RESEARCH PAPER An efficient liquid chromatography-high resolution mass spectrometry approach for the optimization of the metabolic stability of therapeutic peptides Simone Esposito 1 & Riccardo Mele 1 & Raffaele Ingenito 2 & Elisabetta Bianchi 2 & Fabio Bonelli 1 & Edith Monteagudo 1 & Laura Orsatti 1 Received: 23 November 2016 /Revised: 9 January 2017 /Accepted: 16 January 2017 # Springer-Verlag Berlin Heidelberg 2017 Abstract In drug discovery, there is increasing interest in peptides as therapeutic agents due to several appealing char- acteristics that are typical of this class of compounds, includ- ing high target affinity, excellent selectivity, and low toxicity. However, peptides usually present also some challenging ADME (absorption, distribution, metabolism, and excretion) issues such as limited metabolic stability, poor oral bioavail- ability, and short half-lives. In this context, early preclinical in vitro studies such as plasma metabolic stability assays are crucial to improve developability of a peptidic drug. In order to speed up the optimization of peptide metabolic stability, a strategy was developed for the integrated semi-quantitative determination of metabolic stability of peptides and qualita- tive identification/structural elucidation of their metabolites in preclinical plasma metabolic stability studies using liquid chromatography-high-resolution Orbitrap™ mass spectrome- try (LC-HRMS). Sample preparation was based on protein precipitation: experimental conditions were optimized after evaluating and comparing different organic solvents in order to obtain an adequate extraction of the parent peptides and their metabolites and to minimize matrix effect. Peptides and their metabolites were analyzed by reverse-phase liquid chro- matography: a template gradient (total run time, 6 min) was created to allow retention and good peak shape for peptides of different polarity and isoelectric points. Three LC columns were selected to be systematically evaluated for each series of peptides. Targeted and untargeted HRMS data were simul- taneously acquired in positive full scan + data-dependent MS/ MS acquisition mode, and then processed to calculate plasma half-life and to identify the major cleavage sites, this latter by using the software Biopharma Finder™. Finally, as an exam- ple of the application of this workflow, a study that shows the plasma stability improvement of a series of antimicrobial pep- tides is described. This approach was developed for the eval- uation of in vitro plasma metabolic stability studies of pep- tides, but it could also be applied to other in vitro metabolic stability models (e.g., whole blood, hepatocytes). Keywords Peptide metabolite ID . DMPK . LC-HRMS . Qual/Quan . Peptide ADME . Metabolic stability Introduction The constantly increasing number of peptide therapeutics un- der preclinical and clinical development reflects the growing significance of this class of compounds [1–3]. Similarly as for small molecules (SMs), preclinical in vitro and in vivo ADME (absorption, distribution, metabolism, and excretion) studies have pivotal role during the drug discovery process, since structural modification strategies are crucial to improve pep- tide developability [4]. Liquid chromatography coupled to mass spectrometry (LC-MS) is the technique of choice for ADME studies, both for SMs and peptides [ 5 , 6 ]. More recently, liquid chromatography-high-resolution mass spectrometry (LC-HRMS) has gained an important role [7–9] for both quantitative [10, 11] and qualitative analysis in DMPK (drug metabolism and pharmacokinetics) studies [ 12 – 14 ]. LC-HRMS has also been recognized as a valid alternative to * Simone Esposito s.esposito@irbm.it 1 DMPK, IRBM Science Park, Via Pontina km 30,600, 00071 Pomezia, Roma, Italy 2 Peptide Therapeutics, IRBM Science Park, Via Pontina km 30,600, 00071 Pomezia, Roma, Italy Anal Bioanal Chem DOI 10.1007/s00216-017-0213-1