An off-line DPPH-HPLC-PAD method [26 (link)] was applied to screen the main antioxidants from GP. The sample was mixed with 10 mmol/L DPPH at the ratio of 1:1 (v:v) and incubated at room temperature for 30 min. Then the mixture was filtered through a 0.45 μm filter, and injected into the HPLC column. The control sample was prepared by adding methanol instead of DPPH to the sample.
The DPPH-reacted sample and control sample were analyzed using a LC-1200 high performance liquid chromatograph (HPLC) (Agilent, Palo Alto, CA, USA) equipped with an Agilent ZORBAX Eclipse SB-C18 column (250 mm×4.6 mm i.d., 5µm) and a photodiode array detector (PAD). The mobile phase consisted of 0.2% (v/v) acetic acid (A) and acetonitrile (B) using a gradient program of 12–21% B within 0–30 min, 21–32% B within 30–40 min, 32–55% B within 40–50 min, 55–65% B within 50–80 min and 65% B within 80–85 min. The flow rate was 1.0 mL/min, the column temperature was 30°C, the injection volume was 10 μL and the detection wavelength was 265 nm. The main antioxidants could be screened by comparing the chromatographic profiles of DPPH-reacted sample and control sample.
The DPPH-reacted sample and control sample were analyzed using a LC-1200 high performance liquid chromatograph (HPLC) (Agilent, Palo Alto, CA, USA) equipped with an Agilent ZORBAX Eclipse SB-C18 column (250 mm×4.6 mm i.d., 5µm) and a photodiode array detector (PAD). The mobile phase consisted of 0.2% (v/v) acetic acid (A) and acetonitrile (B) using a gradient program of 12–21% B within 0–30 min, 21–32% B within 30–40 min, 32–55% B within 40–50 min, 55–65% B within 50–80 min and 65% B within 80–85 min. The flow rate was 1.0 mL/min, the column temperature was 30°C, the injection volume was 10 μL and the detection wavelength was 265 nm. The main antioxidants could be screened by comparing the chromatographic profiles of DPPH-reacted sample and control sample.