Development of UPLC-MS/MS method for the determination of polar metabolites

Sammanfattning: Trimethylamine-n-oxide (TMAO) is a metabolite found in plasma/serum in humans. Elevated levels of TMAO have been associated with several types of heart disease. It’s therefore of interest to make a simple analytical method to analyse TMAO and other metabolites that are degraded to TMAO, including betaine. In this study, the goal was to develop a method for the sample preparation and analysis of these compounds in human plasma. Sample preparation was performed with an Ostro 96-well method for sample clean-up. The analysis was performed by ultraperformance liquid chromatography – hydrophilic interaction liquid chromatography – tandem masspectrometry (UPLC-HILIC-MS/MS) in multiple reaction monitoring (MRM)-mode using electrospray ionization in positive mode (ESI+)-mode as the ion source. The analytes eluted under five minutes and were all baseline separated in the chromatogram. TMAO and betaine were quantified in quality control (QC) plasma samples using external calibration. Concentration of TMAO ranged from 132 ng/mL – 253 ng/mL and 1025-2084 ng/mL for betaine. Due to the lack of isotopically labelled standards for TMAO and betaine, valine-d8 was tested as an internal standard for the extraction; however, it was not a suitable option due to the low recovery obtained (5-34%) and the low response in ESI+. The recovery needs to be investigated further using isotopically labelled TMAO or betaine. Overall, the developed UPLC-HILIC-MS/MS method was found to be suitable for analysis of TMAO and betaine in human plasma. Further development and validation is required before application to samples from clinical studies.