Determinação de fármacos como contaminantes em águas residuárias utilizando preparo de amostra on-line e LC-MS/MS e aplicação da técnica MRM-profiling na avaliação de compostos de relevância ambiental (Exposoma)
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Data
2020-11-25
Autores
Marasco Júnior, César Augusto [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Compostos farmacêuticos e produtos de cuidado pessoal (PPCPs) são substâncias amplamente presentes na sociedade moderna. Estes compostos, conhecidos como contaminantes emergentes, são encontrados no ambiente em níveis de concentração que variam de ng L-1 a µg L-1. Assim, faz-se necessário o desenvolvimento de estudos e métodos analíticos para determinar compostos psicoativos, antibióticos e marcadores de atividade antropogênica em países em desenvolvimento, como no Brasil, onde as condições de saneamento ainda são precárias, seja pela falta de estações de tratamento de esgoto (ETEs) e/ou tratamento adequado dos efluentes. A cromatografia líquida de alta eficiência acoplada à espectrometria de massa sequencial (LC-MS/MS) tem sido a técnica de escolha para detecção e quantificação desses compostos a baixos níveis de concentração (i.e., ng L-1). Tendo em vista a complexidade das amostras ambientais, é necessário a aplicação da extração em fase sólida (SPE) e/ou de materiais de acesso restrito (RAM) para pré-concentrar os analitos. Estas abordagens, aliado ao modo on-line de preparo de amostras, constituem uma alternativa para diminuir a contaminação cruzada, além de permitir automatização e realização do procedimento de forma sequencial. Os métodos analíticos desenvolvidos foram validados de acordo com diretivas nacionais e internacionais de validação utilizando apenas 50 µL de volume de injeção em água ultrapura e esgoto lab-made, sendo a primeira abordagem considerada para quantificação em virtude do uso de padrões isotópicos individuais. Figuras de mérito como limite de detecção (LD), limite de quantificação (LQ), linearidade, precisão intra- e inter-dia e efeito matriz foram avaliadas. Análise de variância (ANOVA) foi aplicada para verificar possível falta de ajuste. Além disso, a fim de verificar a eficiência cromatográfica, resolução (Rs) e assimetria de pico (As) foram consideradas; o efeito de memória do sistema também foi avaliado a fim de garantir a confiabilidade da análise e reutilização da coluna de pré-concentração. Com tempos de análise de 24 min (para o cartucho OASIS HLB® da Waters) e 16 min (para coluna RAM feita em laboratório), esses métodos mostraram ser alternativas rápidas e viáveis para analise simultânea dos compostos alvo. A linearidade de ambos os métodos mostrou ser adequada, visto que r2 > 0,99 foi obtido para todos os compostos em água ultrapura ou esgoto lab-made (exceto para CBZ em esgoto lab-made no cartucho OASIS e para FLX também em esgoto lab-made na coluna RAM). Os valores de precisão intra- e inter-dia foram inferiores a 10 e 17 %, respectivamente, para o cartucho OASIS e inferiores a 5,8 e 14,1 % para a coluna RAM. Em ambos os métodos, cafeína foi o composto amplamente detectado, atingindo concentração máxima de 715 µg L−1 na análise utilizando o cartucho de pré-concentração OASIS e 392 µg L−1 na análise utilizando a coluna RAM. Uma estratégia exploratória simples e informativa aplicada à análise de produtos químicos de exposição ambiental em amostras biológicas foi desenvolvida utilizando a técnica MRM-profiling, que apontou e confirmou a presença de quatro compostos alvo dentre os 87 avaliados em amostras de urina humana, sendo eles metformina, acrilamida, acetaminofeno e metoprolol.
Pharmaceutical compounds and personal care products (PPCPs) are substances widely present in modern society. These compounds, known as emerging contaminants, are found in the environment at concentration levels ranging from ng L-1 to µg L-1. Thus, it is necessary to develop studies and analytical methods to determine psychoactive compounds, antibiotics and markers of anthropogenic activity in developing countries, such as Brazil, where sanitation conditions are still precarious, either due to the lack of wastewater treatment plants (WWTPs) and/or suitable effluents treatment. High performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has been the technique of choice for detecting and quantifying these compounds at background concentrations (i.e., ng L-1). Due the complexity of the environmental samples, it is necessary to apply solid phase extraction (SPE) and/or restricted access media (RAM) to pre-concentrate the analytes. These approaches combined with the online method of sample preparation are an alternative to decrease cross-contamination, in addition to allowing automation and carrying out the procedure sequentially. The analytical methods developed were validated according to national and international validation guidelines using only 50 µL of injection volume in ultrapure water and lab-made sewage, being the first approach being considered for quantification due to the use of individual isotopic labeled standards. Figures of merit such as limit of detection (LOD), limit of quantification (LOQ), linearity, intra- and inter-day precision and matrix effect were evaluated. Analysis of variance (ANOVA) was applied to verify possible lack of fit. In addition, in order to verify the chromatographic efficiency, resolution (Rs) and peak asymmetry (As) were considered; carryover was also evaluated in order to ensure analysis reliability and reuse preconcentration column reusability. With analysis times of 24 min (for Waters' OASIS HLB® cartridge) and 16 min (for RAM column), the methods proved to be fast and viable alternatives for simultaneous analysis of the target compounds. The methods’ linearity proved to be suitable, since r2 > 0,99 was obtained for all compounds in ultrapure water and lab-made sewage (except for CBZ in lab-made sewage on OASIS cartridge and for FLX also in lab-made sewage on RAM column). The intra- and inter-day precision values were less than 10 and 17 %, respectively, for the OASIS cartridge and less than 5,8 and 14,1 % for the RAM column. In both methods, caffeine was the compound widely detected, reaching a maximum concentration of 715 µg L-1 in the analysis using the OASIS pre-concentration cartridge and 392 µg L-1 in the analysis using the RAM column. A simple and informative exploratory strategy applied to the analysis of environmental exposure chemicals in biological samples was developed using the MRM-profiling mass spectrometry, which suggested and confirmed the presence of four target compounds among the 87 evaluated in human urine samples, being them metformin, acrylamide, acetaminophen and metoprolol.
Pharmaceutical compounds and personal care products (PPCPs) are substances widely present in modern society. These compounds, known as emerging contaminants, are found in the environment at concentration levels ranging from ng L-1 to µg L-1. Thus, it is necessary to develop studies and analytical methods to determine psychoactive compounds, antibiotics and markers of anthropogenic activity in developing countries, such as Brazil, where sanitation conditions are still precarious, either due to the lack of wastewater treatment plants (WWTPs) and/or suitable effluents treatment. High performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has been the technique of choice for detecting and quantifying these compounds at background concentrations (i.e., ng L-1). Due the complexity of the environmental samples, it is necessary to apply solid phase extraction (SPE) and/or restricted access media (RAM) to pre-concentrate the analytes. These approaches combined with the online method of sample preparation are an alternative to decrease cross-contamination, in addition to allowing automation and carrying out the procedure sequentially. The analytical methods developed were validated according to national and international validation guidelines using only 50 µL of injection volume in ultrapure water and lab-made sewage, being the first approach being considered for quantification due to the use of individual isotopic labeled standards. Figures of merit such as limit of detection (LOD), limit of quantification (LOQ), linearity, intra- and inter-day precision and matrix effect were evaluated. Analysis of variance (ANOVA) was applied to verify possible lack of fit. In addition, in order to verify the chromatographic efficiency, resolution (Rs) and peak asymmetry (As) were considered; carryover was also evaluated in order to ensure analysis reliability and reuse preconcentration column reusability. With analysis times of 24 min (for Waters' OASIS HLB® cartridge) and 16 min (for RAM column), the methods proved to be fast and viable alternatives for simultaneous analysis of the target compounds. The methods’ linearity proved to be suitable, since r2 > 0,99 was obtained for all compounds in ultrapure water and lab-made sewage (except for CBZ in lab-made sewage on OASIS cartridge and for FLX also in lab-made sewage on RAM column). The intra- and inter-day precision values were less than 10 and 17 %, respectively, for the OASIS cartridge and less than 5,8 and 14,1 % for the RAM column. In both methods, caffeine was the compound widely detected, reaching a maximum concentration of 715 µg L-1 in the analysis using the OASIS pre-concentration cartridge and 392 µg L-1 in the analysis using the RAM column. A simple and informative exploratory strategy applied to the analysis of environmental exposure chemicals in biological samples was developed using the MRM-profiling mass spectrometry, which suggested and confirmed the presence of four target compounds among the 87 evaluated in human urine samples, being them metformin, acrylamide, acetaminophen and metoprolol.
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Palavras-chave
Preparo de amostras (Química), Cromatografia líquida, Espectrometria de massa, Fármacos, Esgotos - Aspectos ambientais