Avaliação de radicais cátions derivados de aminas aromáticas como sondas espectrofotométricas para determinação da capacidade antioxidante em amostras de bebidas

Abstract

Many well-established methods for antioxidant capacity (CAO) determination are described in literature for a wide samples variety, and these only two methods DPPH• and ABTS•+ exploring radicals as spectrophotometric probes for analytical purpose. However, these methods have some limitations such as slower kinetic, solvent polarity effect, hydro and lipophilicity of compounds, chemical cost, among others. Thus, the aim of this study was the development an alternative spectrophotometric method for CAO determination in beverages exploring cation radicals derived from aromatic amines: DMPD, DEPD, EHPD and EHMPD. The chemical and physical parameters of the method were optimized for DEPD such as amine model and after these steps the optimal conditions was applied for other aromatic amines. The spectrophotometric method proposed was based on aromatic amines oxidation by Fe(III) ions (pH = 4.0) leading to corresponding purple color cation radical with max = 500 nm (DEPD•+) and 510 nm (DMPD•+, EHPD•+ and EHMPD•+). After the radical formation the addition of an antioxidant compound lead to color reduction intensity of solution, proportional to the antioxidant concentration in the medium. In the optimization method the allowing parameters was evaluated: time of the cation radical generation and reaction of different species, pH, reactants and buffer concentration, amine and Fe(III) ratio and organic solvents and counter ions effect. To reactivity radicals cation generated evaluated was employed the EC50 values (ability to inhibit 50% of their radical) of 17 compounds (phenolic, thiolic, glycosylated and reducing salt). The lower EC50 values were obtained for phenolic compound (caffeic acid, tannic acid and L-dopa) and higher for N-propyl gallate, rutin and N-acetylcysteine. Electrochemical studies using cyclic voltammetry and theoretical quantum calculations of amines evaluated were performed for a better processes understanding. For the aromatic amines cathode (Epc) and anodic (Epa) potential were obtained in five sweep speeds, and the voltammetric profile showed quasi-reversible redox process. The electrostatic potential map, HOMO and LUMO energies were calculated indicating the possible reactive sites to oxidation reactions. The main analytical figures were calculated using gallic acid (GA), tannic acid (TA), ascorbic acid (AA) and quercetin (QC) (for DEPD•+), for the other probes AG only was used as reference, except EHMPD•+, which was employed trolox (TR). In the method with DEPD amine AG presented the narrower (0.10 - 1.25 mg L-1) and ascorbic acid (0.50 - 7.5 mg L-1) the widest linear range, the values of limit of detection (LOD) varied from 0.0002 mg L-1 (AG) to 0.002 mg L-1 (AT), while the relative standard deviation (RSD) ranged from 0.9 to 9.2%. The spectrophotometric probes DMPD•+, EHPD•+, EHMPD•+ showed linear ranges variables being the widest to EHMPD•+ (0.50 - 5.0 mg L-1 at TR equivalent) and closer to the DMPD•+ (0.2 to 1.2 mg L-1 at GA equivalent). The values of RSD obtained were all less than 2.0% indicating good analytical precision. The methods developed were applying in wines, teas and infusions samples (14≤ N ≤ 30) and the data obtained compared with Folin-Ciocalteu (FC), Folin-Denis (FD), ABTS and DPPH methods. Linear correlation analysis at the level of 95% confidence was employed for results comparison and was observed good agreement and correlation coefficient r > 0.9000, and the correlations between EHMPD and ABTS (r = 0.8797) and EHMPD and FC (r = 0.8415) showed that the lower linear coefficients. Thus, the method developed exploring the radical cations of aromatic amines as spectroscopic probes proved simple, accurate and consistent with other assays for determination of total phenolic compounds and antioxidant capacity.