Validação de método espectrofotométrico UV-VIS e espectrofluorimétrico para determinação de corante vermelho de origem biotecnológica associado a nanocarreadores
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Data
2019-02-15
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Universidade Estadual Paulista (Unesp)
Resumo
O controle da qualidade dos resultados de análises químicas tem sido cada vez mais exigido devido ao grande prejuízo que dados analíticos não confiáveis podem gerar, principalmente quando se diz respeito à segurança do produto, e às consequências financeiras irreversíveis que pode causar. Para garantir a confiabilidade dos resultados obtidos durante as etapas analíticas é necessário que o método empregado seja validado. Neste contexto, o presente trabalho visa desenvolver e validar um método espectroanalítico para determinação de corantes vermelho e derivados livre e nanoencapsulado, por espectroscopia de absorção e emissão de fluorescência no UV-Vis, como métodos alternativos aos cromatográficos tradicionais. A partir da espectroscopia de absorção e emissão de fluorescência no UV-visível utilizando-se o espectrofotômetro modelo Genesys 10s da Thermo Scientific e espectrofluorímetro Shimadzu RF6000 desenvolveu-se um protocolo experimental para determinação da curva de correlação entre máximo de emissão de absorção e emissão de fluorescência em função de diferentes concentrações de quinizarina. O método foi determinado com número de repetição (n) igual a 3. Os parâmetros de aquisição dos espectros de fluorescência foram fixados com um comprimento de onda de excitação igual a 480 nm, fendas de excitação e emissão igual a 10/10 nm, respectivamente, com emissão na faixa de 520 a 680 nm. As amostras foram preparadas com auxílio de uma micropipeta (10,0 uL Eppendorf) a partir de diluição infinita em acetonitrila (2,0mL), diretamente em cubeta de quartzo de 10 mm de caminho óptico, partindo-se de uma solução inicial da quinizarina 1,00 mg/mL em dimetil-sulfóxido. Os métodos apresentaram linearidade na faixa de 1,00 a 12,00 µg/mL (y = 0,0433[quinizarina, µg.mL-1] + 0,0019, r2 = 0,9999 para espectroscopia de absorção, e de 5,00 a 12,00 µg/mL (y = 31152[quinizarina, µg.mL-1] + 91994, r2 = 0,9964 para espectrofluorimetria. O teste ANOVA demonstrou que as equações da reta são estatisticamente significantes (p < 0,05), e o modelo linear é adequado para o método analítico. O valores de limites de detecção (LD) foram de 0,15 e 1,35 µg/mL e o limite de quantificação (LQ) de 0,47 e 4,10 µg/mL para absorção e fluorescência, respectivamente. Assim, foi possível estabelecer uma relação linear que permitiu a determinação da inclinação e equação da reta a partir dos pontos experimentais. Os métodos apresentaram desvio padrão relativo (DPR ) menor que 5% para os parâmetros de precisão, recuperação dentro da faixa de 95 a 105% e nenhuma interferência dos compostos da matriz, logo, foram considerados precisos, exatos e seletivos. Através do estudo de validação, realizado conforme preconizado pela RDC 166/2017, foi possível comprovar a adequabilidade dos métodos espectro-analíticos para quantificação de quinizarina e corantes vermelhos derivados.
Quality control of chemical analysis results has been increasingly required because of the great harm that unreliable analytical data can generate, especially when it comes to product safety, and the irreversible financial consequences it can cause. To guarantee the reliability of the results obtained during the analytical steps it is necessary that the method employed be validated. In this context, the present work aims to develop and validate a spectroanalytical method for the determination of red dyes and free and nanoencapsulated derivatives, by absorption spectroscopy and UV-Vis fluorescence emission, as alternative methods to traditional chromatographic methods. UV-visible fluorescence emission and absorption spectroscopy using the Genesys 10s spectrophotometer from Thermo Scientific and Shimadzu RF6000 spectrofluorimeter was developed an experimental protocol for determination of the correlation curve between maximum emission of absorption and emission of fluorescence as a function of different concentrations of quinizarin. The method was determined with repeat number (n) equal to 3. The acquisition parameters of the fluorescence spectra were set with an excitation wavelength equal to 480 nm, excitation and emission slots equal to 10/10 nm, respectively, with emission in the range of 520 to 680 nm. The samples were prepared using a micropipette (10.0 μL Eppendorf) from infinite dilution in acetonitrile (2.0 mL), directly into a 10 mm optical path quartz cuvette, starting with an initial solution of quinizarin 1.00 mg / mL in dimethylsulfoxide. The methods showed linearity in the range of 1.00 to 12.00 μg / mL (y = 0.0433 [quinizarin, μg.mL-1] + 0.0019, r2 = 0.9999 for absorption spectroscopy, and 5, 00 to 12.00 μg / mL (y = 31152 [quinizarine, μg.mL-1] + 91994, r2 = 0.9964 for spectrofluorimetry. The ANOVA test showed that the equations of the line are statistically significant (p <0.05), and the linear model is suitable for the analytical method. The limits of detection (LD) were 0.15 and 1.35 μg / mL and the limit of quantification (LQ) of 0.47 and 4.10 μg / mL for absorption and fluorescence, respectively. Thus, it was possible to establish a linear relationship that allowed the determination of slope and equation of the line from the experimental points. The methods showed a relative standard deviation (DPR) of less than 5% for precision parameters, recovery within the range of 95 to 105% and no interference of matrix compounds were therefore considered accurate, accurate and selective. Through the validation study, performed according to RDC 166/2017, it was possible to prove the suitability of the spectroanalytical methods for the quantification of quinizarine and derived red dyes.
Quality control of chemical analysis results has been increasingly required because of the great harm that unreliable analytical data can generate, especially when it comes to product safety, and the irreversible financial consequences it can cause. To guarantee the reliability of the results obtained during the analytical steps it is necessary that the method employed be validated. In this context, the present work aims to develop and validate a spectroanalytical method for the determination of red dyes and free and nanoencapsulated derivatives, by absorption spectroscopy and UV-Vis fluorescence emission, as alternative methods to traditional chromatographic methods. UV-visible fluorescence emission and absorption spectroscopy using the Genesys 10s spectrophotometer from Thermo Scientific and Shimadzu RF6000 spectrofluorimeter was developed an experimental protocol for determination of the correlation curve between maximum emission of absorption and emission of fluorescence as a function of different concentrations of quinizarin. The method was determined with repeat number (n) equal to 3. The acquisition parameters of the fluorescence spectra were set with an excitation wavelength equal to 480 nm, excitation and emission slots equal to 10/10 nm, respectively, with emission in the range of 520 to 680 nm. The samples were prepared using a micropipette (10.0 μL Eppendorf) from infinite dilution in acetonitrile (2.0 mL), directly into a 10 mm optical path quartz cuvette, starting with an initial solution of quinizarin 1.00 mg / mL in dimethylsulfoxide. The methods showed linearity in the range of 1.00 to 12.00 μg / mL (y = 0.0433 [quinizarin, μg.mL-1] + 0.0019, r2 = 0.9999 for absorption spectroscopy, and 5, 00 to 12.00 μg / mL (y = 31152 [quinizarine, μg.mL-1] + 91994, r2 = 0.9964 for spectrofluorimetry. The ANOVA test showed that the equations of the line are statistically significant (p <0.05), and the linear model is suitable for the analytical method. The limits of detection (LD) were 0.15 and 1.35 μg / mL and the limit of quantification (LQ) of 0.47 and 4.10 μg / mL for absorption and fluorescence, respectively. Thus, it was possible to establish a linear relationship that allowed the determination of slope and equation of the line from the experimental points. The methods showed a relative standard deviation (DPR) of less than 5% for precision parameters, recovery within the range of 95 to 105% and no interference of matrix compounds were therefore considered accurate, accurate and selective. Through the validation study, performed according to RDC 166/2017, it was possible to prove the suitability of the spectroanalytical methods for the quantification of quinizarine and derived red dyes.
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Palavras-chave
Corantes, Validação de Métodos, Métodos Espectroanalíticos, Dyes, Validation of Methods, Spectroanalytical Methods