VALIDATION OF A SIMULTANEOUS QUANTIFICATION METHOD OF CAFFEINE AND CHLOROGENIC ACID CONTENTS IN GREEN COFFEE BEAN BY USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Abstract

The aim of study is to validate a quantitative method of caffeine (CAF) and chlorogenic acid (CGA) contents in green coffee bean by high performance liquid chromatography (HPLC). The analysis was performed on HPLC Waters e2695, Reliant C18 column (5 µm; 4.6x250 mm), using the mobile phase of formic acid 0.1% in water (A) and acetonitrile (B). The mobile phase gradients were operated at: initially 5% B, gradually increased to 10% B by 20 mins and remained constant until 30 mins. The sample injection volume was 10 µl. Flow rate was 1 ml/min. Measurement was taken at two wave lengths at 270 nm and 330 nm. The quantification process has been validated to meet the requirements of the ICH (International Conference on Harmonization). Results show that the method meets criteria such as specificity, linearity (R² > 0.99), high repeatability with RSD < 0.7% for peak area and RSD < 0.45% for retention time, limit of detection (≥ 0.06 µg/ml for CAF and 0.36 µg/ml for CGA), limit of quantification (≥ 0.19 µg/ml for CAF and 1.10 µg/ml for CGA). The accuracy of the method also meets requirements through the recovery rate ranging from 97.31 – 104.26%. Thus, the quantification method is suitable for simultaneous determination of caffeine and chlorogenic acid in green coffee beans.