Isomer separation and analysis of amphiphilic polysialogangliosides using reversed‐phase liquid chromatography‐mass spectrometry

Abstract

Gangliosides are amphiphilic, acidic glycosphingolipids possessing one or more sialic acid residues and several isobaric structural isomers with different abundances and bioactivities. Therefore, the distinction between these isomers is crucial for their proper profiling. Although liquid chromatography-mass spectrometry has been successfully employed for this purpose, the distinction process can still be improved, particularly regarding liquid chromatography. Recently, a reversed-phase liquid chromatography method that could separate disialoganglioside isomers was reported; however, the distinction of trisialoganglioside isomers using reversed-phase liquid chromatography has not been demonstrated. Here, we investigated the practicality of a reversed-phase liquid chromatography with an octadecylsilane column for separating polysialoganglioside isomers and successfully achieved the isomer separation of disialogangliosides and trisialogangliosides for the first time. We also confirmed several crucial factors in the mobile-phase composition, which affect the differential retention and mass spectral response of the isomers. First, an organic modifier, acetonitrile, exhibited superior selectivity against polysialogangliosides over methanol. Second, ammonium bicarbonate was the best ammonium salt additive among those tested, in terms of the separation efficiency and mass spectral response. Third, as the ammonium salt concentration increased, the negative electrospray ionization response was extensively suppressed, and the retention of gangliosides increased.

Optimization of chromatographic buffer conditions for the simultaneous analysis of phosphatidylinositol and phosphatidylinositol phosphate species in canola

“…This suggested a potential relationship between the mobile phase pH and the chromatographic performance of PIPs, which is consistent with the literature on the chromatography of phosphorylated compounds [26]. Additionally, changes in retention time such as those observed in Figure 1 have previously been related to the pKb of the buffer anion [28]. However, as shown by the data in Table 1, peak widths and retention time produced by the buffers did not correlate with either buffer pH, or the anions pKb.…”

“…In addition to those advances previously made in the analysis of PIs, various sources throughout the literature have suggested a number of other variables which may be leveraged to pursue this goal. Experimental work regarding the influence of mobile phase pH and solvent composition has suggested that an alkaline and acetonitrile-based mobile phase may be sufficient to enhance the chromatographic retention, resolution, and ionization efficiency for these lipid classes [18,23,[26][27][28]. Furthermore, it is suggested that the chelation behavior and poor chromatographic performance of PIPs may be addressed through the use of certain specialized stationary phase technologies [29] and mobile phase additives [30], circumventing the previous necessity of ion-pairing agents and their potential for instrument contamination [27,31,32].…”