Characterizing EV preparations via gas-phase electrophoresis on a nES GEMMA instrumentation Victor U. Weiss1, Viktoria Weber2, Martina Marchetti-Deschmann1 1 TU Wien, Institute of Chemical Technologies and Analytics, Vienna, Austria 2 University of Continuing Education Krems, Krems, Austria Introduction: As recommended by the European Commission, nanoparticle characterization should be based on particle number concentrations (2011/696/EU from October 18th, 2011, updated in 2022). Gas-phase electrophoresis on a nano electrospray gas-phase electrophoretic mobility molecular analyser (nES GEMMA) allows such an approach. Single-charged (bio-)nanoparticles obtained after a nES process with subsequent surface-drying and charge equilibration are separated based on their electrophoretic mobility diameter (EMD) corresponding to the particle diameter for spherical analytes. By voltage variation, particles of different EMD successively reach the instruments’ detector unit and are counted, resulting in a spectrum in the size range of several few up to several hundred nm EMD. Methods: EVs were either purified via ultracentrifugation or a combination of precipitation and centrifugation. Subsequently, sample buffer was exchanged to volatile ammonium acetate. EV monitoring was via nanoparticle tracking analysis (NTA, Particle Metrix, Inning am Ammersee, Germany), gas-phase electrophoresis was on a nES GEMMA instrumentation (TSI Inc., Shoreview, MN, USA). Results: In previous work, we succeeded in demonstrating the applicability of nES GEMMA for the analysis of liposomes, vesicles consisting of a lipid bilayer encapsulating an aqueous lumen. nES GEMMA enabled the characterization of vesicles in terms of size distribution, particle number concentration and the occurrence of smaller-sized building blocks next to large vesicles. Also, offline hyphenation of gas-phase electrophoresis with orthogonal analysis methods, for instance atomic force microscopy, MALDI mass spectrometry or spectroscopy was shown. In vivo, cell-derived EVs, being part of cell/cell communication, are comparable to liposomes. They are envisioned as pharmaceutical cargo transporters, explaining the need for their characterization. We succeeded in porting gas-phase electrophoresis of liposomes to the characterization of EVs, demonstrating protein co-purification and loss of EV stability upon further polishing of vesicle preparations. Our nES GEMMA results were compared to data from NTA. Summary: We demonstrate gas-phase electrophoresis relating additional characteristics for EV containing samples in comparison to prevailing techniques like e.g. NTA.