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Phosphatidylinositol phosphates (PIPs) are phospholipids (PLs) that play fundamental roles as signaling molecules in numerous cellular processes. However, PIPs have shown to be perhaps some of the most challenging compounds to analyze in bioanalytical samples, as PIPs are strongly adsorbed to surfaces containing glass and metal, and can easily stick to column walls, injectors and tubings, and build up a nonionized layer on the MS ion source. In this study, the suitability of reversed phase (RP) LC and hydrophilic interaction liquid chromatography (HILIC) for identification and separation of the PIPs, with respect to the phosphate groups and fatty acid chains, using electrospray ionization (ESI) MS was investigated. In preliminary studies, HILIC chromatography of a phosphate-containing compound was performed with a ZIC-HILIC (0.3 x 150 mm, 3.5 μm) column, using a mobile phase consisting of acetonitrile (ACN)/20 mM ammonium acetate (NH4Ac) (79/21, v/v). It was discovered that HILIC separations of a phosphate-containing standard could result in a large peak splitting and was dependent on sample solvent. The best peak shape was obtained when the sample was dissolved in more than 60 % ACN and 20 mM NH4Ac. Injectors, loops, tubings, nuts, ferrules, columns and a syringe of PEEK were applied to decrease adsorption and increase the detection of PIPs. MS detection of PIPs is challenging, but by including appropriate additives to the mobile phase, detection improved largely. Additives that provided high pH in the mobile phase were found to be the most appropriate. PIPs containing various fatty acid chains could be separated on a C4 (1.0 x 5.0 mm, 5 μm, 300 Å) column using a mobile phase consisting of MeOH/20 mM TEA at pH 10.0 (80/20, v/v). PIPs containing the same fatty acid chains, but different number of phosphate groups, could not be separated using the same C4 column. However, separation according to number of phosphate groups was possible using a ZIC-pHILIC (2.1 x 150 mm, 5 μm) column with a mobile phase consisting of ACN/10 mM (NH4)2CO3/0.2 % NH4OH at pH 10.0 (55/45, v/v). However, the retention window was small, and optimizing parameters such as temperature, water and the ion concentration in the mobile phase did not increase the retention of the PIPs. Hence, separation of PIPs on a ZIC-pHILIC column using ESI-MS is possible, but with several limitations. Nonetheless, a combination of a ZIC-pHILIC column and a RP column in a two-dimensional (2D) system using ESI-MS may be suitable to determine the different PIP isomers.
