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Materiais em contacto com a água para consumo humano : implementação e validação de um método de ensaio para análise de aminas e nitrosaminas
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O trabalho desenvolvido está centrado na implementação e validação de um método de cromatografia gasosa associado à espectrometria de massa (GC-MS) para a análise de aminas e nitrosaminas nos lixiviados dos ensaios de migração com diferentes materiais. O objetivo do trabalho foi alinhado com a necessidade de desenvolver e validar novas técnicas analíticas para a monitorização de novos parâmetros da qualidade da água (aminas e nitrosaminas), de forma a cumprir os novos requisitos previstos na Diretiva (UE) 2020/2184 do Parlamento Europeu e do Conselho de 16 de dezembro de 2020, relativa à qualidade da água destinada ao consumo humano.
Este trabalho teve início com a otimização de vários parâmetros da GC-MS, nomeadamente, seleção dos fragmentos iónicos (razão massa/carga) característicos de cada composto, tempos de retenção e programa de temperatura, de forma a aumentar a sensibilidade e seletividade do método para 22 compostos alvo.
O método por GC-MS apresentou uma boa gama de trabalho tanto para as nitrosaminas como para as aminas, com coeficientes de determinação (r2) entre 0,9952 (DBuA) e 0,9999 (NDMA), com coeficientes de variação do método (CVm %) inferiores a 3,5 % e PG < F (0,05; 1; N-3). A gama de trabalho variou entre 10 µg/L e 356 µg/L para as nitrosaminas e 17 µg/L e 1359 µg/L para as aminas. Os limites de deteção (LOD) variaram entre os 0,7 µg/L (NDMA e ENBA) e 55,0 µg/L (NMBA) e os limites de quantificação (LOQ) variaram entre os 2,3 µg/L e 183,40 µg/L.
O método de GC-MS apresentou a coeluição de algumas aminas e nitrosaminas. Como a valor limite das aminas é de 250 µg/L testou-se dois métodos espectrofotométricos para a análise de aminas, o método do ácido p-cloranílico (pCA) e o método da ninidrina. O método do pCA não é aplicável a amostras aquosas e no método da ninidrina, o composto mais sensível apresentava uma sensibilidade de 500 mg/L, a qual representa uma concentração 2000 vezes superior ao valor guia das aminas (250 µg/L).
Face à impossibilidade de utilização de um método espetrofotométrico para a quantificação de aminas, implementou-se um método por GC-MS, que foi denominado de Método B, sendo separado do método já desenvolvido para as nitrosaminas (Método A).
Face à baixa concentração destes compostos nos lixiviados (ordem dos µg/L) estudaram-se duas técnicas de pré-tratamento da amostra, a extração em fase sólida (SPE) e a extração líquido-líquido (LLE), com o objetivo de pré-concentração da amostra.
Relativamente à SPE verificou-se a presença de contaminantes provenientes da fase estacionária dos cartuchos interferindo na análise de duas nitrosaminas, nomeadamente a N-metil-N-nitrosobenzenamina (MNBA) e N-nitroso-di-isopropilamina (NDIPA). Como as taxas de recuperação dos compostos alvo por SPE-GC-MS, foram inferiores a 70% para a maioria dos compostos, a SPE como técnica de preparação de amostra foi substituída pela extração líquido-líquido (LLE). Vários parâmetros foram otimizados, entre os quais, pH da amostra, volume de amostra e volume de solvente.
Para as nitrosaminas estudadas, a precisão instrumental foi avaliada em condições de repetibilidade e precisão intermédia. O desvio padrão relativo (DPR), nos ensaios de repetibilidade, variou entre 0,8% e 15% na gama de concentrações superiores e 1,1% e 5,3% na gama inferior de concentrações. O desvio padrão relativo foi inferior a 25% para os ensaios de precisão intermédia.
As recuperações médias do método de LLE-GC-MS variaram entre 49% (NDMA) e 127% (NDBA) com um DPR < 25%.
Os limites de quantificação do método (MQL) variaram entre 0,0045 µg/L (ENBA) e 0,0378 µg/L (NDBA). A soma dos limites de quantificação do método (0,2 µg/L) é inferior ao limite de referência de 0,3 µg/L para as nitrosaminas nos lixiviados dos ensaios de migração.
De acordo com os estudos de estabilidade de amostra e dos extratos, a análise da amostra pode ser realizada até 14 dias após a data da colheita e os extratos podem ser analisados até 21 dias (3 semanas) se armazenados num congelador a – 80 °C.
Os estudos de migração foram realizados em 4 materiais os quais foram submetidos a ensaios de migração sequenciais em água desmineralizada, com e sem cloro, num total de 3 ensaios de migração. Para efeito de controlo dos materiais, apenas é considerado o valor das nitrosaminas no terceiro ensaio de migração. Nas águas de migração destes materiais foram identificadas e quantificadas quatro nitrosaminas, nomeadamente a MNBA, NMOR, NDPA e ENBA. Na água da 3ª migração apenas um dos materiais apresentou nitrosaminas em concentrações superiores ao LOQ. A concentração na água de migração (CTap) foi de 0,070 µg/L e 0,040 µg/L para as águas de migração com e sem cloro, respetivamente. Como o CTap foi inferior ao valor estabelecido como referência (0,3 µg/L), os materiais foram aprovados para utilização na rede de distribuição de água de consumo humano.
The work developed focuses on implementing and validating a gas chromatography–mass spectrometry (GC-MS) method for the analysis of amines and nitrosamines in the leachates from migration assays in different materials. The aim of the work was aligned with the need to develop and validate new analytical techniques for monitoring new water quality parameters (amines and nitrosamines) to fulfill the new requirements set out in Directive (EU) 2020/2184 of the European Parliament and the Council of December 16, 2020, on the quality of water intended for human consumption. This work began with the optimization of various GC-MS parameters, namely the selection of ionic fragments (mass/charge ratio) characteristic of each compound, retention times, and temperature program, to increase the sensitivity and selectivity of the method for 22 target compounds. The GC-MS method showed a good working range for both nitrosamines and amines, with coefficients of determination (r2) between 0.9952 (DBuA) and 0.9999 (NDMA), with method coefficients of variation (CVm %) lower than 3.5 % and PG < F (0.05; 1; N-3). The working range varied between 10 µg/L e 356 µg/L for nitrosamines and 17 µg/L e 1359 µg/L for amines. The detection limits (LOD) ranged from 0.7 µg/L (NDMA and ENBA) and 55.0 µg/L (NMBA) and the limits of quantification (LOQ) ranged from 2.3 µg/L and 183.40 µg/L. The GC-MS method showed coelution of some amines and nitrosamines. As the parametric value for amines is 250 µg/L, two spectrophotometric methods were tested for analysing amines, the p-chloranilic acid (pCA) method and the ninhydrin method. The pCA method is not applicable to aqueous samples and in the ninhydrin method, the most sensitive compound had a sensitivity of 500 mg/L, which represents a concentration 2000 times higher than the guide value for amines (250 µg/L). Since it was impossible to use a spectrophotometric method to quantify amines, a GC-MS method was implemented, called Method B, which was separate from the existing method for nitrosamines (Method A). Given the low concentration of these compounds in the leachates (around µg/L), two sample pre-treatment techniques were studied: solid phase extraction (SPE) and liquid-liquid extraction (LLE), with the aim of pre-concentrating the sample. Regarding SPE, it was verified that the stationary phase of the cartridges analysed showed interference from two nitrosamines, namely N-methyl-N-nitrosobenzenamine (MNBA) and N-nitroso-di-isopropylamine (NDIPA). The recovery rates of the target compounds by SPE-GC-MS were less than 70% for most of the compounds. SPE, as a sample preparation technique, was replaced by liquid-liquid extraction (LLE). Several parameters were optimized, including sample pH, sample volume, and solvent volume. Instrumental precision was assessed under repeatability and intermediate precision conditions for the nitrosamines studied. The relative standard deviation (RSD) in the repeatability tests varied between 0.8% and 15% in the upper concentration range and 1.1% and 5.3% in the lower concentration range. The intermediate precision tests relative standard deviation was less than 25%. The average recoveries of the LLE-GC-MS method ranged from 49% (NDMA) to 127% (NDBA) with an RSD < 25%. The method quantification limits (MQL) ranged from 0.0045 µg/L (ENBA) and 0.0378 µg/L (NDBA). The sum of the method quantification (0.2 µg/L) is lower than the reference limit of 0.3 µg/L for nitrosamines in the leachates from the migration assays. According to the sample and extract stability studies, the sample can be analysed up to 14 days after the collection date, and the extracts can be analysed after 21 days (3 weeks) if stored in a freezer at - 80 °C. The sequential migration waters of 4 materials were analysed and four nitrosamines were identified and quantified, namely MNBA, NMOR, NDPA, and ENBA. At the end of the 3rd migration, the migration water of one of the materials showed nitrosamines in concentrations higher than the LOQ. The concentration in the migration water (CTap) was 0.070 µg/L and 0.040 µg/L for the migration water with and without chlorine, respectively. As the CTap was lower than the value established as a reference for (0.3 µg/L), the materials were approved for use in the drinking water distribution network.
The work developed focuses on implementing and validating a gas chromatography–mass spectrometry (GC-MS) method for the analysis of amines and nitrosamines in the leachates from migration assays in different materials. The aim of the work was aligned with the need to develop and validate new analytical techniques for monitoring new water quality parameters (amines and nitrosamines) to fulfill the new requirements set out in Directive (EU) 2020/2184 of the European Parliament and the Council of December 16, 2020, on the quality of water intended for human consumption. This work began with the optimization of various GC-MS parameters, namely the selection of ionic fragments (mass/charge ratio) characteristic of each compound, retention times, and temperature program, to increase the sensitivity and selectivity of the method for 22 target compounds. The GC-MS method showed a good working range for both nitrosamines and amines, with coefficients of determination (r2) between 0.9952 (DBuA) and 0.9999 (NDMA), with method coefficients of variation (CVm %) lower than 3.5 % and PG < F (0.05; 1; N-3). The working range varied between 10 µg/L e 356 µg/L for nitrosamines and 17 µg/L e 1359 µg/L for amines. The detection limits (LOD) ranged from 0.7 µg/L (NDMA and ENBA) and 55.0 µg/L (NMBA) and the limits of quantification (LOQ) ranged from 2.3 µg/L and 183.40 µg/L. The GC-MS method showed coelution of some amines and nitrosamines. As the parametric value for amines is 250 µg/L, two spectrophotometric methods were tested for analysing amines, the p-chloranilic acid (pCA) method and the ninhydrin method. The pCA method is not applicable to aqueous samples and in the ninhydrin method, the most sensitive compound had a sensitivity of 500 mg/L, which represents a concentration 2000 times higher than the guide value for amines (250 µg/L). Since it was impossible to use a spectrophotometric method to quantify amines, a GC-MS method was implemented, called Method B, which was separate from the existing method for nitrosamines (Method A). Given the low concentration of these compounds in the leachates (around µg/L), two sample pre-treatment techniques were studied: solid phase extraction (SPE) and liquid-liquid extraction (LLE), with the aim of pre-concentrating the sample. Regarding SPE, it was verified that the stationary phase of the cartridges analysed showed interference from two nitrosamines, namely N-methyl-N-nitrosobenzenamine (MNBA) and N-nitroso-di-isopropylamine (NDIPA). The recovery rates of the target compounds by SPE-GC-MS were less than 70% for most of the compounds. SPE, as a sample preparation technique, was replaced by liquid-liquid extraction (LLE). Several parameters were optimized, including sample pH, sample volume, and solvent volume. Instrumental precision was assessed under repeatability and intermediate precision conditions for the nitrosamines studied. The relative standard deviation (RSD) in the repeatability tests varied between 0.8% and 15% in the upper concentration range and 1.1% and 5.3% in the lower concentration range. The intermediate precision tests relative standard deviation was less than 25%. The average recoveries of the LLE-GC-MS method ranged from 49% (NDMA) to 127% (NDBA) with an RSD < 25%. The method quantification limits (MQL) ranged from 0.0045 µg/L (ENBA) and 0.0378 µg/L (NDBA). The sum of the method quantification (0.2 µg/L) is lower than the reference limit of 0.3 µg/L for nitrosamines in the leachates from the migration assays. According to the sample and extract stability studies, the sample can be analysed up to 14 days after the collection date, and the extracts can be analysed after 21 days (3 weeks) if stored in a freezer at - 80 °C. The sequential migration waters of 4 materials were analysed and four nitrosamines were identified and quantified, namely MNBA, NMOR, NDPA, and ENBA. At the end of the 3rd migration, the migration water of one of the materials showed nitrosamines in concentrations higher than the LOQ. The concentration in the migration water (CTap) was 0.070 µg/L and 0.040 µg/L for the migration water with and without chlorine, respectively. As the CTap was lower than the value established as a reference for (0.3 µg/L), the materials were approved for use in the drinking water distribution network.
Descrição
Tese de mestrado, Qualidade Alimentar e Saúde, 2023, Universidade de Lisboa, Faculdade de Farmácia.
Palavras-chave
Água Aminas e nitrosaminas Cromatografia gasosa Espectrometria de massa Materiais Validação estatística Teses de mestrado - 2023