Faculty for Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, 1000 Ljubljana, Slovenia.
Faculty for Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, 1000 Ljubljana, Slovenia; COBIK, Tovarniška 26, 5270 Ajdovščina, Slovenia.
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 May 1;1171:122557. doi: 10.1016/j.jchromb.2021.122557. Epub 2021 Jan 20.
Human insulin and six most used therapeutic analogues are very similar in terms of retention on a reversed-phase column. Thus, the LC methods prescribed in the European Pharmacopoeia monographs for insulin and insulin analogues include many similar separation methods, which tend to be time consuming when separating individual products of insulins or are inadequate when handling a mixture. In this study, we present a simple, robust, versatile and accessible HPLC-UV separation method for identification and quantification of human insulin and its analogues in one run. The simultaneous separation and detection is possible by fine-tuning the mobile phase properties that affect the separation mechanism on a mixed mode column combining anion exchange and reversed-phase characteristics. Also developed was a simple and effective SPE sample cleaning procedure with insulin recoveries ranging from 80 to 100% for all analogues. On the other hand, the concentration of major excipients such as phenol and m-cresol fall below 1%. The two developed and validated separation methods differ in their compatibility with the use of a quaternary or binary pump, thus enabling sample characterisation independent of the HPLC solvent delivery system. The methods are compatible with the use of a mass spectrometric detector for an indisputable identification.
人胰岛素和六种最常用的治疗性类似物在反相柱上的保留行为非常相似。因此，《欧洲药典》胰岛素和胰岛素类似物专论中规定的 LC 方法包含许多类似的分离方法，当分离胰岛素的单个产品时，这些方法往往很耗时，或者在处理混合物时不够有效。在本研究中，我们提出了一种简单、稳健、通用且易于使用的 HPLC-UV 分离方法，可在一次运行中鉴定和定量人胰岛素及其类似物。通过微调影响混合模式柱分离机制的流动相性质，实现了同时分离和检测，该混合模式柱结合了阴离子交换和反相特性。我们还开发了一种简单有效的 SPE 样品净化程序，对于所有类似物，胰岛素的回收率在 80%至 100%之间。另一方面，主要赋形剂如苯酚和间甲酚的浓度低于 1%。这两种开发和验证的分离方法在与使用四元或二元泵的兼容性上有所不同，因此能够独立于 HPLC 溶剂输送系统对样品进行特征描述。这些方法与质谱检测器的使用兼容，可进行无可争议的鉴定。
