Quantification of free amino acids and dipeptides using isotope dilution liquid chromatography and electrospray ionization tandem mass spectrometry

• Published in: Amino acids Vol. 32; no. 2; pp. 203 - 212
• Main Authors: Fonteh, A. N, Harrington, R. J, Harrington, M. G
• Format: Journal Article
• Language: English
• Published: Austria Vienna : Springer-Verlag 01.02.2007; Springer Nature B.V
• Subjects: Amino acids; Amino Acids - blood; Amino Acids - cerebrospinal fluid; Amino Acids - urine; Analytical chemistry; Biological; Body Fluids - metabolism; cerebrospinal fluid; Cerebrospinal Fluid - metabolism; Chloroformate; Chromatography; Chromatography, Liquid - methods; Derivatives; dipeptides; Dipeptides - blood; Dipeptides - cerebrospinal fluid; Dipeptides - urine; Humans; Ionization; Ions; Levodopa - pharmacology; Liquid chromatography; Mass spectrometry; Mass Spectrometry - methods; Plasma; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization - methods; Tandem mass spectrometry; Time Factors; urine
• DOI: 10.1007/s00726-006-0370-6
• ISSN: 0939-4451

Our aim was to develop a liquid chromatography and electrospray ionization tandem mass spectrometry (LCMS²) method to measure free amino acid (FAA) and dipeptide (DP) concentrations in biological fluids. We synthesized chloroformate derivatives of FAA and DP, identified the major precursor ions and used LCMS² to obtain the most intense product ions. Using serial dilutions of unlabeled and labeled standards ([²H₃]-L-Dopa, homoarginine, homophenylalanine, [¹⁵N]-Glutamine and [²H₃]-methionine), we observed linear relationships in MS response that we used to calculate the amounts of FAA and DP in biological samples. This method is sensitive with a limit of detection (LOD) for most of the FAAs and DPs tested in the 0.05-1 pmol range and is linear over 3-5 orders of magnitude when many metabolites were measured simultaneously. Reproducibility and between run or daily variations were <10% for most FAAs and DPs. We applied this method to human samples and quantitatively measured 21 FAAs and 2 DPs in 200 μl CSF, 31 FAAs and 6 DPs in 100 μl plasma, and 23 FAAs and 5 DPs in 200 μl urine. These data demonstrate the potential for using LCMS² to discover changes in FAA and DP metabolic pathways that occur during disease pathogenesis.