Urinary amino acid analysis: A comparison of iTRAQ®-LC-MS/MS, GC-MS, and amino acid analyzer

Hannelore Kaspar, Katja Dettmer, Queenie Chan, Scott Daniels, Subodh Nimkar, Martha L. Daviglus, Jeremiah Stamler, Paul Elliott, Peter J. Oefner*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

121 Scopus citations


Urinary amino acid analysis is typically done by cation-exchange chromatography followed by post-column derivatization with ninhydrin and UV detection. This method lacks throughput and specificity. Two recently introduced stable isotope ratio mass spectrometric methods promise to overcome those shortcomings. Using two blinded sets of urine replicates and a certified amino acid standard, we compared the precision and accuracy of gas chromatography/mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of propyl chloroformate and iTRAQ® derivatized amino acids, respectively, to conventional amino acid analysis. The GC-MS method builds on the direct derivatization of amino acids in diluted urine with propyl chloroformate, GC separation and mass spectrometric quantification of derivatives using stable isotope labeled standards. The LC-MS/MS method requires prior urinary protein precipitation followed by labeling of urinary and standard amino acids with iTRAQ® tags containing different cleavable reporter ions distinguishable by MS/MS fragmentation. Means and standard deviations of percent technical error (%TE) computed for 20 amino acids determined by amino acid analyzer, GC-MS, and iTRAQ®-LC-MS/MS analyses of 33 duplicate and triplicate urine specimens were 7.27 ± 5.22, 21.18 ± 10.94, and 18.34 ± 14.67, respectively. Corresponding values for 13 amino acids determined in a second batch of 144 urine specimens measured in duplicate or triplicate were 8.39 ± 5.35, 6.23 ± 3.84, and 35.37 ± 29.42. Both GC-MS and iTRAQ®-LC-MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines.

Original languageEnglish (US)
Pages (from-to)1838-1846
Number of pages9
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Issue number20-21
StatePublished - Jul 1 2009


  • Amino acid analyzer
  • Amino acids
  • Gas chromatography-mass spectrometry
  • Liquid chromatography-tandem mass spectrometry
  • Precolumn derivatization
  • Propyl chloroformate
  • Stable isotope dilution
  • Urine
  • iTRAQ

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology

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