Development and validation of a simple UHPLC-MS/MS method for the simultaneous determination of trimethylamine N-oxide, choline, and betaine in human plasma and urine

A simple, sensitive, and precise ultra-high performance liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of trimethylamine N-oxide, choline, and betaine in human plasma and urine. Sample preparation involved protein precipitation with methanol containing internal standards. Chromatographic separation was achieved using an Acquity BEH Amide (2.1 mm x 50 mm, 1.7 mu m) analytical column with gradient elution of solvent A (10 mM ammonium formate, pH 3.5) and solvent B (acetonitrile). The flow rate was 0.4 mL/min and the total run time was 5 min. Detection of analytes was performed using heated electrospray ionization (positive mode) and selected reaction monitoring. Excellent linearity was observed over the standard curve concentration ranges of 0.010-5.00 mu g/mL (plasma) and 1.00-150 mu g/mL (urine) for all analytes. The intra- and inter-day accuracy and precision for all quality controls were within +/- 10%. Excellent recovery was observed. The method is rapid, accurate and reproducible, and was successfully applied to a pilot study of markers of atherosclerosis in patients with kidney disease who underwent successful kidney transplantation. (C) 2015 Elsevier B.V. All rights reserved.