# Development of a candidate reference method for the simultaneous quantification of betaine, choline and trimethylamine N-oxide in serum samples by two-dimensional liquid chromatography and isotope dilution tandem mass spectrometry

## TL;DR

A new method was developed to measure betaine, choline, and trimethylamine N-oxide in serum, which could help predict cardiovascular risks.

## Contribution

The novel method combines two-dimensional liquid chromatography and isotope dilution tandem mass spectrometry for simultaneous quantification of three metabolites in serum.

## Key findings

- The method was validated according to CLSI guidelines for accuracy and reliability.
- The technique was applied to 74 serum samples from ischemic stroke patients to study these metabolites as potential biomarkers.
- The approach enables rapid and precise separation of the three analytes using a two-dimensional chromatography setup.

## Abstract

Serum choline, betaine and trimethylamine N-oxide levels are associated with the risk of cardiovascular events. However, no reference procedure for the determination of these compounds in serum has been developed so far. This work describes the combination of IDMS and two-dimensional liquid chromatography operating in multiple heart-cutting mode for the simultaneous quantification of the three compounds in human serum by isotope dilution tandem mass spectrometry. A reversed-phase separation is proposed as the first dimension and coupled with cation exchange chromatography in the second dimension. The online isolation of the single fraction in which the three analytes co-elute from the first dimension enables a rapid chromatographic separation in the second dimension through cation exchange. The method was validated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines and applied to the analysis of 74 serum samples from patients who had suffered from an ischemic stroke in the past 24 h for further study of these metabolites as potential biomarkers to predict an ischemic stroke patient’s prognosis.

The online version contains supplementary material available at 10.1007/s00216-025-05914-z.