Imaging of Triglycerides in Tissues Using Nanospray Desorption Electrospray Ionization (Nano-DESI) Mass Spectrometry

Nonpolar triglycerides (TGs) are rarely detected in mass spectrometry imaging (MSI) experiments unless they are abundant in the sample. Herein, we use nanospray desorption electrospray ionization (nano-DESI) to explore the role of the solvent composition and ionic dopants on the detection of TGs in a murine gastrocnemius muscle tissue used as a model system. We evaluated three solvent mixtures for their ability to extract nonpolar TG species: MeOH:H₂O 9:1 (v/v), MeOH:DCM 6:4 (v/v) and MeOH:AcN:tol 5:3.5:1.5 (v/v/v). We observe that TGs are mainly detected as [M+K]⁺ adducts and their extraction efficiency is improved using less polar solvents: MeOH:DCM and MeOH:AcN:tol. We also explore whether the ionization efficiency of TGs may be improved by doping the MeOH:AcN:tol solvent with ammonium formate (AF) and other ionic additives. However, the formation of [M+NH₄]⁺ adducts of TGs is less efficient than the formation of [M+K]⁺ adducts in the range of AF concentrations from 0.1 to 10 mM. Chemical derivatization using 100 μM of Girard T reagent predominately generates reaction products of phosphatidylcholine rather than TG species. Moreover, the presence of the Girard T reagent suppresses ion signals of all the species in the spectrum including TGs. Nano-DESI MSI experiments performed using MeOH:AcN:tol solvent enable imaging of TGs without any detectable adverse effect on signals of other lipids and metabolites. Specifically, 10 out of 14 TG species were detected exclusively using MeOH:AcN:tol and the sensitivity towards other TGs was improved by at least an order of magnitude. Although polyunsaturated TGs may be detected using both solvents, saturated and monounsaturated TGs are only detected using MeOH:AcN:tol. Our results provide a direct path for the improved detection of TGs in tissue imaging experiments using liquid-based ambient ionization techniques.