Development and Validation of a Simple and Cost-Effective LC-MS/MS Method for the Quantitation of the Gut-Derived Metabolite Trimethylamine N-Oxide in Human Plasma of Healthy and Hyperlipidemic Volunteers

Trimethylamine N-oxide (TMAO) is a gut microbial metabolite of dietary precursors, including choline and carnitine. Elevated levels of TMAO in human plasma have been associated with several diseases such as cardiovascular, diabetes mellitus, chronic kidney disease, neurological disorders, and cancer. This has led to an increased interest in the accurate determination of TMAO in human blood, for which a reliable, cost-effective and sensitive analytical method should be established. LC-MS/MS has emerged as a powerful tool for the determination of TMAO due to its high sensitivity, specificity, and ability to handle complex matrices. Herein, we describe the development and validation of an LC-MS/MS method for the determination of TMAO in human blood plasma. Our method involves a simple sample preparation protocol, involving a protein precipitation step along with a non-deuterated IS, followed by a Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis using a triple quadrupole mass spectrometer. Additionally, the method was adapted and implemented on an UPLC-QTOF/MS. The method was validated using the guidelines set by the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) for assay performance and robustness in human plasma and successfully applied to plasma derived from healthy and hyperlipidemic volunteers. The developed method was found to be specific, sensitive, and accurate for the determination of TMAO in human plasma, with a lower limit of quantification of 0.25 µM. The intra- and inter-assay precision and trueness were within acceptable limits.