Expanding the Measurement Space of Ion Mobility-Mass Spectrometry Using Chemical Derivatizations

Steroid analysis by mass spectrometry remains challenging because many compounds exhibit both poor ESI efficiency and subtle structural differences that limit differentiation by conventional techniques. While chemical derivatization has historically been employed to improve analytical sensitivity, its broader influence on ion mobility-mass spectrometry (IM-MS) measurements has not been systematically investigated. Here, seven hydrazine-based derivatization reagents were evaluated across a panel of 16 isomeric 11-oxosteroids using IM-MS and matrix-assisted laser desorption/ionization (MALDI)-MS. All derivatization chemistries systematically altered both molecular mass and collision cross section (CCS), producing reagent-specific shifts through CCS vs m/z measurement space. Beyond these global shifts, several reactions generated characteristic mobility fingerprints composed of multiple mobility-resolved structural populations, providing substantially richer structural information than CCS alone. The analytical utility of these measurements was demonstrated through improved differentiation of structurally similar steroid isomers using complementary descriptors including CCS, reaction stoichiometry, mobility fingerprints, and reagent-specific isotopic signatures. Finally, successful translation of the derivatization chemistry to MALDI analysis, including direct surface derivatization, established compatibility with alternative ionization methods and supports future integration with mass spectrometry imaging (MSI) workflows. Collectively, these results demonstrate that chemical derivatization fundamentally expands the analytical information accessible by IM-MS and establishes a general framework for engineering molecular measurements through differential, yet controlled, manipulation of molecular mass, CCS, and gas-phase structural diversity.