## 3 MALDI Non-Imaging MS Analysis of GSLs
### Instrumentation in MALDI Non-Imaging MS Analysis of GSLs  
Matrix-assisted laser desorption and ionization mass spectrometry (MALDI MS) uses an ultraviolet absorbing matrix (in most cases) in order to rapidly and accurately determine different chemical compounds [144]. The matrix serves to absorb the laser's emitted energy, averting the aggregation of analyte molecules and thwarting the formation of cluster ions [145]. Principally, when the laser beam interacts with the sample, the matrix absorbs the laser energy, forming co-crystals with the analyte. The matrix exists in a considerably greater excess compared to the analyte (often up to a 100,000-fold surplus). The vaporized matrix transports analyte molecules into the vapor phase. As the gas cloud expands, ions such as H\({}^{+}\) and Na\({}^{+}\) are exchanged between the matrix and analyte, resulting in charged analyte molecules. This process generates both cations and anions [146]. The key distinction between ions found in conventional electron impact (EI) spectra, known as'molecular (radical) ions', and the 'quasimolecular ions' generated by MALDI MS lies in their mass. Molecular ions stem from the analyte by losing one electron, leading to a mass that perfectly corresponds to the analyte's mass. Conversely, quasimolecular ions arise from the addition of a cation to the analyte, causing the mass of the analyte molecule to be lower than that of the quasimolecular ions [144]. These ions are accelerated within a potent electric field, reaching magnitudes up to approximately 20,000 V, before passing through a charged grid. The separation of masses occurs when ions traverse a field-free space in drift mode. Higher-mass ions take longer to reach the detector compared to lower-mass ions [147]. TOF detectors are widely embraced due to their nearly limitless mass range, rendering them particularly suitable for analyzing large molecules, as is often the case in MALDI MS. Furthermore, the pulsed ion generation inherent to MALDI aligns well with the characteristics of the TOF detector [115]. Due to its capacity to handle high masses and the pulsed nature of MALDI, this technique harmonizes well with a TOF analyzer. Increased laser power introduces an additional broadening of peaks due to the heightened energy dispersion of the desorbed ions. Linear TOF instruments usually have low resolution, and isotope peaks, photochemical-, and alkali metal adducts are frequently not resolved. At high laser powers, additional peak broadening to the high mass side for ions having the same \(m/z\) but different velocities is observed. To achieve optimal mass accuracy and resolution with a TOF instrument, it is imperative to maintain the laser power at the lowest feasible level, ideally just above the point where the ions first emerge [114]. The resolution of a TOF instrument can be optimized by using delayed ion extraction to extract the sample ions and a reflectron to minimize the spread of kinetic energy of ions with the same \(m/z\). The coupling of a second TOF instrument to perform tandem MS analysis is sometimes necessary for the investigation of specific components in complex mixtures, in which case the first TOF mass analyzer performs the selection of the precursor ions, which are further subjected to fragmentation in a collision cell, while the second TOF mass analyzer is used to analyze the obtained fragment ions [148]. For MALDI, some of the most noteworthy advantages are that MALDI analysis is very simple, very fast--taking less than one minute to investigate one sample--and that MALDI can tolerate higher amounts of impurities, which minimizes the need for sample purification [144]. There are also disadvantages, especially concerning the sample preparation: in MALDI-TOF MS a solid sample is used, and the homogeneity of co-crystalscreated between the matrix and analyte is never entirely attained, resulting in inherent irregularities.