Recovery rates of TMA, TMAO, d9-TMA and d9-TMAO. Here, 500 μL of 40 μM (TMA + TMAO) was mixed with 20 μL of 1 mM (d9-TMA + d9-TMAO) and extracted with 2 mL hexane and different volumes of butanol in the presence of 1 mL NaOH at varying concentrations. This step was followed by the transfer to the aqueous phase by adding 0.2 mL of 0.2 N formic acid. Then, 5 μL was injected into the liquid chromatography–mass spectrometry (LC/MS). The recovery rate (%) was calculated by the peak area of the standard after extraction preparation divided by the peak area of the standard, 100 μM (TMA + TMAO + d9-TMA + d9-TMAO), without undergoing extraction. Data were acquired in a Thermo Quantiva mass spectrometer with a Vanquish auto-sampler and a LC pump system.