Siva Kumar R, Sravan Kumar K V, Kondareddy L, Yogeshwara K R, Manish Gangrade, Jeenet Jayachandran, Nitesh Kanyawar
Analytical Development Laboratory, Cipla Limited, Virgonagar, Bangalore, Karnataka 560 049, India.
J Chromatogr Sci. 2018 May 1;56(5):383-395. doi: 10.1093/chromsci/bmx084.
A new sensitive and stability indicating reverse phase high-performance liquid chromatography method has been developed for the quantitative determination of potential impurities of Ledipasvir, active pharmaceutical ingredient. Efficient chromatographic separation was achieved on waters × select column contains C18 stationary phase in gradient mode, quantitation at wavelength of 325 nm and mobile phase A is 50 mM ammonium formate pH 3.5 and mobile phase B is acetonitrile with flow rate 1.2 mL. The method was validated according to International Council for Hormonisation guideline, linearity studies has shown correlation coefficient value greater than 0.999 for Ledipasvir and all known impurities. Limit of detection and Limit of quantification were determined to be 0.01% and 0.03%, respectively, for all the impurities. The consistent recoveries were obtained for all impurities (95-105%). Various stress conditions were applied to study the degradation behavior of the drug substance. LC-MS was used to analyse the degraded samples and possible structural identifications were assigned based upon known reactivity of the drug. The method was found to be specific, selective and robust to the degradation products. Major degradation of the drug substance was found to occur under photolytic stress conditions resulting a novel impurity (impurity F). The formed degradant was identified and characterized by FT-IR, 1HNMR and LC-MS/MS. The stress samples quantified against qualified working standard and the mass balance found above 98%.
已开发出一种新的灵敏且具有稳定性指示功能的反相高效液相色谱法，用于定量测定活性药物成分雷迪帕韦的潜在杂质。在含有C18固定相的waters × select柱上以梯度模式实现了高效的色谱分离，在325 nm波长处进行定量，流动相A为50 mM甲酸铵（pH 3.5），流动相B为乙腈，流速为1.2 mL。该方法根据国际协调理事会指南进行了验证，线性研究表明雷迪帕韦和所有已知杂质的相关系数值均大于0.999。所有杂质的检测限和定量限分别确定为0.01%和0.03%。所有杂质的回收率均一致（95 - 105%）。施加了各种应力条件以研究原料药的降解行为。采用LC-MS分析降解样品，并根据药物的已知反应性对可能的结构进行鉴定。结果发现该方法对降解产物具有特异性、选择性和稳健性。发现原料药在光解应力条件下主要发生降解，产生一种新杂质（杂质F）。通过FT-IR、1HNMR和LC-MS/MS对形成的降解产物进行了鉴定和表征。对照合格的工作标准对应力样品进行定量，质量平衡高于98%。
