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
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.
KeywordsTandem mass spectrometry Valine Leucine Isoleucine TMAO
Compliance with ethical standards
The collection and analysis of serum samples used in this study was approved by Edith Cowan University’s Human Research Ethics Committee (13402). The volunteer participants provided written informed consent to participate in this study.
Conflict of interest
The authors declare that they have no conflict of interest.
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