Trimethylamine N-oxide (TMAO), a small molecular amine oxide, is generated from dietary choline and carnitine through gut microbial metabolism. It has been proved as a potential biomarker or therapeutic target for many disease, such as type 2 diabetes, cardiovascular diseases and chronic kidney diseases. The plasma concentration of TMAO was found positively correlated with the increased risks of the disease progression. Therefore, quantifying TMAO in clinical samples with high accuracy and sensitivity is crucial.

In this study, we established and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for TMAO quantitation and applied it in a clinical pharmacology study. To avoid the possible endogenous interference in the typical biological matrix, we introduced an artificial surrogate matrix instead of human plasma for a more sensitive quantitation.

The constructed eight-point calibration curve in the range of 1–5000 ng/mL achieved excellent linearity and reproducibility with R² ranged between 0.9962 and 0.9979. Both the intra-day and inter-day assays achieved satisfactory precision and accuracy results ranging from 1.65–7.15% and 96.36–111.43%, respectively. In addition, we also applied this quantitation method to clinical samples to evaluate the variations in TMAO levels, which denoted the clinical applicability of this method. Overall, these results demonstrate that the described method is applicable in determining disease risk and monitoring disease progression in clinical therapeutics.