A hybrid linear ion trap/Orbitrap mass spectrometer was used to perform tandem mass spectrometry (MS/MS) experiments and high-resolution mass analysis of lipids desorbed from nerve tissue. A dramatic improvement in mass spectral resolution and a decrease in background are observed in the spectra collected from the Orbitrap mass analyzer, which allows generation of more accurate mass spectrometric images of the distribution of lipids within nerve tissue. Employment of both mass analyzers provides a rapid and reliable means of compound identification based on MS/MS fragmentation and high-resolution mass spectrometry (HRMS) accurate mass.