HPLC Analysis of Organic Acids in Plants

Individual organic acids were determined by HPLC, based on procedures optimized by [33 ] and applied to different types of plant samples by [34 (link),35 (link),36 (link)]. Extraction was performed with 0.5 g of dried sample in 25 mL of 4.5% m-phosphoric acid and analyzed using an HPLC-UV methodology.
The HPLC equipment was a liquid chromatographer equipped with an isocratic pump (model PU-II, Micron Analítica, Madrid, Spain), an AS-1555 automatic injector (Jasco, Tokyo, Japan), a Sphereclone ODS (2) 250 × 4.60, 5 mm Phenomenex column (Torrance, CA, USA), a UV detector (Thermo Separation Specta Series UV100, San Jose, CA, USA) working at 215 nm. The mobile phase was 1.8 mmol/L H₂SO₄ (pH 2.6) at 0.4 mL/min flow rate. Data were analyzed using Chromonec XP software (Micronec, Madrid, Spain). Identification was performed comparing retention times with those obtained from commercial pure standards of oxalic, malic, citric, and succinic acids. Quantification was based on the UV signal response, and the resultant peak areas in the chromatograms were plotted against concentrations obtained from standards (Figure 1). Organic acids contents in S. patula samples were expressed as mg/100 g of fresh plant material.