title: Development and validation of a novel stability-indicating HPLC method for the quantitative determination of eleven related substances in ezetimibe drug substance and drug product.
abstract: Ezetimibe is a novel lipid-lowering agent that inhibits intestinal absorption of dietary and biliary cholesterol. In the present work, a simple, sensitive and reproducible gradient reverse phase high performance liquid chromatographic (RP-HPLC) method for separation and determination of the related substances of ezetimibe was developed and validated. Eleven potential process-related impurities (starting materials, (3S,4S,3'S)-isomer, degradants and byproducts) were identified in the crude samples. Tentative structures for all the impurities were assigned primarily based on comparison of their retention time and mass spectrometric data with that of available standards and references. This method can be applied to routine analysis in quality control of both bulk drugs and commercial tablets. Separation of all these compounds was performed on a Phenomenex Luna Phenyl-Hexyl (100mm×4.6mm, 5μm) analytical column. The mobile phase-A consists of acetonitrile-water (pH adjusted to 4.0 with phosphoric acid)-methanol at 15:75:10 (v/v/v), and mobile phase-B contains acetonitrile. The eluted compounds were monitored at 210nm. Ezetimibe was subjected to hydrolytic, acid, base, oxidative, photolytic and thermal stress conditions as per ICH serves to generate degradation products that can be used as a worst case to assess the analytical method performance. The drug showed extensive degradation in thermal, acid, oxidative, base and hydrolytic stress conditions, while it was stable to photolytic degradation conditions. The main degradation product formed under thermal, acid, oxidative, base and hydrolytic stress conditions corresponding to (2R,3R,6S)-N, 6-bis(4-fluorophenyl)-2-(4-hydroxyphenyl)-oxane-3-carboxamide (Ezetimibe tetrahydropyran impurity) was characterized by LC-MS/MS analysis. The degradation products were well resolved from the main peak and its impurities, thus proved the stability-indicating power of the method. The developed method was validated as per international conference on harmonization (ICH) guidelines with respect to specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision and robustness.
authors.affiliation: School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, PR China.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Luo
authors.affiliation: DDLD (Beijing) Pharmaceutical Technology Co., Ltd., Beijing, PR China.
authors.forename: Zhongqing
authors.initials: Z
authors.lastname: Deng
authors.affiliation: School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, PR China. Electronic address: liuyang@bucm.edu.cn.
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
authors.affiliation: School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, PR China.
authors.forename: Guopeng
authors.initials: G
authors.lastname: Wang
authors.affiliation: School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, PR China.
authors.forename: Wenning
authors.initials: W
authors.lastname: Yang
authors.affiliation: School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, PR China.
authors.forename: Chengbo
authors.initials: C
authors.lastname: Hou
authors.affiliation: School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, PR China.
authors.forename: Minming
authors.initials: M
authors.lastname: Tang
authors.affiliation: School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, PR China.
authors.forename: Ruirui
authors.initials: R
authors.lastname: Yang
authors.affiliation: DDLD (Beijing) Pharmaceutical Technology Co., Ltd., Beijing, PR China. Electronic address: zhm_zhlw@sina.com.
authors.forename: Huaming
authors.initials: H
authors.lastname: Zhou
mesh_terms: D000924:Anticholesteremic Agents / Q000032:analysis / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004340:Drug Contamination; D004355:Drug Stability; D000069438:Ezetimibe / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D006868:Hydrolysis; D057230:Limit of Detection; D015203:Reproducibility of Results; D013607:Tablets; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000924:Anticholesteremic Agents; D013607:Tablets; D000069438:Ezetimibe
keywords: Ezetimibe; HPLC; Method development; Related substances; Stability-indicating; Validation