Gas chromatography-atmospheric-pressure chemical ionization-time-of-flight mass spectrometry (GC-APCI-TOFMS) was compared to GC × GC-electron ionization (EI)-TOFMS, GC-EI-TOFMS, GC-chemical ionization (CI)-quadrupole mass spectrometry (qMS), and GC-EI-qMS in terms of reproducibility, dynamic range, limit of detection, and quantification using a mix of 43 metabolites and 12 stable isotope-labeled standards. Lower limits of quantification for GC-APCI-TOFMS ranged between 0.06 and 7.81 μM, and relative standard deviations for calibration replicates were between 0.4% and 8.7%. For all compounds and techniques, except in four cases, R(2) values were above 0.99. Regarding limits of quantification, GC-APCI-TOFMS was inferior to only GC × GC-EI-TOFMS, but outperformed all other techniques tested. GC-APCI-TOFMS was further applied to the metabolic fingerprinting of two Escherichia coli strains. Of 45 features that differed significantly (false discovery rate < 0.05) between the strains, 25 metabolites were identified through highly accurate and reproducible (Δm ± SD below 5 mDa over m/z 190-722) mass measurements. Starting from the quasimolecular ion, six additional metabolites were identified that had not been found in a previous study using GC × GC-EI-TOFMS and an EI mass spectral library for identification purposes. Silylation adducts formed in the APCI source assisted the identification of unknown compounds, as their formation is structure-dependent and is not observed for compounds lacking a carboxylic group.