Mangiferin is a pharmacologically active and a natural xanthone C-glycoside,which widely distributed in the bark,fruits,roots and leaves of Mangifera indica L.and many higher plants.Mangiferin has been reported to possess important pharmacological activity,including antioxidant,antitumor,antiviral,immunomodulatory,and antidiabetic activities.As the active pharmaceutical ingredients,mangiferin product extracted from M.indica L.leaves can be used to prepare medicines,such as antivirus and anti-inflammation medicament.In order to ensure the quality and safety of pharmaceuticals,it becomes important to determine the contents of mangiferin and related substance in the mangiferin products.In this paper,an unknown impurity in the prepared mangiferin extracted from Mangifera indica L.leaves was isolated and determined by a newly developed reverse phase high performance liquid chromatographic (HPLC) method.The unknown impurity was identified by LC/MS/MS and its structure was confirmed by spectrometric studies of IR and NMR.Based on the spectral data,the molecular structure of the unknown impurity was characterized as 2-C-β-D-glucopyranosyl-1,6,7-trihydroxy-3-methoxy-xanthen-9-one.The fragmentation mechanisms of the impurity and mangiferin were proposed.From the comparison of the HPLC chromatogram between the prepared mangiferin product and the crude methanol extract of Mangifera indica L.leaves,it can be seen that the unknown impurity is natural occurring compound.The validated HPLC method was used to determine the amounts of the impurity in nine batches of prepared mangiferin products.The results shown the contents of the irnpurity in nnine batches of mangiferin sample (mangiferin content >90%) were range from 13.3mg·g1~75.9 mg·g-1.