2.11. Determination of Malic Acid and Pyruvic Acid Content through High-Performance Liquid Chromatography (HPLC)

A total of 1.0 g leaf tissue was extracted with 80% methanol and filtered through a nylon membrane filter (0.22 µm), Thermofisher Scientific, Waltham, USA, and subjected to HPLC analysis (Agilent 1260 equipped with a diode array detector (DAD), Agilent Technologies, Santa Clara, USA). A Luna C18 column (4.6 × 250 mm) (Phenomenex, Torrance, USA) (set at 25 °C) coupled with reversed-phase (60% acetonitrile with 0.1% acetic acid as solvent) was used with a retention time of 7–8 (malic acid) min and 3–3.5 (pyruvic acid) min, with a constant flow rate of 1.0 mL min⁻¹, sample injection volume of 20 µL, and was detected at 210 nm by the DAD. The malic acid and pyruvic acid contents were analyzed and calculated from the chromatogram, with standards of malic acid and pyruvic acid (sodium pyruvate) from the recorded peak area through OpenLab Chem Station software (Agilent Technologies, Santa Clara, USA) [21].