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- 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).
- Microextraction by packed sorbent clean-up for the determination of selected synthetic cathinones in hair samplesPublication . Simão, A.; Margalho, Cláudia; Dinis, P.; Andraus, M.; Barroso, Mário; Gallardo, E.Background and aims: The use of synthetic cathinones (SC), also known as "bath salts," has been increasing in recent years, leading to a growing concern about their effects on public health. Different clean-up approaches have already been studied by different authors. However, microextraction by packed sorbent (MEPS), a miniaturized form of solid-phase extraction, has not been explored extensively for the determination of SC in hair samples. Therefore, we propose the optimization of the MEPS procedure as sample clean-up method followed by gas chromatography mass spectrometry for the determination of these analytes in hair samples. Methods: Hair samples were washed consecutively with dichloromethane, deionized water and methanol to remove dirt and externally deposited material. After drying, hair was scissor-cut into small fragments. Fifty milligrams of hair were incubated overnight in the presence of 1 M NaOH at 45 °C, after which the extracts were neutralized and subjected to MEPS using a C8-SCX cartridge. The compounds were analyzed underivatized by GC-MS in the SIM mode. MEPS conditions were conditioning (250 μL of methanol followed by 250 μL of water), sample loading (24 x 700 μL of hair extract) and elution (7 x 100 μL of 2% NH4OH in acetonitrile). Results and discussion: The procedure resulted in acceptable recoveries (15-59% for all analytes), and allowed reaching lower limits of quantification (LLOQs) of 100 pg/mg for 4-MDMB, 4-MNEB and 4- MDMP and of 200 pg/mg for the remaining analytes (4-CEC, 4-MNEP, 4-MDEB, 4-MDMH, ethylone, alpha-PVP, penthylone, 4-Cl-PVP, MPHP and MDPV). Conclusions: In this work the optimization of the MEPS technique coupled to GC-MS for the determination of selected synthetic cathinones in hair samples is presented. The method was considered suitable for the analysis of these compounds in hair, providing acceptable recoveries and obtaining low limits of quantification.
- Validação de um método analítico para a determinação de antidepressivos em fluido oral com recurso a MEPS e análise por GC-MS/MSPublication . Soares, Sofia; Rosado, Tiago; Barroso, Mário; Gallardo, EugeniaO consumo de antidepressivos representa uma problemática a nível mundial, apresentando Portugal uma das taxas de prevalência de doenças mentais mais elevadas da Europa. A monitorização terapêutica é uma prática instituída para um pequeno número de fármacos para os quais há uma relação direta entre a concentração e o efeito farmacológico, envolvendo habitualmente a determinação de drogas no soro, plasma ou sangue. O presente trabalho descreve uma metodologia para a determinação de antidepressivos (fluoxetina, venlafaxina, sertralina, citalopram, paroxetina e metabolitos) em fluido oral (250 μL) com recurso à microextração em seringa empacotada (MEPS) e à cromatografia gasosa acoplada à espectrometria de massa em tandem (GC-MS/MS). A técnica MEPS foi otimizada utilizando a ferramenta estatística Design of Experiments (DoE). Previamente à MEPS procedeu-se à precipitação proteica do fluido oral com 500 μL de acetonitrilo gelado, após a qual se centrifugou, decantou e se evaporou o sobrenadante. Posteriormente, dissolveu-se o resíduo em 1 mL de tampão fosfato 25 mM (pH 6). A técnica de MEPS otimizada consistiu no acondicionamento com 250 μL de metanol (MeOH) e 250 μL de 0,1% HCOOH em H2O; load de 150 μL da amostra (12×); lavagem com 4x50 μL de HCOOH a 1% em H2O; secagem por aspiração de 4x50 μL de ar; eluição com 4x100 μL 1% NH4OH em MeOH. O eluato foi evaporado e procedeu-se à derivatização por 2 min em microondas a 800 W, após o qual se injetaram 2 μL no sistema cromatográfico. O método foi validado de acordo com as diretrizes internacionalmente aceites da Food and Drug Administration para o intervalo de linearidade entre 10 e 500 ng/mL. Obtiveram-se valores de coeficiente de determinação de pelo menos 0,99 e os valores de erro relativo e coeficiente de variação foram definidos de acordo com os critérios de validação. Os limites de quantificação foram estabelecidos entre os 10 e os 100 ng/mL e as recuperações absolutas variaram entre 12 e 93%. A estabilidade foi estudada para amostras processadas, estabilidade de bancada e ciclos de congelação/descongelação e a metodologia foi aplicada na análise de amostras autênticas de fluido oral. De salientar que este é o primeiro trabalho que utiliza MEPS para determinação de antidepressivos e metabolitos em amostras de fluido oral.
- 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.
- Desenvolvimento e otimização de uma nova metodologia para determinação de antidepressivos em plasma com extração por MEPS e análise por GC-MS/MSPublication . Soares, Sofia; Rosado, Tiago; Barroso, Mário; Gallardo, EugeniaO consumo de antidepressivos constitui uma problemática mundial e Portugal apresenta uma das taxas de doenças mentais mais elevadas da Europa1. A monitorização terapêutica está estabelecida para um pequeno número de medicamentos para os quais se verifica uma relação direta entre concentração e efeito farmacológico. Este trabalho tem como objetivo desenvolver uma metodologia para deteção de antidepressivos (fluoxetina, venlafaxina, citalopram, sertralina, paroxetina e metabolitos) em 250 μL de plasma por microextração em seringa empacotada (MEPS) e cromatografia gasosa acoplada à espectrometria de massa em tandem (GC-MS/MS). A técnica de MEPS foi otimizada utilizando a ferramenta estatística Design of Experiments e o número de strokes foi o único fator significativo. O procedimento final de extração foi: adicionar 250 μL de amostra com 500 μL de acetonitrilo, centrifugar, decantar e evaporar; dissolver o resíduo com 1 mL de tampão fosfato 25 mM (pH 5), adicionar o padrão interno e homogeneizar; acondicionamento com 250 μL de metanol e 250 μL de 0,1% de ácido fórmico em H2O; loading de 12x150 μL de amostra; lavagem de 4x50 μL com 1% de ácido fórmico em H2O; secagem por aspiração de 4x50 μL de ar; eluição de 6x100 μL com 1% de hidróxido de amónia em metanol; evaporar até a secura, derivatizar durante 2 minutos com microondas a 800 W e injetar 2 μL no sistema cromatográfico. Foram obtidas recuperações entre 12-93% e limites de quantificação entre 10-100 ng/mL. Este é o primeiro trabalho a utilizar a MEPS e GC-MS/MS na determinação de antidepressivos e metabolitos em amostras de plasma.
- 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.
- Microextraction by Packed Sorbent as a Clean-up Approach for the Determination of Ketamine and Norketamine in Hair by Gas Chromatography--Tandem Mass SpectrometryPublication . Simão, Ana Y.; Oliveira, Patrik; Rosendo, Luana; Rosado, Tiago; Andraus, Maristela; Barroso, Mário; Gallardo, EugeniaThe use of new psychoactive substances has been increasing and constitutes a social and public health problem, and hence, toxicological analysis has become of utmost importance for the detection of such substances. In this article, we present the development and full validation of a simple, user and environmentally friendly, cheap and suitable method for the determination of ketamine and its main metabolite norketamine in hair samples. The procedure included using a miniaturized procedure-microextraction by packed sorbent with mixed-mode sorbent-for sample clean-up. Organic solvents use was minimal, and it was possible to obtain a linear method (0.05-10 ng/mg for both analytes). The extraction efficiency ranged from 32 to 61%, which did not impair sensitivity. The method proved to be selective, precise, accurate and suitable for routine analysis for the determination of said compounds in 50-mg hair samples.
- Desenvolvimento e validação de um método para a determinação de canabinóides em urina por microextracção em seringa empacotada e GC-MSPublication . Rosendo, Luana; Rosado, Tiago; Oliveira, Patrik; Simão, Ana; Margalho, Cláudia; Costa, Suzel; Passarinha, Luís; Gallardo, Eugenia; Barroso, MárioDe acordo com o Observatório Europeu da Droga e da Toxicodependência, no último ano (dados de junho de 2022), existiam aproximadamente 22,2 milhões consumidores de canábis na Europa. A urina é uma das amostras mais utilizadas para a deteção e quantificação de canabinóides. (...)