Simultaneous Determination of Multiple Intracellular Primary Metabolites by Ultrahigh Performance Liquid Chromatography Coupled with a Q Exactive HF Mass Spectrometer.

In this study, we established an ultrahigh-performance liquid chromatography-Q Exactive HF MS (UHPLC-HF MS) method for the simultaneous determination of 25 targeted metabolites relating to a broad coverage of central metabolic pathways, such as glycolysis pathway, tricarboxylic acid cycle (TCA), serine biosynthesis pathway (SSP), glutaminolysis pathway, and closely related biosynthetic reactions. A Shodex Asahipak NH2P-50 2D column was used to separate the targeted compounds, and Full MS + PRM detection using an electrospray ionization source in negative mode was employed. The method also integrated a sample purification step by passing through a Waters Sirocco 96 plate to remove protein impurities, ensuring the better resolution and sensitivity of the proposed method. The calibration curves of the method showed good linearity within the range of 1-10 000 μg L-1 with the correlation coefficient no less than 0.99. The method can be used for routine quantification of primary metabolites in a wide variety of cell extract samples. With the help of the method, for the first time, we successfully separate the isomers of 3-phosphoglycerate (3-PG) and 2-phosphoglycerate (2-PG), which lay the groundwork for the accurate quantification of metabolites of the tumor cells, the study of PGAM1 inhibitors, and the development of neotype anticancer drugs.