Simultaneous determination of trimethylamine N-oxide (TMAO) and its 15 related metabolites by UPLC-MS/MS in human...

Trimethylamine N-oxide (TMAO) is increasingly recognized for its role in the pathogenesis and progression of cardiovascular and metabolic diseases by disturbing fatty acid metabolism. Concurrently, the acylcarnitine profile serves as a critical indicator of associated mitochondrial dysfunction. However, present ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methodologies often focus on a few targets such as betaine, choline, TMAO, and trimethylamine and lack the integrated capability to monitor simultaneous changes in the acylcarnitine profile. This analytical gap restricts the depth of toxicological investigations into the effects of environmental pollutants as well as the thorough assessment of metabolic homeostasis. In this study, an optimized UPLC-MS/MS method was developed for the simultaneous measurement of TMAO and its 15 related metabolites in human serum. Serum samples underwent pretreatment through targeted ethyl bromoacetate-derivatization and protein precipitation. Using 4% bovine serum albumin as a surrogate matrix, matrix-matched calibration was performed to reduce matrix effects and ensure analytical accuracy. Using 10 mmol/L ammonium formate (pH 4.0) and acetonitrile containing 0.1% formic acid as mobile phases, chromatographic separation was achieved on a HILIC column within a 5.5 min runtime. Positive electrospray ionization and multiple reaction monitoring mode were used to monitor the target analytes. The assay demonstrated good linearity (R² > 0.992) with limits of detection ranging from 0.012 to 5.2 μg/L. Spike recoveries in real samples were 84.5%-114.9%, with relative standard deviations below 10%. In conclusion, this study presents a rapid and validated UPLC-MS/MS method for simultaneous profiling of a targeted panel of TMAO, its precursors, and functionally related acylcarnitines in human serum. It may support studies investigating their associations with cardiometabolic disorders, chronic kidney disease, and metabolic dysfunction linked to gut microbial metabolism.