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- Evaluation of the Cytotoxicity of Ayahuasca BeveragesPublication . Simão, Ana Y.; Gonçalves, Joana; Gradillas, Ana; García, Antonia; Restolho, José; Fernández, Nicolás; Rodilla, Jesus M.; Barroso, Mário; Duarte, Ana Paula; Cristóvão, Ana C.; Gallardo, EugeniaAyahuasca is a beverage consumed at shamanic ceremonies and currently has gained popularity on recreational scenarios. It contains beta-carboline alkaloids and N,N-dimethyltryptamine, which possesses hallucinogenic effects. Only a few studies have elicited the psychoactive effects and the dose of such compounds on neurological dopaminergic cells or animals. In this work, we aimed to study the cytotoxic effects of these compounds present in ayahuasca beverages and on five different teas (Banisteriopsis caapi, Psychotria viridis, Peganum harmala, Mimosa tenuiflora and Dc Ab (commercial name)) preparations on dopaminergic immortalized cell lines. Moreover, a characterization of the derivative alkaloids was also performed. All the extracts were characterized by chromatographic systems and the effect of those compounds in cell viability and total protein levels were analyzed in N27 dopaminergic neurons cell line. This is the first article where cytotoxicity of ayahuasca tea is studied on neurological dopaminergic cells. Overall, results showed that both cell viability and protein contents decreased when cells were exposed to the individual compounds, as well as to the teas and to the two mixtures based on the traditional ayahuasca beverages. View Full-Tex
- An Update on the Implications of New Psychoactive Substances in Public HealthPublication . Simão, Ana Y.; Antunes, Mónica; Cabral, Emanuel; Oliveira, Patrik; Rosendo, Luana M.; Brinca, Ana Teresa; Alves, Estefânia; Marques, Hernâni; Rosado, Tiago; Passarinha, Luís A.; Andraus, Maristela; Barroso, Mário; Gallardo, EugeniaThe emergence of new psychoactive substances has earned a great deal of attention, and several reports of acute poisoning and deaths have been issued involving, for instance, synthetic opiates. In recent years, there have been profound alterations in the legislation concerning consumption, marketing, and synthesis of these compounds; rapid alert systems have also been subject to changes, and new substances and new markets, mainly through the internet, have appeared. Their effects and how they originate in consumers are still mostly unknown, primarily in what concerns chronic toxicity. This review intends to provide a detailed description of these substances from the point of view of consumption, toxicokinetics, and health consequences, including case reports on intoxications in order to help researchers and public health agents working daily in this area.
- Analysis of opiates in urine using microextraction by packed sorbent and gas Chromatography- Tandem mass spectrometryPublication . Simão, Ana Y.; Monteiro, Catarina; Marques, Hernâni; Rosado, Tiago; Margalho, Cláudia; Barroso, Mário; Andraus, Maristela; Gallardo, EugeniaOpiates recreational consumption has always been a concern in society, public health, and in clinical toxicology analysis. The aim of this study was to develop and fully validate an analytical method, which was simple and rapid for the determination of tramadol, codeine, morphine, 6- acetylcodeine, 6-monoacetylmorphine and fentanyl using gas chromatography coupled to tandem mass spectrometry. The procedure includes the use of microextraction by packed sorbent for sample clean-up. A mixed mode sorbent was used, allowing the minimal use of solvents. The method was validated in urine samples, with the ability to detect and quantify all analytes with satisfactory linearity (in the range of 1 – 1000 ng/mL for all analytes, except for fentanyl (10–1000 ng/mL)). Extraction efficiency varied from 17 to 107%, which did not impair sensitivity, taking into account the low LLOQs obtained (1 ng/ mL for all analytes; and 10 ng/mL for fentanyl). The developed procedure proved to be fast, selective, and accurate for use in routine analysis, with a low volume of sample (250 µL).
- The Determination of Cannabinoids in Urine Samples Using Microextraction by Packed Sorbent and Gas Chromatography-Mass SpectrometryPublication . Rosendo, Luana M.; Rosado, Tiago; Oliveira, Patrik; Simão, Ana Y.; Margalho, Cláudia; Costa, Suzel; Passarinha, Luís A.; Barroso, Mário; Gallardo, EugeniaCannabis is the most consumed illicit drug worldwide, and its legal status is a source of concern. This study proposes a rapid procedure for the simultaneous quantification of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), cannabidiol (CBD), and cannabinol (CBN) in urine samples. Microextraction by packed sorbent (MEPS) was used to pre-concentrate the analytes, which were detected by gas chromatography–mass spectrometry. The procedure was previously optimized, and the final conditions were: conditioning with 50 µL methanol and 50 µL of water, sample load with two draw–eject cycles, and washing with 310 µL of 0.1% formic acid in water with 5% isopropanol; the elution was made with 35 µL of 0.1% ammonium hydroxide in methanol. This fast extraction procedure allowed quantification in the ranges of 1–400 ng/mL for THC and CBD, 5–400 ng/mL for CBN and 11-OH-THC, and 10–400 ng/mL for THC-COOH with coefficients of determination higher than 0.99. The limits of quantification and detection were between 1 and 10 ng/mL using 0.25 mL of sample. The extraction efficiencies varied between 26 and 85%. This analytical method is the first allowing the for determination of cannabinoids in urine samples using MEPS, a fast, simple, and low-cost alternative to conventional techniques
- Determination of Ketamine and Norketamine in hair samples using MEPS as sample clean-upPublication . Simão, Ana Y.; Oliveira, Patrik; Rosendo, Luana M.; Rosado, Tiago; Gallardo, Eugenia; Andraus, Maristela; Barroso, MárioIntroduction and Aim: The use of hair samples to determine ketamine (K) and its metabolite, norketamine (NK), has been studied by several authors, using different clean-up approaches. However, microextraction by packed sorbent (MEPS), a miniaturized form of the classic solid-phase extraction has not been applied to date for the purpose. Therefore, a method to determine K and NK in hair samples was developed, optimized, and validated using MEPS as sample clean-up. Materials & Methods: Hair samples were scissor-cut into small fragments, and rinsed with methanol to remove dirt and externally deposited material. Following an overnight incubation with methanol at 65 ºC (no stirring), the compounds were analysed by GC-MS/MS without the need of derivatization procedures; MEPS conditions were: conditioning (5 x 250 µL of methanol and 4 x 250 µL of deionized water); load (10 x 150 µL of hair extract); washing (50 µL of 0.1% acetic acid and 50 µL of 10% methanol); and elution (100 µL of 3% ammonium hydroxide in methanol). Results & Discussion: The procedure resulted in acceptable recoveries, 39-61% for K and 32-43% for NK, and allowed reaching limits of quantification (LOQs) of 50 pg/mg for both analytes. The analytical method presented acceptable accuracy and precision with coefficients of variation typically lower than 15% and BIAS within ± 15%, except at the LOQ (20%). The method was successfully applied to 2 authentic samples, and ketamine concentrations were below 0.05 and 0.18 ng/mg. Norketamine was not detected. Conclusions: This work is the first analytical method using MEPS coupled to GC-MS/MS for the determination of K and NK in hair samples. Following a comparison with a SPE-based method using authentic samples, it was considered rapid and suitable for routine analysis. Acknowledgments: FCT (UIDB /00709/2020 and UIDP/00709/2020). A.Y. Simão acknowledges the PhD fellowship from FCT (2020.09070.BD).
- Optimization and validation of an analytical method for the determination of opiates in urine using microextraction by packed sorbentPublication . Simão, Ana Y.; Monteiro, Catarina; Marques, Hernâni; Rosado, Tiago; Barroso, Mário; Andraus, Maristela; Gallardo, EugeniaAccording to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), approximately 1.3 million individuals have used opiates, both for medical and illicit purposes1, presenting a significant public health challenge2,3. To address this issue, methods for quantifying these substances are needed. Urine is a commonly employed matrix in clinical and forensic toxicological analyses due to its ease of collection and ample availability. Its short detection window is particularly effective for monitoring recent drug exposure4,5. This study aimed to optimize a method for determining tramadol, codeine, morphine, 6-acetylmorphine, 6- acetylcodeine, and fentanyl in urine samples (250 μL). The process involved centrifugation, acid hydrolysis, and extraction using microextraction by packed sorbent (MEPS). MEPS offered a rapid, environmentally friendly, and reusable extraction technique6. All parameters that influence the extraction were previously optimized. The method was validated following international guidelines, demonstrating excellent linearity [1 to 1000 ng/mL for all compounds, except for fentanyl (10 to 1000 ng/mL), with coefficients of determination of at least 0.99], and presenting coefficients of variation and bias ≤ 15% for precision and accuracy, except for the lowest calibrator (≤ 20%). Recoveries obtained ranged from 17 to 107%, with lowest percentages for morphine (12 to 17%). Despite the low extraction efficiency obtained for morphine, it was possible to detect concentrations as low as 1 ng/mL for all compounds, except for fentanyl (10 ng/mL). The method was successfully applied to real samples from consumers of these substances. This is the first method to use MEPS and GC-MS/MS for the simultaneous determination of these six opioids in urine samples.
- Otimização de um método para a determinação de anfetaminas e derivados em cabelo por microextração em seringa empacotadaPublication . Pires, Bruno; Simão, Ana Y.; Rosado, Tiago; Gallardo, Eugenia; Barroso, MárioTêm sido utilizados diferentes protocolos de extração para a determinação de compostos relacionados com anfetaminas em cabelo. A microextração em seringa empacotada (MEPS) é uma versão miniaturizada da extração em fase sólida que já tem sido aplicada para determinar diversas drogas em cabelo (por exemplo, opiáceos, cocaína, ketamina, etc.). No entanto, no que diz respeito às anfetaminas, a MEPS só foi ainda utilizada em cabelo para a determinação de anfetamina (AMP) e metanfetamina (MAMP). O objetivo deste trabalho foi o desenvolvimento e otimização de um método para determinar não só a AMP e a MAMP, como também MDA, MDMA, MDE e MBDB em amostras cabelo, utilizando a MEPS como técnica de extração. O cabelo pulverizado (50 mg) foi incubado com NaOH 1M a 45 ºC durante a noite, após o que se adicionou HCl 10M para neutralizar. De seguida, a solução foi centrifugada, recolhendo cuidadosamente o sobrenadante. Após se realizar a extração recorrendo ao MEPS, adicionou-se 30 μL de MBTFA ao eluato obtido, e as amostras foram evaporadas à secura. Posteriormente foram derivatizadas com recurso a microondas e utilizando 50 μL de MBTFA. O produto derivatizado foi então analisado por GC-MS em modo SIM. O procedimento de MEPS foi optimizado usando os softwares MiniTab e SPSS, mediante dois testes de Desenho Experimental (DOE) e uma análise não paramétrica, respetivamente. Inicialmente, foi conduzido um DOE fatorial, com o estudo dos seguintes parâmetros: número de aspirações de amostra (entre 5 e 15 aspirações de 100 μL); número de aspirações para a lavagem [entre 1 e 3 aspirações com 50 μL de H2O ultrafiltrada e 50 μL de H2O:MeOH (95:5)]; e número de aspirações para a eluição (entre 2 e 6 eluições com 100 μL de 2% NH4OH em ACN). Foi necessário implementar um Desenho Experimental de Superfície de Resposta (SR), estendendo os limites dos parâmetros mencionados para uma análise mais detalhada. Os intervalos ampliados foram os seguintes: 0 a 26,1 aspirações de 100 μL de amostra; 0 a 7,7 aspirações de lavagem; e 0 a 10,3 aspirações de eluição. Após esta optimização, o método final de MEPS foi o seguinte: 18 aspirações de 100 μL de amostra; 0 aspirações para a lavagem; 7 aspirações com 100 μL de 2% NH4OH em ACN para a eluição. Este método permite a análise sensível de compostos derivados das anfetaminas em amostras de cabelo, podendo ser utilizado em situações onde estejam envolvidos.
- Determination of amphetamine-type psychostimulants in hair samples using MEPS as sample clean-up and gas-chromatography coupled with mass spectrometryPublication . Pires, Bruno; Simão, Ana Y.; Rosado, Tiago; Gallardo, Eugenia; Barroso, MárioNumerous protocols for the analysis of amphetamine-type psychostimulants (ATS) in hair have been employed over the years. Microextraction by packed sorbent (MEPS), a downsized version of solid-phase extraction (SPE), has been effectively applied in hair drug analysis, including substances like opiates, cocaine, and ketamine. However, concerning ATS, MEPS has primarily been used to the determination of amphetamine (AMP) and methamphetamine (MAMP) in hair1-4. The main objective of this study was to develop and validate a method using MEPS as a sample clean-up, for the determination of AMP, MAMP, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), 1-(1,3-benzodioxol-5-yl)propan-2-yl(ethyl)amine (MDE), and N-methyl-1-(1,3-benzodioxol-5-yl)-2-aminobutane (MBDB) in hair. The extraction procedure involved incubating 50 mg of hair with 1M NaOH at 45 ºC overnight, followed by neutralization with 10M HCl and centrifugation5. A Design of Experiments (DoE) approach was employed to optimize MEPS clean-up process, encompassing conditioning, loading, and elution steps. The eluted extract underwent derivatization and was analysed using gas chromatography coupled to mass spectrometry (GC-MS). The developed MEPS method yielded recoveries ranging from 8 to 52% for the different analytes in hair samples and linearity was obtained between 0.2 (cut-off proposed by SoHT) and 5.0 ng/mg. The precision and accuracy were in accordance to international method validation standards. This study introduces the first analytical method integrating MEPS with GC-MS for the detection of these specific amphetamines in hair samples. Notably, this method offers a viable alternative to conventional procedures, characterized by its speed, eco-friendliness, and cost-effectiveness.
- Determination of amphetamine-related drugs in hair using MEPS as sample clean-upPublication . Pires, Bruno; Simão, Ana Y.; Rosado, Tiago; Gallardo, Eugenia; Barroso, MárioIntroduction and aims: Different extraction and clean-up protocols have been used for amphetamine-related compounds determination in hair. Microextraction by packed sorbent (MEPS) is a miniaturized version of solidphase extraction (SPE) which has been applied to determine a number of drugs in hair (e.g., opiates, cocaine, ketamine, etc.) [1-4]. However, concerning amphetamine-related drugs, MEPS has only been used in hair for the determination of amphetamine (AMP) and methamphetamine (MAMP). The aim of this work was the development and validation of a method to determine AMP, MAMP, MDA, MDMA, MDE and MBDB in hair using MEPS for sample clean-up. Materials and methods: Hair (50 mg) was incubated with NaOH 1M at 45 °C overnight [5], after which HCl 10M was added for neutralization and the solution was centrifuged. The MEPS procedure for extract clean-up was optimized using the Design of Experiments (DoE) approach, and the final steps were: conditioning (1 x 250 μL of methanol and 1 x 250 μL of deionized water); loading (18 x 100 μL); and elution (7 x 100 μL of 2% NH4OH in acetonitrile). To the eluted extract, 30 μL of MBTFA was added (to avoid loss of analytes), and extracts were evaporated to dryness, following microwave-assisted derivatization with 50 μL of MBTFA. A gas chromatography coupled to mass spectrometry (GC-MS) system was used for determination. Results and discussion: The MEPS procedure resulted in recoveries of 8-14% for AMP, 14-20% for MAMP, 10-15% for MDA, 18-28% for MDMA, 25-43% for MDE and 34-52% for MBDB, in the linear range of 0.2-5.0 ng/mg. Precision and accuracy of the developed method were in accordance with the statements of international guidelines for method validation. Conclusions: This is the first analytical method using MEPS coupled to GC-MS to determine the selected amphetamines in hair samples, proving to be a great alternative to the classic procedures, being rapid, eco-friendly and less expensive.
- A review of publications in hair testing: 2020-2023Publication . Simão, Ana Y.; Antunes, Mónica; Pires, Bruno; Catarro, Gonçalo; Soares, Sofia; Matos, Francisco; Rosado, Tiago; Gallardo, Eugenia; Barroso, MárioIntroduction and Aims: Since its first use until now, the relevance of hair in the toxicological expertise has evolved, mainly due to its advantages over traditional samples. Despite the fact that it is one of the most used alternative specimens, hair still presents important challenges. The objective of this study was to perform a bibliographic review of scientific publications on toxicological applications of hair testing between the years 2020 and 2023. Materials & Methods: The Scopus database was used for literature search, for papers published from 2020 to 2023 (10th April), and only articles written in English were considered. The search strings for terms included in the title, abstract or keywords were “hair testing” and “hair samples”. The papers were independently reviewed by four of the authors to determine their relevance in the context of this study. Results & Discussion: Taking into consideration these criteria and search strings, 218 articles were found, from which 18 were excluded since animal hair specimens had been used. The papers were classified according to type (e.g. review, research paper), analytes, extraction and clean-up approaches. Each paper could be placed under more than one category, except review papers (32 articles). Concerning analytes, the highest number of papers involved drugs of abuse (175, from which 45 involving new psychoactive substances), drugs (44) and alcohol biomarkers (17). Other biomarkers included hormones, endocannabinoids, etc. (44 papers, 25 of which involved cortisol). Most studies involve protein precipitation and direct injection (91), and only 12 studies address the issues of cosmetic treatments or drug adulterants. Conclusions: Hair testing is still mainly directed to the determination of drugs of abuse, and new psychoactive substances are still seldom included. Additionally, other applications are becoming more relevant, for instance the use of hair to determine biomarkers in some diseases.