Development of Novel Reverse Phase High Performance Liquid Chromatography Method for Simultaneous Estimation of Gallic Acid, Protocatechuic Acid, Vanillic Acid and Syringic Acid in Sugarcane Roots

A novel, simple, accurate and precise isocratic high performance liquid chromatographic method was developed for the estimation of four phenolic acids, namely gallic acid, protocatechuic acid, vanillic acid and syringic acid in the sugarcane roots. The selected phenolic acids were analyzed using shim-pack high performance liquid chromatography C18 column (250×4.6 mm, 5 μm) by isocratic elution. The mobile phase used was methanol:water containing 0.2 % ortho-phosphoric acid in the ratio of 20:80 at 28°. The flow rate was 1 ml/min and detection wavelength was set to 220 nm. The run time was kept 30 min. The retention time of gallic acid, protocatechuic acid, vanillic acid and syringic acid was found to be 4.864, 8.741, 20.298 and 25.268 min, respectively. Linearity was established for all the selected phenolic acids in the range of 0.25-20 μg/ml. The percentage recoveries of gallic acid, protocatechuic acid, vanillic acid and syringic acid were found to be in the range of 98-102 %. The method was found to be robust. Therefore, the method was successfully applied for the determination of the above mentioned phenolic acids in the sugarcane rootss. Key words: Sugarcane roots.