Abstract: A high performance liquid chromatographic method was established for the quantitative determination of free ellagic acid content in guava leaves. Hot ethanol reflux (containing 0.01% HCl) extraction was applied for the extraction of samples. The analyte was separated on an inertsil ODS-SP column (250 mm × 4.5 mm, 5μm) held at 35 ℃ using a mobile phase composed of 3% glacial acetic acid (phase A) and net methanol (phase B) with the following gradient elution program: 0－4 min, 20%－45% B; 4－10 min, 45% B; 10－15 min, 45%－70% B; 15－20 min 70%－100% B; 20－21 min 100% B. The injection volume used was 10μl. The detection wavelength was set as 254 nm. The relative standard deviation of retention time for 5 replicate injections was 0.6%. Free ellagic acid content displayed an excellent linear relationship with pear area over the range of 0.5 to 200 mg/L, with a correlation coefficient of more than 0.9998. The spike recoveries for free ellagic acid were between 95.86% and 98.31 %. This method was used to determine free ellagic acid content in the real samples of young, mature and aged guava leaves. The results proved its good feasibility and showed that young, mature and aged guava leaves contained 0.814, 0.023 mg/g and 0 mg/g free ellagic acid, respectively, suggesting that free ellagic acid content in guava leaves is related to their maturity.

Key words: high performance liquid chromatography (HPLC), guava leaf, ellagic acid