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J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Mar 1;951-952:69-77. doi: 10.1016/j.jchromb.2014.01.019. Epub 2014 Jan 25.

Quantitative amino acid profiling and stable isotopically labeled amino acid tracer enrichment used for in vivo human systemic and tissue kinetics measurements.

Author information

1
Clinical Metabolomics Core Facility (CMCF), Rigshospitalet, Section 7652, Ole Maaløesvej 26, 2100 Copenhagen, Denmark. Electronic address: a.bornoe@ClinicalMetabolomics.dk.
2
Clinical Metabolomics Core Facility (CMCF), Rigshospitalet, Section 7652, Ole Maaløesvej 26, 2100 Copenhagen, Denmark; Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark. Electronic address: gvanhall@ClinicalMetabolomics.dk.

Abstract

An important area within clinical functional metabolomics is in vivo amino acid metabolism and protein turnover measurements for which accurate amino acid concentrations and stable isotopically labeled amino acid enrichments are mandatory not the least when tissue metabolomics is determined. The present study describes a new sensitive liquid chromatography tandem mass-spectrometry method quantifying 20 amino acids and their tracer(s) ([ring-(13)C6]/D5Phenylalanine) in human plasma and skeletal muscle specimens. Before analysis amino acids were extracted and purified via deprotonization/ion exchange, derivatized using a phenylisothiocyanate reagent and each amino acid was quantitated with its own stable isotopically labeled internal standard (uniformly labeled-(13)C/(15)N). The method was validated according to general recommendations for chromatographic analytical methods. The calibration curve correlations for amino acids were on average; r(2)=0.998. Interday accuracy for amino acids determined in spiked plasma was on average 97.3% and the coefficient of variation (CV) was 2.6%. The ([ring-(13)C6]/D5Phenylalanine) enrichment CV's for machine reproducibility in muscle tissue fluid and plasma were 4.4 and 0.8%, and the interday variability was 3.4% and the recovery was 90.5%, respectively. In conclusion, we have developed and validated a method for quantitative amino acid profiling that meets the requirements for systemic and tissue human in vivo amino acid and protein turnover kinetics measurements. Moreover, citrulline, ornithine, π-methyl-histidine, τ-methyl-l-histidine, hydroxy-proline and carnitine were analysed but when similar precision and accuray are required an additional stable istopically labeled internal standard for these meatablites should be be added.

KEYWORDS:

Amino acids; Liquid chromatography; Metabolomics; Quantification; Stable isotopes; Tracer kinetics

PMID:
24513911
DOI:
10.1016/j.jchromb.2014.01.019
[Indexed for MEDLINE]

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