Application of High-Performance Liquid Chromatography with Diode Array Detection to Simultaneous Analysis of Reference Antioxidants and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) in Free Radical Scavenging Test

Antioxidant activity can be analyzed by various methods, both in vitro and in vivo. The widely used colorimetric method using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging reaction has many limitations, such as interference from photosynthetic pigments naturally found in plant extracts. The DPPH-HPLC eliminates these troubles by enabling the separation of the DPPH free radical (DPPH-R) peak and its reduced form (DPPH-H) from other extract components. However, simultaneous analysis of antioxidants and evaluation of their activity is more complicated. To date, a post-column reaction with DPPH has been used for this purpose. The aim of the current study was the elaboration on a DPPH-RP-HPLC in gradient elution mode for simultaneous evaluation of the antioxidant activity of standards on the basis of DPPH-R peak inhibition, together with the identification of standards, as well as the products of redox reactions. The following antioxidants (AOs) were used as standards: quercetin, resveratrol, Trolox, chlorogenic acid, hesperetin, and coumarin. Flavone was used as the control chemical without antioxidant activity. The separation of the DPPH-R/DPPH-H pair, together with standards and reaction products, was studied on a C18 column using a gradient of acetonitrile from 5 to 60% within 20 min. The stability of DPPH was evaluated with different gradient profiles. The influence of the addition of acetic acid in concentrations of 0.05 to 1%, the duration of the analysis, and the radiation emitted by the UV lamp of a diode array detector on the induction of DPPH decomposition processes were investigated. The most significant parameter affecting DPPH stability appeared to be the acidic environment and water content in the mobile phase. An increase in the water content from 70 to 95% worsened the LOD of DPPH-R from 31.64 nM to 107.31 nM, as measured at 517 nm, and from 189.41 to 1677.05 nM at 330 nm. Each gradient profile provided good linearity (R 2 = 0.9790–0.9977) of the relationship between the DPPH-R as well as DPPH-H peak areas, and a wide concentration range from 0.5 to 2.5 mM for UV-vis detection. Free radical scavenging activity was expressed by the percentage of DPPH-R peak inhibition at 517 nm. This simple method is suitable for monitoring DPPH radical scavenging by AO standards.