Sugar phosphate analysis with baseline separation and soft ionization by gas chromatography-negative chemical ionization-mass spectrometry improves flux estimation of bidirectional reactions in cancer cells.

Precise measurement of sugar phosphates in glycolysis and the pentose phosphate (PP) pathway for C-metabolic flux analysis (C-MFA) is needed to understand cancer-specific metabolism. Although various analytical methods have been proposed, analysis of sugar phosphates is challenging because of the structural similarity of various isomers and low intracellular abundance. In this study, gas chromatography-negative chemical ionization-mass spectrometry (GC-NCI-MS) is applied to sugar phosphate analysis with o-(2,3,4,5,6-pentafluorobenzyl) oxime (PFBO) and trimethylsilyl (TMS) derivatization. Optimization of the GC temperature gradient achieved baseline separation of sugar phosphates in 31 min. Mass spectra showed the predominant generation of fragment ions containing all carbon atoms in the sugar phosphate backbone. The limit of detection of pentose 5-phosphates and hexose 6-phosphates was 10 nM. The method was applied to C-labeling measurement of sugar phosphates for C-MFA of the MCF-7 human breast cancer cell line. C-labeling of sugar phosphates for C-MFA improved the estimation of the net flux and reversible flux of bidirectional reactions in glycolysis and the PP pathway.