title: 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.
abstract: 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 (2.1mm×50mm, 1.7μm) analytical column with gradient elution of solvent A (10mM ammonium formate, pH 3.5) and solvent B (acetonitrile). The flow rate was 0.4mL/min and the total run time was 5min. 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 0.010-5.00μg/mL (plasma) and 1.00-150μg/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.
authors.affiliation: Center for Clinical Pharmaceutical Sciences, Department of Pharmacy and Therapeutics, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Ocque
authors.affiliation: The Kidney Institute, University of Kansas Medical Center, Kansas City, KS, USA.
authors.forename: Jason R
authors.initials: JR
authors.lastname: Stubbs
authors.affiliation: Center for Clinical Pharmaceutical Sciences, Department of Pharmacy and Therapeutics, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, USA. Electronic address: nolin@pitt.edu.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Nolin
mesh_terms: D001622:Betaine / Q000097:blood* / Q000652:urine*; D002138:Calibration; D002794:Choline / Q000097:blood* / Q000652:urine*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007202:Indicators and Reagents; D007674:Kidney Diseases / Q000378:metabolism; D013058:Mass Spectrometry; D008744:Methylamines / Q000097:blood* / Q000652:urine*; D010865:Pilot Projects; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results
chemical_list: D007202:Indicators and Reagents; D008744:Methylamines; D001622:Betaine; C005855:trimethyloxamine; D002794:Choline
keywords: Choline; Human plasma; Human urine; LC–MS/MS; Trimethylamine N-oxide