Extracted-ion LC chromatograms from the multiple reaction monitoring (MRM) in positive-ion mode of trimethylamine (TMA), deuterated trimethylamine (d9-TMA), trimethylamine-N-oxide (TMAO) and deuterated trimethylamine-N-oxide (d9-TMAO) in a typical human urine sample by two different processing methods. (A): Mixed with 4 volumes of methanol and internal standards d9-TMA and d9-TMAO; (B): Extracted with hexane/butanol after spiking with internal standards d9-TMA and d9-TMAO, under alkaline pH followed by acidification to transfer to the aqueous phase. Then, 5 μL sample was injected onto a Silica column followed by a solvent elution as described in the methods. The eluate was monitored for TMA, TMAO, d9-TMA and d9-TMAO in a Thermo Quantiva mass spectrometer. The precursor-to-product transitions were m/z 60 → 44, m/z 69 → 49, m/z 76 → 58 and m/z 85 → 66 for TMA, d9-TMA, TMAO and d9-TMAO, respectively. The concentrations of TMAO and TMA in this urine sample were measured by the hexane/butanol extraction method as 314 μM and 2.37 μM, respectively. The data were acquired in Thermo Quantiva mass spectrometer with a Vanquish auto-sampler and LC pump system.