It is challenging to have comprehensive spatial lipidomic analysis by mass spectrometry imaging (MSI) due to the strong ion suppression and peak interference from high-abundance polar lipids to low-abundance poorly ionizable lipids. In this work, we proposed a new MSI approach via ambient liquid extraction techniques assisted by a new mixed-mode adsorptive material, graphene oxide (GO)/TiO 2 nanocomposite. The material combines chelation affinity from TiO 2 and hydrophobic interaction from GO. By finely tuning the adsorption solvent as 9% H 2 O−6% ammonia−85% methanol/acetonitrile (1:1, v/v), simultaneous enrichment of poorly ionizable glycolipids and glycerides with separation from high-abundance phospholipids was achieved on the material. In 10 mg/mL glucose−6% ammonia−94% methanol, all of the adsorbed glycolipids and glycerides could be desorbed from the material efficiently. Then, GO/TiO 2 nanocomposite was coated onto the sample plate for thaw-mounting the tissue section, and ambient liquid extraction probe was used to have pixel-to-pixel desorption of the lipids on the section in two steps with the above two solvents. The results show that most of the phospholipids were imaged in the first step MSI, and glycolipids and glycerides were selectively imaged in the second step MSI, largely reducing ion suppression and peak interference. Compared with direct ambient liquid extraction MSI, much more glycolipid species (22 vs 9), glyceride species (10 vs 5), phosphatidylethanolamines (11 vs 3), and lysophospholipids (12 vs 2) were detected via GO/TiO 2 nanocomposite-assisted two-step MSI. The ion images of most lipids show much higher signals and imaging quality with the new method than with the traditional method. Thus, comprehensive enhancement of lipid coverage in MSI by on-tissue separation was achieved here for the first time, providing more information about spatial lipidomics studies.
