The lung is the predominant target organ for chemically induced damage by tobacco aerosol constituents. While metabolism by oxidative enzymes and subsequent conjugation with endogenous compounds generally results in their detoxification and excretion from the body, it can also lead to the production of more toxic metabolites. In vitro metabolic investigations using microsomes, a well-established test system, were used to investigate the potential toxification of tobacco aerosol related xenobiotics. In addition, parent compound kinetics were also investigated using comprehensive two-dimensional gas chromatography coupled to fast acquisition time-of-flight mass spectrometry (GCxGC-TOF-MS). GCxGC-TOF-MS is a widely established method for non-targeted analyses, suitable for the analysis of highly complex matrices such as tobacco aerosols. Aqueous trapping solutions perfused with tobacco aerosol as well as microsomal incubate were injected directly on-column and analyzed by GCxGC-TOF-MS using a combination of water resistant ionic liquid based analytical columns. In the absence of a solvent front, this setup enables the detection of highly volatile constituents, including furans. This method represents a novel approach, which defies the common belief that aqueous media are incompatible with gas chromatography. Furan compounds are naturally present in tobacco leaves and are further generated during the smoking of tobacco products by thermal degradation. Furan and alkylfurans are rapidly metabolized by microsomes as a substrate of cytochrome P450 2E1-catalyzed oxidation, which leads to the generation of reactive intermediates (like cis-1,4-butendial by furan oxidative CYP activation), whereas furfural and furfuryl alcohol are predominantly detoxified. A mixture of furan, alkylfurans, furfural, furfuryl alcohol and 5-methylfurfuryl alcohol was investigated for phase i metabolism using human liver and lung microsomes. The kinetics for parent compound transformation rates were determined by GCxGC-TOF-MS measurements in parallel with a LC high resolution MS approach for gaining complementary information regarding any reactive metabolites. In addition to that, we analysed reactive intermediate metabolites using nucleophile trapping. Potentially unknown furan metabolites were investigated using non-targeted data analysis complemented by in silico metabolism prediction using meteor Nexus®. The impact of microsomal activation upon aqueous tobacco aerosol fractions was also investigated using a non-targeted workflow for parent compound kinetics.