pSIRM workflow including sample preparation, identification, and quantification of metabolites. (A) Scheme of the experimental workflow from cell culture and cell harvest to GC-MS measurement. (B) A representative GC-MS selected ion chromatogram obtained from a cell culture sample using temperature-controlled and split injection. Due to the method, the peaks of pyruvate and lactate are baseline separated, detected, and quantified. The majority of phosphates are distinguishable by their retention behavior. (C) Distribution of the coefficient of variation (CoV) of the measured metabolite quantities from five biological replicates; distinct compound classes are indicated. (D) Spike-in experiment in order to test recovery of metabolites (see also Additional file1: Table S5). Therefore, the samples were measured alone and with spiked-in quantification mixtures at seven dilutions. (E) Absolute quantities of metabolites of T98G cells ranked by concentrations.