title: Quality control of cationic cell-penetrating peptides.
abstract: During fundamental research, it is recommended to evaluate the test compound identity and purity in order to obtain reliable study outcomes. For peptides, quality control (QC) analyses are routinely performed using reversed-phase liquid chromatography coupled to an ultraviolet (UV) detector system. These traditional QC methods, using a C18 column and a linear gradient with formic acid (FA) as acidic modifier in the mobile phase, might not result in optimal chromatographic performance for basic peptides due to their cationic nature and hence may lead to erroneous results. Therefore, the influence of the used chromatographic system on the final QC results of basic peptides was evaluated using five cationic cell-penetrating peptides and five C18-chromatographic systems, differing in the column particle size (high performance liquid chromatography (HPLC) versus ultra-high performance liquid chromatography (UHPLC)), the acidic modifier (FA versus trifluoroacetic acid (TFA)), and the column temperature (30 °C versus 60 °C). Our results indicate that a UHPLC system with the C18 column thermostated at 30 °C and a mobile phase containing TFA, provides the most suitable routine QC analysis method for cationic peptides, outperforming in sensitivity and resolution compared to the other systems. We also demonstrate the use of a single quad mass spectrometry (MS) detector system during QC analysis of (cationic) peptides, allowing identification of the peptide and its impurities, as well as the evaluation of the peak purity.
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.
authors.forename: Sofie
authors.initials: S
authors.lastname: Stalmans
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.
authors.forename: Bert
authors.initials: B
authors.lastname: Gevaert
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.
authors.forename: Frederick
authors.initials: F
authors.lastname: Verbeke
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.
authors.forename: Matthias
authors.initials: M
authors.lastname: D'Hondt
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Bracke
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.
authors.forename: Evelien
authors.initials: E
authors.lastname: Wynendaele
authors.affiliation: Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium. Electronic address: Bart.DeSpiegeleer@UGent.be.
authors.forename: Bart
authors.initials: B
authors.lastname: De Spiegeleer
mesh_terms: D000595:Amino Acid Sequence; D002412:Cations; D057846:Cell-Penetrating Peptides / Q000032:analysis* / Q000235:genetics / Q000592:standards*; D002626:Chemistry, Pharmaceutical / Q000379:methods / Q000592:standards*; D002851:Chromatography, High Pressure Liquid / Q000379:methods / Q000592:standards; D008969:Molecular Sequence Data; D010316:Particle Size; D011786:Quality Control
chemical_list: D002412:Cations; D057846:Cell-Penetrating Peptides
keywords: Cell-penetrating peptides; HPLC–UV; Impurity profiling; Quality control; Single quad mass Spectrometer; UHPLC–UV