Li Jingyi, Shao Shan, Jaworsky Markian S, Kurtulik Paul T
Analytical Research and Development, Celgene Corporation, Summit, NJ 07091, USA.
J Chromatogr A. 2008 Mar 28;1185(2):185-93. doi: 10.1016/j.chroma.2008.01.083. Epub 2008 Feb 12.
A novel mixed-mode reversed-phase and cation-exchange high-performance liquid chromatography (HPLC) method is described to simultaneously determine four related impurities of cations, zwitterions and neutral compounds in developmental Drug A. The commercial column is Primesep 200 containing hydrophobic alkyl chains with embedded acidic groups in H(+) form on a silica support. The mobile phase variables of acid additives, contents of acetonitrile and concentrations of potassium chloride have been thoroughly investigated to optimize the separation. The retention factors as a function of the concentrations of potassium chloride and the percentages of acetonitrile in the mobile phases are investigated to get an insight into the retention and separation mechanisms of each related impurity and Drug A. Furthermore, the elution orders of the related impurities and Drug A in an ion-pair chromatography (IPC) are compared to those in the mixed-mode HPLC to further understand the chromatographic retention behaviors of each related impurity and Drug A. The study found that the positively charged Degradant 1, Degradant 2 and Drug A were retained by both ion-exchange and reversed-phase partitioning mechanisms. RI2, a small ionic compound, was primarily retained by ion-exchange. RI4, a neutral compound, was retained through reversed-phase partitioning without ion-exchange. Moreover, the method performance characteristics of selectivity, sensitivity and accuracy have been demonstrated to be suitable to determine the related impurities in the capsules of Drug A.
本文描述了一种新型的混合模式反相和阳离子交换高效液相色谱（HPLC）方法，用于同时测定研发中的药物A中的四种阳离子、两性离子和中性化合物相关杂质。所用的商业柱为Primesep 200，它在硅胶载体上含有带嵌入酸性基团的疏水烷基链，呈H(+)形式。对酸添加剂的流动相变量、乙腈含量和氯化钾浓度进行了深入研究，以优化分离效果。研究了保留因子与流动相中氯化钾浓度和乙腈百分比的函数关系，以深入了解各相关杂质和药物A的保留和分离机制。此外，还比较了离子对色谱（IPC）中相关杂质和药物A的洗脱顺序与混合模式HPLC中的洗脱顺序，以进一步了解各相关杂质和药物A的色谱保留行为。研究发现，带正电荷的降解产物1、降解产物2和药物A通过离子交换和反相分配机制保留。RI2是一种小离子化合物，主要通过离子交换保留。RI4是一种中性化合物，通过反相分配保留，不涉及离子交换。此外，该方法在选择性、灵敏度和准确性方面的性能特征已被证明适用于测定药物A胶囊中的相关杂质。
