Chromatographic Quantification of DPPH Radical Scavenging Activity by HPLC–DAD: Application to the Colored Berberis libanotica Extract

Evaluation of the antioxidant activity in plant extracts is often hindered by spectral interference in conventional colorimetric assays; particularly, direct measurements at ۵۱۷ nm are prone to spectral interference in colored matrices. This study describes a methodological innovation through a pigment-tolerant, rapid (۱.۶ min) readout by quantifying the ۱,۱-diphenyl-۲-picrylhydrazyl (DPPH) consumption via high-performance liquid chromatography with diode-array detection (HPLC-DAD). A standard HPLC system with a C۸ column and isocratic MeOH (۱ mL/min) monitored DPPH at ۵۱۷ nm. Main analytical performance parameters demonstrated high robustness, including linearity (۵–۵۰۰ µM) with R۲≈۰.۹۹۹. The method showed excellent precision with a repeatability %RSD of ≈۰.۳۵ and inter-day precision of ≈۱.۸۶. On the other hand, the limit of detection was found to be ۱۰.۴۱ µM, and the limit of quantification was ۳۱.۵۳ µM. To demonstrate the significance of the method, HPLC readouts were compared to traditional colorimetry, showing tight agreement across ascorbic acid (AA) and sodium metabisulfite (SMB) dose ranges. Determined IC۵۰ values ranked AA ≪ B. libanotica extract ≪ SMB; with AA was ~۶۰ times more potent than SMB. Accordingly, the method minimizes spectral interference by quantifying the chromatographically resolved DPPH peak. It is suitable for colored matrices where direct absorbance is problematic, delivering a fast, precise, and high-throughput antioxidant assessment on standard hardware.

کلیدواژه ها:

Antioxidant ، Berberis libanotica ، DPPH ، HPLC-DAD ، hydroalcoholic extract ، Radical Scavenging Activity