Evaluation of hair hydrolysis and MEPS columns for the detection of amphetamines derivatives in hair

Comunicação apresentada no 10º Congresso Iberoamericano de Ciências Farmacêuticas, Coimbra, Outubro de 2023. Abstract da comunicação publicado na Acta Farmacêutica Portuguesa. vol. de Outubro 2023. ISSN 2182-3340.Introduction: Different extraction and cleanup procedures have been used to identify amphetamines in hair samples. The microextraction by packed sorbent (MEPS), a miniaturized approach of the classic solid-phase extraction (SPE), has only been used so far to determine the presence of amphetamine (AMP) and methamphetamine (MAMP) in hair. The aim of this work was to evaluate different hair sample extraction conditions, namely hydrolysis, to maximize the detection of AMP, MAMP, MDA, MDMA, MDE, and MBDB in combination with MEPS and GC-MS. Methodology: Pulverized authentic hair (50mg) was incubated with 500μL of NaOH 1M at 80ºC for 1H, 60ºC overnight and 45ºC overnight1. After incubation, the extracts were neutralized with 50μL of 10M HCl and subsequently centrifuged. The supernatant was tested with several conditions to access the most suitable when paired with MEPS clean-up. The studied conditions were as follows: the addition of 500μL of H2O; supernatant filtration followed by addition of H2O; and precipitation with cold ACN followed by centrifugation. After clean-up, 30μL of MBTFA were added for evaporation under nitrogen gas. After being fully dried, 50μL of MBTFA was added and derivatization was done with a microwave oven. Finally, the extract was injected into a GC-MS and analysed. Results: Results obtained from these tests indicated that the best hydrolysis for this purpose was obtained with 500μL NaOH 1M at 45ºC overnight, with the addition of H2O before MEPS clean-up. The clean-up procedure was previously optimized by design of experiments (not being the scope of this study). Furthermore, M1 and C18 MEPS sorbents were tested to check which one would provide cleaner extracts and greater analyte signals. A concentration of 2.00 ng/mg of AMP, MAMP, MDA, MDMA, MDE, and MBDB was used for this test. Discussion: Considering the results, both columns presented similar recoveries for each amphetamine, when using equal hydrolysis and MEPS conditions.N/

Determination of amphetamine-related drugs in hair using MEPS as sample clean-up

Comuniicação apresentada no 27th Meeting of the Society of Hair Testing, Lisboa, 2023Introduction 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.N/

Determination of amphetamine-type psychostimulants in hair samples using MEPS as sample clean-up and gas-chromatography coupled with mass spectrometry

Comunicação apresentada no 13º Encontro Nacional de CromatografiaNumerous 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.N/

Optimization and validation of an analytical method for the determination of opiates in urine using microextraction by packed sorbent

Comunicação apresentada no 13º Encontro Nacional de Cromatografia, 2023According 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.N/

Evaluation of the Cytotoxicity of Ayahuasca Beverages

Ayahuasca 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-Texinfo:eu-repo/semantics/publishedVersio

Determination of Ketamine and Norketamine in hair samples using MEPS as sample clean-up

Palestra realizada no encontro SoHT-GTFI Joint Meeting, Verona, Junho 2022Introduction 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).N/

Analysis of opiates in urine using microextraction by packed sorbent and gas Chromatography- Tandem mass spectrometry

Opiates 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).info:eu-repo/semantics/publishedVersio

A review of publications in hair testing: 2020-2023

Comunicação apresentada no 27th Meeting of the Society of Hair Testing.Introduction 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.N/

Otimização de um método para a determinação de anfetaminas e derivados em cabelo por microextração em seringa empacotada

Poster apresentado no 21º Congresso Nacional de Medicina Legal e Ciências Forenses, Lamego, 2023Têm 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.N/

Insights into the Bioactivities and Chemical Analysis of Ailanthus altissima (Mill.) Swingle

ReviewMany species of the so-called exotic plants coexist with native species in a balanced way, but others thrive very quickly and escape human control, becoming harmful—these are called invasive alien species. In addition to overcoming geographic barriers, these species can defeat biotic and abiotic barriers, maintaining stable populations. Ailanthus altissima is no exception; it is disseminated worldwide and is considered high risk due to its easy propagation and resistance to external environmental factors. Currently, it has no particular use other than ornamental, even though it is used to treat epilepsy, diarrhea, asthma, ophthalmic diseases, and seborrhoea in Chinese medicine. Considering its rich composition in alkaloids, terpenoids, sterols, and flavonoids, doubtlessly, its use in medicine or other fields can be maximised. This review will focus on the knowledge of the chemical composition and the discovery of the biological properties of A. altissima to understand this plant better and maximise its possible use for purposes such as medicine, pharmacy, or the food industry. Methods for the extraction and detection to know the chemical composition will also be discussed in detailinfo:eu-repo/semantics/publishedVersio