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

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.