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Epirubicin (EPI), daunorubicin (DNR) and mitoxantrone (MXT), are mainly used in the treatment of acute or chronic leukemia, malignant lymphoma and other malignant tumor, belong to a group of important anthracycline anticancer antibiotics. Their effect is exerted by their inhibition of DNA and RNA synthesis, when they intercalate into the double helix of DNA and combine with the base pairs. Among them, NNR is the first generation anthracycline antibiotic, and its main defect lies on serious cardiac toxicity and bone marrow toxicity. EPI and MXT are the second generation anthracycline antibiotics, which are used more widely due to their higher efficacy and lower side effect. Especially, MXT is one of important drugs to cure breast cancer because of its outstanding activity and much lower cardiac toxicity. Therefore, it is important to determine quantificationally anthracycline antibiotics for both drug analysis and clinical assay.