Identification of four new degradation products of epirubicin through forced degradation, LC-UV, MSn and LC-MS-TOF studies.

Abstract

Epirubicin (EPI) was subjected to International Conference on Harmonization recommended forced degradation under the conditions of hydrolysis, oxidation, dry heat and photolysis to characterize its possible impurities and/or degradation products. The drug was found highly unstable to alkaline hydrolysis even at room temperature, unstable to acid hydrolysis at 80°C and to oxidation at room temperature. The hydrolytic and oxidative degradation products were resolved on an Agilent RP8 (150 mm × 4.6 mm; 5 µm) column with isocratic elution using mobile phase composed of ammonium formate (10 mM, pH 3.0), acetonitrile and methanol. The drug degraded to four oxidative products (O-I, O-II, O-III and O-IV) and to one acid hydrolyzed product (A-I). Purity of each peak in liquid chromatography-ultraviolet (LC-UV) chromatogram was ascertained through photodiode array (LC-PDA) analysis. The products were characterized through electrospray ionization-mass spectrometry (+ESI-MS(n)) studies on EPI and liquid chromatography-time of flight mass spectrometry (LC-MS-TOF) studies on degraded drug solutions. The products, O-I-O-IV, were characterized as 2-hydroxy-8-desacetylepirubicin-8-hydroperoxide, 4-hydroxy-8-desacetylepirubicin-8-hydroperoxide, 8-desacetylepirubicin-8-hydroperoxide and 8-desacetylepirubicin, respectively, and product A-I was characterized as deglucosaminylepirubicin. While A-I was found to be a pharmacopoeial impurity, all oxidative products were found to be new degradation impurities. The mechanisms and pathways of degradation of EPI were discussed and outlined.