Rapid determination of eight organic acids in plant tissue by sequential extraction and high performance liquid chromatography

Abstract

A sequential extraction method was developed to determine different forms of oxalate and seven oxalate-metabolism-related organic acids (glyoxylic acid, tartaric acid, gly-colic acid, malic acid, acetic acid, citric acid, succinic acid) in plant tissue. The ultra-pure water was used as the extraction medium to obtain water-soluble oxalic acid and the other seven water-soluble organic acids. After the extraction of the water-soluble organic acids, the resi-dues were extracted by dilute hydrochloric acid successively to get the acid-soluble oxalate which entered the liquid phase. A Hypersil ODS column was used with 5 mmol / L potassium dihydrogen phosphate buffer solution (pH 2.8) as the mobile phase. The diode array detector was set at 210 nm and the column temperature at 30 °C with the injection volume of 5 μL. The flow rate was controlled at different times which allowed a good and rapid separation of the organic acids and hydrochloric acid. Under these conditions, the linear ranges of the method were 1-2 000 mg / L for oxalic acid, 25-2 000 mg / L for acetic acid, and 10-2 000 mg / L for glyoxylic acid, tartaric acid, glycolic acid, malic acid, citric acid and succinic acid, with the correlation coefficients of the eight organic acids ≥ 0.999 6. The average recoveries of the eight organic acids in leaves and roots were 93.5% -104.4% and 85.3% -105.4% with RSDs of 0.15% -2.43% and 0.31% -2.9% (n =7), respectively. The limits of detection ranged from 1 to 10 ng (S / N = 3). The results indicated that the method is accurate, rapid and reproducible for the determination of organic acids in plant samples.