University of Würzburg, Institute for Pharmacy and Food Chemistry, 97074, Würzburg, Germany.
European Directorate for the Quality of Medicines & HealthCare, Council of Europe, 7 allée Kastner, CS 30026, 67081, Strasbourg, France.
J Pharm Biomed Anal. 2019 Mar 20;166:71-82. doi: 10.1016/j.jpba.2018.12.034. Epub 2018 Dec 23.
The LC methods for related proteins prescribed in the European Pharmacopoeia monographs for insulins and insulin analogues are very similar and present some drawbacks including long run time and low resolution. LC to UHPLC-UV geometrical transfer was attempted to overcome such drawbacks. With the new UHPLC method, additional substances were separated in bovine and porcine insulins. A UHPLC MS-compatible method was developed using a mixed-mode C18 stationary phase with charged surface hybrid technology and a mobile phase containing a low concentration of trifluoroacetic acid and acetonitrile. An unknown peak was detected and identified as being B30-des-Alanine-insulin which was also confirmed by microTOF direct infusion and specific digestion with bovine carboxypeptidase A. Based on the results obtained during geometrical method transfer, a single UHPLC-UV method for human, bovine, porcine insulins and insulin lispro and aspart was developed and validated according to ICH Q2 guidelines. The new method is superior to the current European Pharmacopoeia LC methods with improved selectivity and shorter run time. The method is based on gradient elution and employs a commonly available stationary phase (conventional C18 column) which makes it an appropriate method for pharmacopoeial public quality standards. It may therefore represent a valid alternative to the LC methods currently described in the European Pharmacopoeia for insulins.
欧洲药典中胰岛素和胰岛素类似物专论中规定的相关蛋白的 LC 方法非常相似，但存在一些缺点，包括运行时间长和分辨率低。尝试将 LC 转换为 UHPLC-UV 以克服这些缺点。使用具有带电表面混合技术的混合模式 C18 固定相和含有低浓度三氟乙酸和乙腈的流动相，开发了一种与 UHPLC-MS 兼容的方法。检测到并鉴定了一个未知峰，其为 B30-去丙氨酸胰岛素，该物质也通过微 TOF 直接进样和牛羧肽酶 A 的特异性消化得到了证实。基于几何方法转移过程中获得的结果，根据 ICH Q2 指南，开发并验证了用于人胰岛素、牛胰岛素、猪胰岛素和赖脯胰岛素及门冬胰岛素的单一 UHPLC-UV 方法。该新方法优于当前的欧洲药典 LC 方法，具有更高的选择性和更短的运行时间。该方法基于梯度洗脱，使用常用的固定相（常规 C18 柱），使其成为药典公共质量标准的合适方法。因此，它可能是目前欧洲药典中描述的胰岛素 LC 方法的有效替代方法。
