LC–MS quantification of TMAO
SEPARATION, RESOLUTION, INTERACTION
Urine samples were specific gravity normalised and prepared as previously described [Trimethylamine N-oxide (TMAO) was then quantified by Hydrophilic Liquid Interaction Chromatography (HILIC) in positive mode, performed on a Vanquish liquid chromatography chromatographic separation system connected to an IDX High Resolution Mass Spectrometer (Thermo Scientific). The HILIC positive data set was processed via Compound Discoverer 3.2 according to the following settings: untargeted metabolomic workflow with online database: mass tolerance 10 ppm, maximum shift 0.3 min, alignment model adaptive curve, minimum intensity 500 K, S/N threshold 3, compound consolidation, mass tolerance 10 ppm, RT tolerance 0.3 min. Database matching was performed at MS2 level using Thermo scientific m/z cloud with a similar index of 80% or better. TMAO intensity was then identified in the overall metabolite dataset and log transformed for statistical analysis.