Hydro 80 a rp column

The analysis of l-ascorbic acid (l-ASC) and dehydroascorbic acid (DHA) was performed using High Performance Liquid Chromatography (HPLC) method. As a solvent, 2 mL of 5% meta-phosphoric acid were added to the sample. Then, sample was mixed by vortex mixer (5 seconds), incubated in an ultrasonic bath (15 min, 20 °C) and centrifuged (6000 rpm, 0 °C). The supernatant (1mL) was transferred to dark HPLC vials. The Phenomenex Hydro 80-A RP column (250 × 4.6 mm), the mobile phase of 50 mM phosphate buffer (pH 4.4), and 0.1 mM sodium acetate were used for analysis. Time of the analysis was 18 min, and the detection wavelength was 255–260 nm. The compounds were identified based on the retention time of the external Sigma-Aldrich l-ASC and DHA standards. The applied extraction and analytical method was based on the study of Nojovan et al. [47 (link)], optimised based on Van de Velde et al. [48 (link)], with our own modifications [49 (link)].

The content of vitamin C in the apples was conducted by the HPLC method (high-performance liquid chromatography) (HPLC), as previously described by Kazimierczak et al. [22 (link)], using a Shimadzu HPLC-set (Shim-pol, Warsaw, Poland) with two LC-20AD pumps, a CMB-20A set controller, a SIL-20AC autosampler and an SPD-20AV visible light detector with spectrum identification. The 100 mg freeze-dried powder was extracted with 5% metaphosphoric acid. The samples were mixed by a vortex mixer, incubated in an ultrasonic bath (15 min, 20 °C) abd and centrifuged (6000 rpm, 10 min, 0 °C). An amount of 100 μL of supernatant was injected onto a Phenomenex Hydro 80-A RP column (250 × 4.6 mm). The analysis parameters were as follows: analysis time of 18 min, mobile phase of 50 mM phosphate buffer (pH 4.4) and 0.1 mM sodium acetate and detection wavelength of 255–260 nm. L-ascorbic acid (L-Asc) and dehydroascorbic acid (DHA) were identified based on Fluka and Sigma–Aldrich (Warsaw, Poland) standards with HPLC purity (99%).