ADME/DMPK Department, IRBM S.p.A.,, Pomezia, Rome, Italy.
Department of Chemical Sciences and Technologies, Tor Vergata University of Rome, Rome, Italy.
J Pept Sci. 2020 Sep;26(9):e3272. doi: 10.1002/psc.3272. Epub 2020 Jul 6.
LC-HRMS-based identification of the products of peptide catabolism is the key to drive the design of more stable compounds. Because the catabolite of a given peptide can be very different from the parent compound and from other catabolites in terms of physicochemical properties, it can be challenging to develop an analytical method that allows recovery and detection of the parent and all parent-related catabolites. The aim of this study was to investigate how the recovery and the matrix effect of peptidic drugs and their catabolites are affected by different protein precipitation (PP) and solid-phase extraction (SPE) protocols. To this purpose, four model peptides representative of different classes (somatostatin, GLP-2, human insulin and liraglutide) were digested with trypsin and chymotrypsin to simulate proteolytic catabolism. The resulting mixtures of the parent peptides and their proteolytic products covering a wide range of relative hydrophobicity (H ) and isoelectric points (pI) were spiked in human plasma and underwent different PP and SPE protocols. Recovery and matrix effect were measured for each peptide and its catabolites. PP with three volumes of ACN or EtOH yielded the highest overall recoveries (more than 50% for the four parent peptides and all their catabolites) among all the tested PP and SPE protocols. Mixed-mode anion exchange (MAX) was the only SPE sorbent among the five tested that allowed to extract all the peptides with recoveries more than 20%. Matrix effect was generally lower with SPE. Overall, it was observed that peptides with either high hydrophilicity (e.g., somatostatin catabolites) or hydrophobicity (GLP-2 and lipidated liraglutide catabolites) had a much narrower choice of PP solvent or SPE protocol. Simulation of catabolism using recombinant enzymes together with in silico calculation of the H and the pI of potential proteolysis products is recommended to select the optimal extraction conditions for the study of peptide catabolism.
基于 LC-HRMS 的肽代谢产物鉴定是推动更稳定化合物设计的关键。由于给定肽的代谢产物在物理化学性质方面可能与母体化合物和其他代谢产物非常不同，因此开发一种允许回收和检测母体和所有母体相关代谢产物的分析方法可能具有挑战性。本研究旨在研究不同蛋白质沉淀 (PP) 和固相萃取 (SPE) 方案如何影响肽类药物及其代谢产物的回收率和基质效应。为此，选择了四种不同类别（生长抑素、GLP-2、人胰岛素和利拉鲁肽）的代表性模型肽进行胰蛋白酶和糜蛋白酶消化，以模拟蛋白水解代谢。所得的母体肽及其广泛的相对疏水性 (H) 和等电点 (pI) 范围内的蛋白水解产物混合物被掺入人血浆中，并经历了不同的 PP 和 SPE 方案。测量了每种肽及其代谢产物的回收率和基质效应。与所有测试的 PP 和 SPE 方案相比，用 ACN 或 EtOH 沉淀 3 倍体积可获得最高的总回收率（四种母体肽及其所有代谢产物均超过 50%）。在测试的五种 SPE 吸附剂中，混合模式阴离子交换 (MAX) 是唯一一种能够以回收率超过 20%提取所有肽的 SPE 吸附剂。SPE 通常会降低基质效应。总体而言，观察到具有高亲水性（例如，生长抑素代谢产物）或疏水性（GLP-2 和脂质化利拉鲁肽代谢产物）的肽的 PP 溶剂或 SPE 方案选择范围要窄得多。建议使用重组酶模拟代谢，并通过计算潜在蛋白水解产物的 H 和 pI 进行计算机模拟，以选择用于肽代谢研究的最佳提取条件。
