## 3 MALDI Non-Imaging MS Analysis of GSLs
### Instrumentation in MALDI Non-Imaging MS Analysis of GSLs
However, when introducing a substantial surplus of molecules beyond gangliosides into an MS ion trap, they may interfere with ganglioside detection due to ionization suppression. MRM's capability to select and analyze ions, coupled with the detection of product ions derived from selected precursor molecules, effectively eliminates excessive non-ganglioside molecules from the analytical system. This confers an advantage for analyzing small content molecules [150]. Negative ion mode MSn analysis provides valuable insights into molecular weights through MS1, oligosaccharide sequences deduced from ions resulting from successive oligosaccharide moiety elimination from deprotonated ions by MS2, and ceramide structure details from ions arising from fatty acids and sphingosine through MS3 [140]. Moreover, the MALDI-QIT system smoothly conducts sequential MS2 and MS3 analyses, offering precise structural elucidation of GSLs. While heterogeneity in ceramide and sugar moieties can yield intricate MS spectra, MS/MS effectively surmounts this challenge [151]. An essential aspect of this procedure is the utilization of a matrix solution composed of DHB dissolved in acetonitrile/water (1:1 \(v/v\)), yielding MS/MS spectra of satisfactory quality for highly sensitive structural analysis. Some GSLs, such as GD3 species, often present challenges in observation when employing MALDI-TOF due to the TOF analyzer's suboptimal resolution. However, this limitation was surmounted by conducting structural elucidation through MS/MS, employing both collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) [122]. Another strategy for enhancing the structural characterization of GSLs involves integrating MALDI MS methods with distinct separation techniques. For instance, Ruh et al. [126] found Gb4 Cer detection in kidney extracts was unfeasible without TLC separation. The TLC-MALDI-QIT-TOF MS system offers advantages such as generating simpler spectra with monosodiated ions, as well as circumventing mass accuracy and resolution issues that could arise from the uneven silica gel plate surface [151]. In the realm of FTMS analysis, which involves long detection times (seconds), a notable drawback is the elevated levels of analyte metastable fragmentation. A devised approach involves directly coupling TLC with vibrational cooling MALDI-FTMS, amalgamating the straightforward sample handling of TLC with high-resolution FT and accurate mass determination. This amalgamation also opens doors to tandem MS for detailed structural investigations [131]. High-performance thin-layer chromatography (HPTLC) can be employed in tandem with MALDI MS, offering the advantage of detecting signals sharing similar Rf values, such as GM1b and GD3 [152]. Several strategies are available to enhance the resolution beyond that which linear TOF instruments can provide. Firstly, incorporating a reflection involving a reflecting electric field, which is arranged such that ions with higher energy penetrate deeper into the field, compensating for their increased velocity through a longer path. This approach aims to enhance resolution [114]. Secondly, employing a magnetic sector instrument equipped with an array detector captures signals from the pulsed ion source. Ions with different values of \(m/z\) can be collected across numerous laser shots and subsequently read out into a data system. Resolutions within the range of 1000-3000 have been achieved using this approach. Notably, a downside of sector instruments lies in their restricted upper mass limit imposed by the magnet, often limited to a few thousand Da [114].