ECO-FRIENDLY SPOT TEST WITH LIQUID-LIQUID MICROEXTRACTION FOR SELECTIVE DETERMINATION OF FLAVONOIDS IN BERRIES

Berries are sources of a wide variety of nutrients and bioactive compounds, such as flavonoids (e.g. anthocyanins and flavonols). These substances are the main functional compounds found in berries and are often used as food colorants and antioxidant agents. The quantification of these compounds can be carried out through different analytical techniques, but the most widely applied procedure for screening of bioactive compounds is UV-vis spectrophotometry based on complex formation with aluminum. This method is not specific and results may be affected by e.g. anthocyanins and phenolic acids (chlorogenic acid). Moreover, it consumes high reagent
amounts (10 mg of AlCl3 per determination) prepared in methanol and require a long reaction time (30 to 60 min). Aiming to overcome these hindrances, this work proposed a spot test exploring smartphone-based digital-image photometry for the determination of flavonoids in berries. It was associated with salt-assisted liquid-liquid microextraction (SALLE) and adjustment of acidity of the complexant solution to avoid interferences from anthocyanins and chlorogenic acid, respectively. Flavonoids were extracted from fresh fruit samples using ethanol (1:10 m/v) under sonication and 1 mL of the extract was submitted to SALLE in 15 mL Falcon tubes, after addition of 300 µL of 1.0 mol L-1 HCl, 500 µL of 2.0 mol L-1 of Na2SO4, 2 mL of ethyl acetate, and 10 mL of water. Then,
500 µL of the supernatant organic phase was dried on a white porcelain plate (5 min, 35 °C), followed by the addition of 100 µL ethanol and 200 µL of 13 mmol L-1 AlCl3 (in buffer solution pH 3.5) for complex formation. Digital-images were acquired with a smartphone camera (Xiaomi Mi A3, 48 MP) under controlled illumination and RGB values were acquired using ImageJ® software. The analytical responses were based on the intensity of reflected radiation, taken at channel B, due to the complementarity with the color of the Al-flavonoid complexes. Inaccurate results (flavonoid amounts up to 7-fold higher than the expected) were achieved when the reference procedure was applied to berry samples, indicating the need for sample pretreatment to avoid interferences from anthocyanins. Under the optimized conditions, a linear response was obtained up to
8.0 mg L-1 quercetin (r > 0.992), with limits of detection and quantification of 0.2 and
0.5 mg L-1, and a coefficient of variation of 3.9% (n=10). Suitable recoveries were achieved for strawberry (102±2 and 86±3), raspberry (106±2 and 98.6±0.7), blackberry (86±3 and 110±4), and blueberry (98±3 and 88±2) after spiking with 5.0 or 7.0 mg L-1 quercetin, respectively. These results indicate that matrix effects and interferences from anthocyanins were avoided. The accuracy was assessed in relation to the spectrophotometric reference procedure, with results in agreement at the 95% confidence level for samples pretreated as proposed herein. These analytical features demonstrate that the proposed procedure is a simple, selective, and reliable approach for determination of flavonoid content in berries, with low reagent consumption (e.g. 350 µg AlCl3 per determination), a 12-fold lower solvent consumption, and low waste generation.