(P17-020-24) A New, Accurate LC-MS/MS Method for Assessment of the Choline Status of the US Population

Objectives: Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of liver disease. It is linked to higher mortality rates and has increased worldwide with about 24% of US adults being diagnosed with the disease. Adequate intake of choline reduces the risk of fatty liver and liver damage. Our objective was to develop a sensitive analytical method for simultaneous detection of choline and 3 of its metabolites in order to conduct a first-time assessment of the choline status of the US population in the upcoming NHANES survey.

Methods: We developed a novel isotope dilution LC-MS/MS method to simultaneously measure plasma choline, betaine, dimethyl glycine (DMG), and trimethylamine N-oxide (TMAO) using a small sample volume (50 µL). The assay uses a fast and simple sample preparation (protein precipitation with internal standard mixture in trichloroacetic acid and acetonitrile performed on ice), centrifugation, and LC-MS/MS analysis (5 min). Blood-based reference materials with certified concentrations for choline and its metabolites are not available. We therefore compared our results to those from Cornell University’s choline LC-MS/MS method (currently retired) using a legacy quality control (QC) sample.

Results: The new LC-MS/MS method achieved excellent accuracy (spike recovery: 91%-111%) and low imprecision (2.5%-4.3%). The established limits of detection (choline: 0.1 uM, betaine: 0.09 uM, DMG and TMAO: 0.02 uM) and the assay linear range (choline: 0-100 uM, betaine: 0-150 uM, DMG and TMAO: 0-50 uM) cover the concentration ranges observed in 30 EDTA plasma single donor samples (choline: 7.7-22 uM, betaine: 23.5-107 uM, DMG: 1.43-10 uM, TMAO: 0.56-10 uM). The CDC method compared well to the Cornel University method with concentration differences varying from 2.3% (betaine) to 16% (TMAO) across the 4 analytes.

Conclusions: We developed a novel, sensitive, and accurate LC-MS/MS method suitable for population monitoring of plasma choline and 3 of its metabolites. It achieves a fast throughput while using a small sample volume.

Funding Sources: This work was supported by direct appropriations from US Congress.

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.