Comprehensive\nCoverage of Glycolysis and Pentose Phosphate\nMetabolic Pathways by Isomer-Selective Accurate Targeted Hydrophilic\nInteraction Liquid Chromatography-Tandem Mass Spectrometry Assay

The accurate liquid chromatography-tandem mass spectrometry\nanalysis\nof phosphorylated isomers from glycolysis and pentose phosphate pathways\nis a challenging analytical problem in metabolomics due to extraction\nproblems from the biological matrix, adherence to stainless steel\nsurfaces leading to tailing in LC, and incomplete separation of hexose\nand pentose phosphate isomers. In this study, we present a targeted\nHILIC-ESI-MS/MS method based on a BEH amide fully porous 1.7 μm\nparticle column with an inert surface coating of column hardware and\nmultiple reaction monitoring (MRM) acquisition fully covering the\nglycolysis and pentose phosphate pathway metabolites. To minimize\ncontact of the phosphorylated analytes with stainless steel surfaces,\na μ-ESI-MS probe with a hybrid electrode made of PEEKsil was\nemployed. Optimized HILIC gradient elution conditions with 100 mM\nammonium formate (pH 11) provided the separation of hexose monophosphate\nand pentose phosphate isomers. To ensure good retention time repeatability\nin HILIC, perfluoroalkoxy alkane bottles were used for the mobile\nphase (with sd over 60 runs between 0.01 and 0.02 min). For the quantitative\nassay, the U-<sup>13</sup>C-labeled cell extract was spiked prior\nto extraction by metal oxide-based affinity chromatography (MOAC)\nwith TiO<sub>2</sub> beads. The concentrations of the 24 targets were\nquantified in HeLa and human embryonic kidney (HEK293) cells. Erastin-induced\nferroptosis in HEK293 cells was accompanied by enhanced levels of\nfructose-1,6-bis-phosphate, 2- and 3-phosphoglycerate, and 2,3-bis-phosphoglycerate.