Determination of homogentisic acid in bamboo shoots by column chromatography on sephadex G-10 and reversed-phase high-performance liquid chromatography

A method for the separation and quantification of homogentisic acid (HGA) in bamboo shoots has been developed by the use of Sephadex G-10 column chromatography and high-performance liquid chromatography (HPLC). For the first purification step, the HGA in bamboo shoots was extracted with methanol: water (70:30, v/ v) and then fractionated into 5ml-fractions from Sephadex G-10 column with 0.05 M phosphate buffer (pH=8.0). The final purification was performed on HPLC (strong cation resin column), eluted with 0.2% phosphoric acid (pH=2.0) at the flow rate of 0.7ml/min. The column temperature was maintained at 55°C and the column effluent was monitered by uv spectrometry at 210nm. The identification of HGA was performed on gas chromatography-mass spectroscopy. 1. From the result of the elution pattern of authentic HGA, it was found that HGA was eluted in fractions 16-26. 2. Partially purified extract of HGA from the Sephadex column was separated and quantificated on HPLC. The peak of HGA from HPLC was well separated with the retention time of 13min. 3. It was found that the overall recovery of HGA was 92.4% and also that HGA was fairly unstable above 100°C. 4. By the determination of HGA in the three sections of bamboo shoots by Sephadex G-10 column and HPLC, it was found that the HGA content was the highest in the top section and also that the content decreased from the top to the bottom section.