Analytical procedures based on digital images and liquid-liquid microextraction for the determination of flavonols in fruits

Author(s)

Luis Claudio Martins

• Document type: Master's Dissertation
• Press: Piracicaba.
• Institution: Universidade de São Paulo (USP). Centro de Energia Nuclear na Agricultura (CENA/STB)
• Defense date: 2023-06-16

Examining board members

Fabio Rodrigo Piovezani Rocha; João Flávio da Silveira Petruci; Vagner Bezerra dos Santos

• Advisor: Fabio Rodrigo Piovezani Rocha

Abstract

Flavonoids are secondary plant metabolites, responsible for their defense and signaling under stress conditions. The flavonols are one of the most representative classes, due to their antioxidant properties and health benefits, which are widely found in fruits and vegetables. Berries are source of bioactive compounds, including anthocyanins, flavonols, and phenolic acids. Analytical procedures for quantification of these species are generally based on UV-vis absorption spectrophotometry after formation of Al(III) complexes (total flavonoids) or individual quantification by liquid chromatography coupled with either spectrophotometric or mass spectrometric detectors. The spectrophotometric procedures lack selectivity, whereas the chromatographic ones demand high-cost equipment and consume high amounts of organic solvents. Aiming at more accessible, practical, selective, and greener alternatives, this Dissertation deals with the development of two analytical methods for the determination of total flavonols in berries (blackberry, raspberry, blueberry, and strawberry) based on salt-assisted liquid-liquid microextraction (SALLME) and digital-image photometry. The first method was based on flavonol extraction with acetonitrile for the separation of more polar species (e.g. anthocyanins), followed by SALLME of the Al(III) complexes to the aqueous phase, thus separating the analytes from less polar species. Method II exploited extraction with ethanol and subsequent SALLME with ethyl acetate for separation of the flavonols from anthocyanins. In both procedures, a smartphone camera and free software/applications were used for the acquisition and conversion of digital images into the RGB color system. Measurements were based on the intensities of the reflected radiation taken as channel B values, which corresponds to the complementary color of the measurement product. For method I, the standard addition calibration method was applied to circumvent the matrix effect, a linear responses were obtained within 1.7-10.0 mg L1 quercetin equivalent (QE). Coefficient of variation (CV, n = 10) and detection limit (LOD, 95% confidence level) were estimated at 3.1% and 0.5 mg L1 QE, respectively. The procedure requires as low as 105 g Al(III), 14.6 mg NaCl, 1,2 mL acetonitrile and generates ca. 1.5 mL residue per determination. Method II yielded quercetin recoveries (1.0 to 2.0 mg L1) ranging from 90±3% to 125±2%, linear response from 0.3 to 5.0 mg L1 QE, CV (n = 10) of 3.9% and LOD of 0.1 mg L1 QE. In this procedure, 70 g Al(III), 142 mg Na2SO4, ca. 9.2 L of HCl, and 2 mL of ethyl acetate were consumed, generating ca. 15 mL of waste per determination, composed of water and ethanol. The results obtained by the proposed approaches agreed with the spectrophotometric reference procedure at the 95% confidence level. Both approaches are efficient, cost-effective, and environmental friendly alternatives for the determination of flavonols in berries. 1 MARTINS, L. C.; SOARES, S.; ROCHA, F. R. P. Digital-image photometry and salting-out assisted liquid-liquid microextraction for determination of flavonols in berries. J. Food Compost. Anal., v. 123, p. 105515, 2023. 2 MARTINS, L. C.; SOARES, S.; ROCHA, F. R. P. Digital-image eco-friendly spot test with liquid-liquid microextraction for selective determination of flavonols in berries. Microchem. J., v. 193, p. 109207, 2023 (AU)

• FAPESP's process: 21/02309-5 - Analytical procedure based on digital images and liquid-liquid microextraction for the determination of flavonoids in fruits
• Grantee: Luís Claudio Martins
• Support Opportunities: Scholarships in Brazil - Master