HPLC Analysis of Organic Acids

Organic acids, including oxalic acid, tartaric acid, malic acid, α-ketoglutaric acid, lactic acid, citric acid, fumaric acid and succinic acid, were measured by the HPLC method according to He et al. [21 (link)]. The system was carried out on a Agilent 1260 Infinity II HPLC equipped with a DAD detector (Agilent Technologies Inc., Santa Clara, CA, USA) and a Agilent Poroshell 120 EC-C18 column (4.6 × 150 mm, 4 µm). Two eluents, filtered through a 0.45 µm durapore membrane, were used as mobile phases: KH₂PO₄ (0.5%, w/v, pH 2.3, eluent A), methanol (eluent B). The elution condition was isostatic elution with A:B of 97:3 (v/v). The flow rate for mobile phase was 0.7 mL/min, and the injection volume was 10 μL. The column oven temperature was maintained at 35 °C. Wavelength for the detection was 210 nm. Organic acid contents in samples were calculated by regression equation and expressed as g/100 g DW.