Abstract: A novel HPLC method was established，which was carried out on a Lichrospher C₁₈ column with a mobile phase consisting of acetonitrile - 0.5% formic acid aqueous solution (5∶95) and a detection wavelength of 280 nm.Two major impurities in bulk material of sodium levofolinate were prepared by this method.Their exact molecular weights were acquired by LC-Q-TOF.Their full scan mass spectra and product ion scan mass spectra were obtained by LC-MS/MS.The fragment profiles of two target impurities were explained and their chemical structures were elucidated.Impurity 1 was identified as 7-hydroxy-8-hydrofolic acid in sodium levofolinate bulk material for the first time.Impurity 2 was identified as 10-formylfolic acid.This method is effective in identifying two major impurities in bulk material of sodium levofolinate detected by the current HPLC method,and the results are useful for its quality controll.