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

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Summary.

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.

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Abbreviations

LCMS2 :

Liquid chromatography and electrospray ionization tandem mass spectrometry

SRM:

selected reaction monitoring

FAA:

free amino acid

DP:

dipeptide

GABA:

γ-aminobutyric acid

βABA:

β-aminobutyric acid

ABA:

α-aminobutyric acid

DABA:

2,4-diaminobutyric acid

Dopa:

3,4-dihydroxyphenyl-L-alanine

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Authors and Affiliations

  1. Molecular Neurology Program, Huntington Medical Research Institutes, Pasadena, CA, U.S.A.

    A. N. Fonteh, R. J. Harrington & M. G. Harrington

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  1. A. N. Fonteh
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  2. R. J. Harrington
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  3. M. G. Harrington
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Fonteh, A., Harrington, R. & Harrington, M. Quantification of free amino acids and dipeptides using isotope dilution liquid chromatography and electrospray ionization tandem mass spectrometry. Amino Acids 32, 203–212 (2007). https://doi.org/10.1007/s00726-006-0370-6

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