Sheathless CE-MS and comparison with nanoRP-

22 Purification and quality control of therapeutic peptides is often performed by one single method, RP23 HPLC. As usage of an orthogonal technique is highly advisable for quality assurance, capillary 24 electrophoresis (CE) employing a coated capillary coupled via a sheathless interface to a mass 25 spectrometer was applied in parallel. The basic therapeutic peptide aviptadil served as a model 26 substance to study the impurity profiles revealing 15 detectable impurities using CE-MS, two were 27 detected by an appropriate nanoRP-HPLC-MS method. None of the impurities detected by CE were 28 observed in LC and vice versa. The LOD in CE-MS was determined in the base peak 29 electropherogram at ~1 fmol, a value 2500 times smaller than the LOD found in nanoRP-HPLC-MS 30 (3 pmol). In nanoRP-HPLC-MS only 0.2 % of the extrapolated CE-MS signal for a 25 ng aviptadil 31 load was observed. We conclude that both, the LOD as well as the impurity profile of aviptadil, as 32 analyzed by nanoRP-HPLC are influenced by both, the ligand-derivatized silica matrix and the flow33 rate. Peptides may disappear completely and their variable emergence may lead to the determination 34 of incorrect ratios as present in the sample. 35