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

Author(s): Kaspar H, Dettmer K, Chan Q, Daniels S, Nimkar S, Daviglus ML, Stamler J, Elliott P, Oefner PJ

Publication: J Chromatogr B Analyt Technol Biomed Life Sci, 2009, Vol. 877, Page 1838-46

PubMed ID: 19481989 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of analyzing urine specimens using LC-MS with iTRAQ labeling, GC-MS or an amino acid analyzer on the detection of amino acids. Specimens were preserved with boric acid and snap-frozen in liquid nitrogen. For use in this study, specimens were thawed, aliquoted, shipped on dry ice and stored at -20 degrees C. Blinded biological replicates were included in the analysis.

   Summary of Findings:

   While GC-MS could only detect 26 amino acids, the amino acid analyzer could detect 40 amino acids and LC-MS with iTRAQ labels could detect 42 amino acids. The mean technical error for the measurement of 20 amino acids in 34 specimens using the amino acid analyzer was only 7.27%, but was 21.18% and 18.34% for GC-MS and LC-MS with iTRAQ labels, respectively. The mean technical error rates in the second cohort of 144 specimens for 13 selected amino acids were 8.39%, 6.23% and 35.37% using the amino acid analyzer, GC-MS and LC-MS with iTRAQ labels, respectively. Correlations for the 12 amino acids measured by both the amino acid analyzer and GC-MS ranged from r=0.800 (tryptophan) to r= 0.980 (glycine). Correlation of cysteine measured by GC-MS and LC-MS with iTRAQ labels was only r=0.822, but for the other 18 amino acids measured by GC-MS and LC-MS with iTRAQ labels correlations ranged from r=0.934 (glutamic acid) to r=0.988 (tyrosine). The correlation of levels measured by amino acid analyzer and LC-MS with iTRAQ labels was only r=0.561 for arginine, but ranged from r=0.764 (tryptophan) to r=0.951 (lysine) for the other 19 amino acids measured by both methods. Only measured levels of glycine and tyrosine showed an excellent agreement (difference of less than 15%) between the three methods. For all other amino acids, comparisons of values obtained using at least 2 of the 3 methods showed either a systemic difference of more than 15% (15.7% of comparisons), a proportional error (27.5% of comparisons), or an increase in standard deviation with concentration (19.6% of comparisons). The authors conclude that all three methods are suitable for amino acid analysis; however, GC-MS covered fewer amino acids, and LC-MS with iTRAQ labeling had lower reproducibility compared to the other two methods.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Other Preservative

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Small molecule	LC-MS or LC-MS/MS
   Small molecule	GC-MS
   Small molecule	Amino acid analyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   LC-MS or LC-MS/MS Specific	Technology platform	GC-MS Amino acid analyzer

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