Multi-Laboratory Assessment Reveals Variable Ion Species Profiles in Electrospray Ionization Mass Spectrometry

Variation in electrospray (ESI) mass spectrometry (MS) instrument design and local ionization environments influences the formation of ion species, affecting analyte detection and quantitation. Here, we assessed ion species formation variability by analyzing a standardized internal retention time standard (IRTS) mixture across ten laboratories employing high-resolution ESI-MS instrumentation from four different instrument vendors (Agilent Technologies, ThermoFisher Scientific, Shimadzu Corporation, and Waters Corporation). Despite the use of standardized extraction and chromatographic protocols, significant differences in ion species formation were observed across vendors, among laboratories using the same vendor instruments, and even within individual labs. These findings highlight the critical need to consider platform-dependent ESI behavior when interpreting LC-MS small molecule data to improve reproducibility and comparability across studies.