 3-me thylbutanal (wort), ethyl alcohol and ethyl octanoate (fermentation, maturation and filtration), or ethyl alcohol and isoamyl acetate (final product). These VOMs can influence the beer final flavour. Ethyl alcohol contributes to its strong and pungent smell and taste, while isoamyl acetate adds intense ‘fruity’ and ‘banana’ odours. These beer volatile fingerprints constitute a valuable tool to obtain insights on the impact of each brewing step on the final product, being also very useful for certification purposes.
- Microextraction using packed sorbent as an effective and high-throughput sample extraction technique: recent applications and future trendsPublication . Pereira, J.; Gonçalves, J.; Alves, V.; Câmara, J. S.In the last decade, several developments, particularly in the liquid chromatography and hyphenated techniques fields, have allowed researchers to reach analytical limits that are becoming very close to the single molecule level. Sample preparation, however, despite being a key step in the analytical methodology, did not track these developments and very few approaches able to cope with these stringent analytical requirements were developed. One such approach is microextraction by packed sorbent (MEPS), a sophisticated and miniaturized form of solid phase extraction that has reduced to the microliter scale every step of the sample extraction methodology including sample volume and solvent usage. Simultaneously, the amount of extraction phase used was also reduced and, more importantly, the sorbent is reusable dozens of times which significantly lowers the cost of analysis versus other solid-phase extraction approaches. In this review, we will update the state-of-the-art of the MEPS technique, focusing on the trends and applications reported since 2010 and future perspectives and developments that in our view will further improve the high-throughput potential and applications of this sample preparation methodology.
- Re-exploring the high-throughput potential of microextraction techniques, SPME and MEPS, as powerful strategies for medical diagnostic purposes. Innovative approaches, recent applications and future trendsPublication . Pereira, Jorge; Silva, Catarina Luís; Perestrelo, Rosa; Gonçalves, João; Alves, Vera; Câmara, José S.The human population continues to grow exponentially in the fast developing and most populated countries, whereas in Western Europe it is getting older and older each year. This inevitably raises the demand for better and more efficient medical services without increasing the economic burden in the same proportion. To meet these requirements, improvement of medical diagnosis is certainly a key aspect to consider. Therefore, we need powerful analytical methodologies able to go deeper and further in the characterization of human metabolism and identification of disease biomarkers and endogenous molecules in body fluids and tissues. The ultimate goal is to have a reliable and early medical diagnosis, mitigating the disease complications as much as possible. Microextraction techniques (METs) represent a key step in these analytical methodologies by providing samples in the suitable volumes and purification levels necessary for the characterization of the target analytes. In this aspect, solid-phase microextraction (SPME) and, more recently, microextraction by packed sorbent (MEPS), are powerful sample preparation techniques, characterized by their reduced time of analysis, low solvent consumption, and broad application. Moreover, as miniaturized techniques, they can be easily automatized to have a high-throughput performance in the clinical environment. In this review, we explore some of the most interesting MEPS and SPME applications, focusing on recent trends and applications to medical diagnostic, particularly the in vivo and near real time applications.
- Development of MEPS–UHPLC/PDA methodology for the quantification of clozapine, risperidone and their major active metabolites in human urinePublication . Gonçalves, João L.; Alves, Vera L.; Conceição, Carlota J.F.; Teixeira, Helena M.; Câmara, José S.A new, fast and reliable methodology using a digitally controlled microextraction by packed sorbent (MEPS), followed by ultrahigh pressure liquid chromatography (UHPLC) separation combined with a photodiode array (PDA) detection, has been developed and validated for the simultaneous determination of clozapine and risperidone, and their active metabolites, in urine samples. Important parameters affecting MEPS performance, including type of sorbent material (C2, C8, C18, SIL, M1, R-AX, R-CX, PGC, PEP, HLB-DVB and H-DVB), elution solvent system, number of extraction cycles, sample volume, elution volume, and sample pH, were evaluated. The optimal experimental conditions involved the loading of 500 μl of sample through a C18 sorbent in a five extraction cycles and in a short time period (5 min for the entire sample preparation step). The obtained extract was analyzed by UHPLC, using a binary mobile phase, composed of aqueous 0.1% formic acid and methanol in the gradient elution mode (10 min total analysis). Good results were obtained under the optimized conditions, in terms of linearity with correlation coefficients (r2) values higher than 0.9986. Low detection limits, ranging between 0.052 and 0.055 μg ml− 1 were achieved, whereas the lowest quantification limit was 0.1 μg ml− 1, corresponding to the lowest concentration of the standard curve. Satisfactory results were found with accuracy values ranging from 83% to 116%, and with precision results from 2.65% to 18%. Recoveries ranged between 76% and 96%, which was in agreement with results reported by other studies.
- Structure assignment of seized products containing cathinone derivatives using high resolution analytical techniquesPublication . Gonçalves, João L.; Alves, Vera L.; Aguiar, Joselin; Caldeira, Maria J.; Teixeira, Helena M.; Câmara, José S.The innovation of the new psychoactive substances (NPS) market requires the rapid identification of new substances that can be a risk to public health, in order to reduce the damage from their use. Twelve seized products suspected to contain illicit substances were analyzed by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), gas chromatography coupled to mass spectrometry (GC-MS), and nuclear magnetic resonance spectroscopy (NMR). Synthetic cathinones (SCat) were found in all products, either as a single component or in mixtures. Infrared spectra of all products were consistent with the molecular structure of SCat, showing an intense absorption band at 1700–1674 cm−1 , corresponding to the carbonyl stretching, a medium/strong peak at 1605–1580 cm−1 , indicating stretching vibrations in the aromatic ring (C=C) and bands with relative low intensity at frequencies near 2700–2400 cm−1 , corresponding to an amine salt. It was possible to identify a total of eight cathinone derivatives by GC-MS and NMR analysis: 40 -methyl-α pyrrolidinohexanophenone (MPHP), α-pyrrolidinohexanophenone (α-PHP), 3-fluoromethcathinone (3-FMC), methedrone, methylone, buphedrone, N-ethylcathinone, and pentedrone. Among the adulterants found in these samples, caffeine was the most frequently detected substance, followed by ethylphenidate. These results highlight the prevalence of SCat in seized materials of the Portuguese market. Reference standards are usually required for confirmation, but when reference materials are not available, the combination of complementary techniques is fundamental for a rapid and an unequivocal identification of such substances.
- The synthetic cannabinoids phenomenon: from structure to toxicological properties. A reviewPublication . Alves, Vera L.; Gonçalves, João L.; Aguiar, Joselin; Teixeira, Helena M.; Câmara, José S.The word “cannabinoid” refers to every chemical substance, regardless of structure or origin, that joins the cannabinoid receptors of the body and brain and that have similar effects to those produced by the Cannabis plant and based on their source of production, cannabinoids can be classified into endocannabinoids, phytocannabinoids and synthetic cannabinoids. Synthetic cannabinoids represent the largest class of drugs detected through the EU Early Warning System with a total of 190 substances notified from 2008 to 2018 and about 280 have been reported worldwide to the United Nations Office on Drugs and Crime. Sprayed on natural herb mixtures with the aim to mimic the euphoria effect of cannabis and sold as “herbal smoking blends” or “herbal incense” under brand names like “Spice” or “K2”, synthetic cannabinoids are available from websites for the combination with herbal materials or more recently, for the use in e-cigarettes. Currently labeled as “not for human consumption” to circum vent legislation, their legal status varies by country with many government institutions currently push ing for their control. However, due to the emergence of new substances, it requires a constant update of the list of controlled drugs. Little is known about how these substances work and their toxic effects in humans and the same product could vary not only in the amount and in the type of substance added. In the last years, synthetic cannabinoids have been associated with deaths and acute intoxica tions in Europe and, despite a range of new measures introduced in this area, continue to represent a challenge to current drug policy models. These synthetic substances are much more potent than nat ural cannabis, as well as displayed greater efficacy, acting as full agonists at the cannabinoid receptors. It is possible that, along with being highly potent, some may also have long half-lives, potentially lead ing to a prolonged psychoactive effect. The present work provides a review on existing literature about the development of synthetic cannabinoids as substances of abuse, current patterns of abuse and their legal status, chemical classification, and some pharmacological and toxicological properties.
- Improved analytical approach based on QuECHERS/UHPLC-PDA for quantification of fluoxetine, clomipramine and their active metabolites in human urine samplesPublication . Alves, Vera; Conceição, Carlota; Gonçalves, João; Teixeira, Helena M.; Câmara, JoséThis paper reports, for the first time, the development of a modified Quick, Easy, Cheap Effective, Rugged and Safe (QuEChERS) combined with a dispersive SPE (d-SPE) based clean-up procedure as a new and powerful strategy for the simultaneous and efficient isolation of two different antidepressants, fluoxetine and clomipramine, and their active metabolites in human urine samples. A univariate experimental design with four independent variables such as sample volume, extraction solvent, buffered salts and clean-up step, was performed and used to investigate the effect of process variables on the extraction efficiency. Good linearity was achieved at the studied concentration range (0.1-5.0 µg mL-1), with correlation coefficients (R2) higher than 0.9961. Low detection limits, ranging between 0.060 and 0.092 μg mL-1 were obtained for all analytes, whereas the lowest quantification limit was 0.1 μg mL-1, corresponding to the lowest concentration of the standard curve. The method also showed good results for accuracy, with values ranging from 91% to 105%. Intra- and inter-day precision, expressed as the relative standard deviation (RSD), were also satisfactory (<10%). Consistent recoveries of antidepressants ranging from 86% to 109% were observed when urine samples were fortified at three concentrations, namely 0.1, 2.5 and 5.0 μg mL-1 In order to evaluate the proposed method for clinical use, the QuEChERS/UHPLC-PDA method was applied to analysis of 12 urine samples from depressed patients.
- An improved analytical strategy combining microextraction by packed sorbent combined with ultra high pressure liquid chromatography for the determination of fluoxetine, clomipramine and their active metabolites in human urinePublication . Alves, Vera; Gonçalves, João; Conceição, Carlota; Teixeira, Helena M.; Câmara, José S.A powerful and sensitive method, by microextraction packed sorbent (MEPS), and ultra-high performance liquid chromatography (UHPLC) with a photodiode array (PDA) detection, is described for the determination of fluoxetine, clomipramine and their active metabolites in human urine samples. The MEPS variables, such as sample volume, pH, number of extraction cycles (draw-eject), and desorption conditions (solvent and solvent volume of elution) were optimized. The analysis were carried out using small sample volumes (500μL) and in a short time period (5min for the entire sample preparation step). Good linearity was obtained for all antidepressants with the correlation coefficients (R(2)) above 0.9965. The limits of detection (LOD) ranged from 0.068 to 0.087μgmL(-1). The recoveries were from 93% to 98%, with relative standard deviations less than 6%. The inter-day precision, expressed as the relative standard deviation, varied between 3.8% and 8.5% while the intra-day precision between 3.0% and 7.1%. In order to evaluate the proposed method for clinical use, the MEPS/UHPLC-PDA method was applied to analysis of urine samples from depressed patients.
- Relationship between Volatile Composition and Bioactive Potential of Vegetables and Fruits of Regular Consumption—An Integrative ApproachPublication . Aguiar, Joselin; Gonçalves, João L.; Alves, Vera L.; Câmara, José S.In recent years, there has been a growing interest in studying and exploring the potential health benefits of foods, mainly from vegetables and fruits from regular intake. The presence of secondary metabolites, namely polyphenols, carotenoids and terpenes, in certain food matrices seems to contribute to their functional properties, expressed through an increased prevention in the development of certain chronic diseases, namely coronary heart diseases, neurodegenerative diseases, cancer and diabetes. However, some foods’ volatile secondary metabolites also present important bioactive properties, although this is a poorly scientifically explored field. In this context, and in order to explore the potential bioactivity of volatile metabolites in different vegetables and fruits from regular consumption, the volatile composition was established using a green extraction technique, solid phase microextraction in headspace mode (HS-SPME), combined with gas chromatography tandem mass spectrometry (GC-MS). A total of 320 volatile metabolites, comprising 51 terpenic compounds, 45 organosulfur compounds, 31 aldehydes, 37 esters, 29 ketones, 28 alcohols, 23 furanic compounds, 22 hydrocarbons, 19 benzene compounds, 13 nitrogenous compounds, 9 carboxylic acids, 7 ethers, 4 halogenated compounds and 3 naphthalene derivatives, were positively identified. Each investigated fruit and vegetable showed a specific volatile metabolomic profile. The obtained results revealed that terpenic compounds, to which are associated antimicrobial, antioxidant, and anticancer activities, are the most predominant chemical family in beetroot (61%), orange carrot (58%) and white carrot (61%), while organosulfur compounds (antiviral activity) are dominant in onion, garlic and watercress. Broccoli and spinach are essentially constituted by alcohols and aldehydes (enzyme-inhibition and antimicrobial properties), while fruits from the Solanaceae family are characterized by esters in tamarillo and aldehydes in tomato.