Quantifying Ascorbic Acid in Samples

Measurement of total AsA concentration was performed following the method as described previously, with minor modifications (Queval & Noctor, 2007 (link); Li et al., 2016 (link); Liu et al., 2021 (link)). Approximately 1.0 g of sample was harvested and immediately frozen in liquid nitrogen. The frozen samples were ground to a fine powder and extracted with 5 ml 0.1% metaphosphoric acid solution. After centrifuging at 8000 g for 10 min at 4°C, the supernatants and AsA standards (GWL8‐54KE, China) were neutralized with NaOH and the final pH of all samples was between 5 and 6. The neutralized supernatants were pre‐treated with 5 mM DL‐dithiothreitol (DTT) for 30 min at room temperature. Next, the supernatants were filtered through a 0.22‐μm filter and 10.0 μl of the supernatant was injected into Accela 1250 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) on a monomeric C18 column (Wondasil C18, Columns 5 µm, 4.6 × 150 mm; GL Sciences Inc., Shanghai, China) with a mobile phase of 0.1% metaphosphoric acid and acetonitrile (98 : 2, v/v). The flow rate of 0.5 ml min⁻¹ and injection volume of 10.0 μl. The standard curve was drawn from the measured values of the AsA standard sample. The standard curves for AsA were generated as references to quantify the AsA content in the samples.