Determination of energy metabolites in cancer cells by porous graphitic carbon liquid chromatography electrospray ionization mass spectrometry for the assessment of energy metabolism

Abstract

A high performance liquid chromatography (HPLC) tandem mass spectrometric (MS/MS) method has been developed for the simultaneous determination of fifteen glucose, or acetate derived metabolites isolated from tumor cells. Glycolytic and tricarboxylic acid (TCA) cycle metabolites as well as acidic amino acids were separated on a HPLC porous graphitic carbon (PGC) column and simultaneously determined by means of triple quadrupole MS/MS using multiple reaction monitoring (MRM). Target compounds were eluted within 10min with 8% v/v formic acid as an electronic modifier added to a 4:1 v/v methanol water mobile phase. The calibration is linear in the 1–100μM concentration range for each analyte. The limit of detection ranges between 0.39 and 2.78μM for the analytes concerned. To test the PGC–HPLC–MS/MS method in metabolomic studies, ZR-75.1 human mammary adenocarcinoma cells were labeled with U-13C glucose or 1-13C acetate. Applying the MRM mode, the incorporation of 13C into metabolites, isolated from the tumor cells, and derived from glucose or acetate, could be properly identified.