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USAMET/UHPLC-FLR como nova abordagem analítica para a quantificação de aminas biogénicas em tunídeos
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Abstract(s)
O crescente número de casos de intoxicações alimentares, motivadas pela presença
de aminas biogénicas (ABs), tem originado uma crescente preocupação a nível da
segurança alimentar. Neste contexto, este trabalho teve como principal objetivo a
determinação quantitativa e qualitativa de ABs (triptamina, cadaverina, putrescina,
espermina, histamina, tiramina e espermidina) presentes em diferentes amostras de
tunídeos, através da microextração assistida por ultrassons (USAMET), seguida da
derivatização com cloreto de dansilo (DnsCl) e da análise por cromatografia líquida de
ultra pressão, com deteção por fluorescência (UHPLC-FLR), usando um gradiente
trifásico (ácido fosfórico (0,1%): metanol (100%): acetonitrilo (100%)), com um fluxo de
200 µL/min.
Para a otimização do processo extrativo testaram-se diferentes condições
experimentais, nomeadamente, o tempo de agitação por ultrassons (US), (1, 5, 10min)
e a natureza do solvente extrator (acetonitrilo e metanol). Similarmente foram testados
diferentes parâmetros analíticos, com influência no processo de derivatização com
DnsCl, incluindo a concentração do DnsCl (1, 2, 3 g/L), o tempo de derivatização sem
agitação e com agitação por US (10 e 20 min) e ainda a temperatura de derivatização
(temperatura ambiente, 40, 60 °C).
A validação da performance do método analítico foi realizada pela determinação
da seletividade, linearidade, limite de deteção (LOD), limite de quantificação (LOQ),
precisão e ainda efeito de matriz (EM).
Relativamente à linearidade obtiveram-se valores do coeficiente de correlação (R2) superiores a 0,9809. Os resultados da percentagem de recuperação foram
satisfatórios variando entre 76-106%. O LOD variou de 0,98 mg/kg (putrescina) a 8,57
mg/kg (tiramina), enquanto o LOQ variou de 3,20 mg/kg (putrescina) a 25,64 mg/kg
(tiramina). Os resultados revelaram uma precisão elevada com valores de precisão
intradia, a variarem entre 5,24-11,64%, e de precisão interdia a oscilarem entre 8,21
14,20%. Relativamente ao EM, verifica-se que tem uma influência significativa no processo extrativo, de acordo com os valores obtidos (2% para a espermina; 50% para a
tiramina), para algumas das ABs em estudo.
A aplicação da metodologia validada a diferentes amostras de atum (posta de
atum fresca, bife de atum congelado, paté de atum, atum em conserva de azeite, atum
conservado em óleo e atum em conserva ao natural) revelou a ausência da maioria das
ABs em estudo e níveis de histamina abaixo do limite máximo de 500 mg/kg estipulado
pela Food and Drugs Administration (FDA).
The increasing number of cases that induce food poisoning due to the presence of biogenic amines (ABs) in food has given rise to a growing concern about food safety. In this context, the main objective of this work was the quantitative and qualitative determination of ABs (tryptamine, cadaverine, putrescine, spermine, histamine, tyramine and spermidine) present in different tuna samples through ultrasound-assisted microextraction (USAMET), followed by derivatization with dansyl chloride (DnsCl) and analysis by ultra-higth performance liquid chromatography with fluorescence detection (UHPLC-FLR) using a three-phase gradient (phosphoric acid (0.1%): methanol (100%):acetonitrile (100%)) and a flow rate of 200 μL/min. For the optimization of the extraction process, different experimental conditions were tested, namely ultrasonic agitation time (1, 5, 10 min) and the nature of the extracting solvent (acetonitrile and methanol). Similarly, different analytical parameters influencing the DnsCl derivatization process were optimized, including the concentration of DnsCl (1, 2, 3 g/L), derivatization time without stirring and ultrasonic agitation (10, 20 min) and derivatizing temperature (ambient temperature, 40, 60 °C). For the validation of the analytical method, the evaluation of parameters like selectivity, linearity, limit of detention (LOD), limit of quantification (LOQ), precision and matrix effect (ME) were taken into account. Regarding to the correlation coefficients (R2) the obtained values were above 0.9809, suggesting a good linearity between the analytes concentration and FLR signal. The results of the recovery were satisfactory ranging from 76-106%. The LOD ranged from 0.98 mg/kg (putrescine) to 8.57 mg/kg (tyramine), while LOQ ranged from 3.20 mg/kg (putrescine) to 25.64 mg/kg (tyramine). The results showed a good precision with intraday precision values varying between 5.24 to 11.64% and of interday precision between 8.21 to 14.20%. Regarding to matrix, a significant influence on the extractive process was shown, according to the values obtained for ME for some of the ABs under study (i.e. 2% for spermine, 50% for tyramine). The application of the validated methodology to different tuna samples (fresh tuna steak, frozen tuna steak, tuna pate, canned tuna of oil, preserved tuna in oil and canned tuna in the wild) revealed the absence of most ABs in study and Histamine levels below the maximum limit of 500 mg/kg stipulated by the Food and Drugs Administration (FDA).
The increasing number of cases that induce food poisoning due to the presence of biogenic amines (ABs) in food has given rise to a growing concern about food safety. In this context, the main objective of this work was the quantitative and qualitative determination of ABs (tryptamine, cadaverine, putrescine, spermine, histamine, tyramine and spermidine) present in different tuna samples through ultrasound-assisted microextraction (USAMET), followed by derivatization with dansyl chloride (DnsCl) and analysis by ultra-higth performance liquid chromatography with fluorescence detection (UHPLC-FLR) using a three-phase gradient (phosphoric acid (0.1%): methanol (100%):acetonitrile (100%)) and a flow rate of 200 μL/min. For the optimization of the extraction process, different experimental conditions were tested, namely ultrasonic agitation time (1, 5, 10 min) and the nature of the extracting solvent (acetonitrile and methanol). Similarly, different analytical parameters influencing the DnsCl derivatization process were optimized, including the concentration of DnsCl (1, 2, 3 g/L), derivatization time without stirring and ultrasonic agitation (10, 20 min) and derivatizing temperature (ambient temperature, 40, 60 °C). For the validation of the analytical method, the evaluation of parameters like selectivity, linearity, limit of detention (LOD), limit of quantification (LOQ), precision and matrix effect (ME) were taken into account. Regarding to the correlation coefficients (R2) the obtained values were above 0.9809, suggesting a good linearity between the analytes concentration and FLR signal. The results of the recovery were satisfactory ranging from 76-106%. The LOD ranged from 0.98 mg/kg (putrescine) to 8.57 mg/kg (tyramine), while LOQ ranged from 3.20 mg/kg (putrescine) to 25.64 mg/kg (tyramine). The results showed a good precision with intraday precision values varying between 5.24 to 11.64% and of interday precision between 8.21 to 14.20%. Regarding to matrix, a significant influence on the extractive process was shown, according to the values obtained for ME for some of the ABs under study (i.e. 2% for spermine, 50% for tyramine). The application of the validated methodology to different tuna samples (fresh tuna steak, frozen tuna steak, tuna pate, canned tuna of oil, preserved tuna in oil and canned tuna in the wild) revealed the absence of most ABs in study and Histamine levels below the maximum limit of 500 mg/kg stipulated by the Food and Drugs Administration (FDA).
Description
Keywords
Aminas biogénicas Thunnus OBesus USAMET Cloreto de Dansilo UHPLC-FLR Validação Biogenic amines Dansyl chloride Validation Bioquímica Aplicada . Faculdade de Ciências Exatas e da Engenharia