Development and validation of a simple LC-MS/MS method for the simultaneous quantitative determination of trimethylamine-N-oxide and branched chain amino acids in human serum

• Published in: Analytical and bioanalytical chemistry Vol. 411; no. 5; pp. 1019 - 1028
• Main Authors: Le, Thao T., Shafaei, Armaghan, Genoni, Angela, Christophersen, Claus, Devine, Amanda, Lo, Johnny, Wall, Philippa Lyons, Boyce, Mary C.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.02.2019; Springer; Springer Nature B.V
• Subjects: Amino acids; Amino Acids, Branched-Chain - blood; Analytical Chemistry; Biochemistry; Blood plasma; Branched chain amino acids; Chain branching; Chains; Characterization and Evaluation of Materials; Chemical properties; Chemistry; Chemistry and Materials Science; Chromatography; Chromatography, Liquid - methods; Diabetes mellitus; Food Science; Health; Humans; Identification and classification; Ions; Isoleucine; Laboratory Medicine; Leucine; Limit of Detection; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Methods; Methylamines - blood; Monitoring/Environmental Analysis; Reproducibility of Results; Research Paper; Retention time; Sample preparation; Spectroscopy; Tandem Mass Spectrometry - methods; Trimethylamine; Trimethylamine-N-oxide; Valine; TMAO; Tandem mass spectrometry; Leucine; Valine; Isoleucine
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-018-1522-8

Serum branched chain amino acids and trimethylamine-N-oxide are monitored as potential indicators of diabetes and cardiovascular health respectively. A rapid method for their simultaneous determination using liquid chromatography and tandem mass spectrometry is described here. Branched chain amino acids and trimethylamine-N-oxide were quantified based on their specific MS/MS fragments using a selected reaction monitoring approach. A number of columns were tested for their ability to separate the analytes. A C18-PFP column separated the analytes in just 4 minutes, and resulted in excellent peak shape and retention time repeatability, and was therefore chosen as the optimal column. A second column, the Intrada Amino Acid column, was chosen for comparison and validation experiments as it provided an orthogonal separation mechanism. The intra-day and inter-day precision and accuracy were less than 12% for trimethylamine-N-oxide and less than 6% for the branched chain amino acids. Recoveries, where serum was spiked with three different concentrations of the analytes, ranged from 97 to 113%. The LODs and LOQs for trimethylamine-N-oxide were 1 and 6 ng/mL, for leucine and isoleucine were 4 and 8 ng/mL, and for valine were 5 and 15 ng/mL, respectively. The C18-PFP column method was validated using the Intrada Amino Acid column method and percentage agreement for all four analytes was within 10%. Sample preparation was minimal, and use of labelled internal standards accounted for matrix effects. The method was successfully applied to human plasma samples. Graphical abstract ᅟ