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.

就保留在反相色谱柱上而言，人胰岛素和六种最常用的治疗类似物非常相似。因此，在《欧洲药典》专着中针对胰岛素和胰岛素类似物规定的液相色谱方法包括许多类似的分离方法，当分离单个胰岛素产品时往往会很费时，或者在处理混合物时可能会不够用。在这项研究中，我们提出了一种简单，耐用，通用且可访问的HPLC-UV分离方法，可一次鉴定和定量人胰岛素及其类似物。通过对影响阴离子分离和反相特性的混合模式色谱柱上分离机理的流动相特性进行微调，可以实现同时分离和检测。还开发了一种简单有效的SPE样品清洗程序，所有类似物的胰岛素回收率在80％到100％之间。另一方面，主要赋形剂（例如苯酚和间甲酚）的浓度降至1％以下。两种经过开发和验证的分离方法与使用四元泵或二元泵的兼容性不同，因此可以独立于HPLC溶剂输送系统进行样品表征。该方法与使用质谱检测器进行无可辩驳的识别是兼容的。因此，可以独立于HPLC溶剂输送系统进行样品表征。该方法与使用质谱检测器进行无可辩驳的识别是兼容的。因此，可以独立于HPLC溶剂输送系统进行样品表征。该方法与使用质谱检测器进行无可辩驳的识别是兼容的。