Combination of an AccQ·Tag-Ultra Performance Liquid Chromatographic Method with Tandem Mass Spectrometry for the Analysis of Amino Acids

  • Carolina Salazar
  • Jenny M. Armenta
  • Diego F. Cortés
  • Vladimir ShulaevEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 828)


Amino acid analysis is a powerful tool in life sciences. Current analytical methods used for the detection and quantitation of low abundance amino acids in complex samples face intrinsic challenges such as insufficient sensitivity, selectivity, and throughput. This chapter describes a protocol that makes use of AccQ∙Tag chemical derivatization combined with the exceptional chromatographic resolution of ultra performance liquid chromatography (UPLC), and the sensitivity and selectivity of tandem mass spectrometry (MS/MS). The method has been fully implemented and validated using different tandem quadrupole detectors, and thoroughly tested for a variety of samples such as Plasmodium falciparum, human red blood cells, and Arabidopsis thaliana extracts. Compared to currently available methods for amino acid analysis, the AccQ∙Tag UPLC-MS/MS method presented here provides enhanced sensitivity and reproducibility, and offers excellent performance within a short analysis time and a broad dynamic range of analyte concentration. The focus of this chapter is the application of this improved protocol for the compositional amino acid analysis in A. thaliana leaf extracts using the Xevo TQ for mass spectrometric detection.

Key words

6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate AccQ∙Tag Amino acid analysis UPLC-ESI-MS/MS 


  1. 1.
    Stein WH et al (1957) Observation on the amino acid composition of human hemoglobins. Biochim Biophys Acta 24: 640–642PubMedCrossRefGoogle Scholar
  2. 2.
    Ullmer R, Plematl A, and Rizzi A (2006) Derivatization by 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate for enhancing the ionization yield of small peptides and glycopeptides in matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom 20: 1469–1479PubMedCrossRefGoogle Scholar
  3. 3.
    Badiou S et al (2004) Determination of plasma amino acids by fluorescent derivatization and reversed-phase liquid chromatographic separation. Clin Lab 50:153–158PubMedGoogle Scholar
  4. 4.
    Bernal JL et al (2005) A comparative study of several HPLC methods for determining free amino acid profiles in honey. J Sep Sci 28:1039–1047PubMedCrossRefGoogle Scholar
  5. 5.
    Cohen SA (2000) Amino acid analysis using precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Methods Mol Biol 159:39–47PubMedGoogle Scholar
  6. 6.
    Cohen SA (2003) Amino acid analysis using pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Analysis of hydrolyzed proteins and electroblotted samples. Methods Mol Biol 211:143–154Google Scholar
  7. 7.
    Hou S et al (2009) Determination of soil amino acids by high performance liquid chromatography-electro spray ionization-mass spectrometry derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Talanta 80:440–447PubMedCrossRefGoogle Scholar
  8. 8.
    Callahan DL et al (2007) Relationships of nicotianamine and other amino acids with nickel, zinc and iron in Thlaspi hyperaccumulators. New Phytol 176: 836–848PubMedCrossRefGoogle Scholar
  9. 9.
    Armenta JM et al (2010) Sensitive and rapid method for amino acid quantitation in malaria biological samples using AccQ.Tag ultra performance liquid chromatography-electrospray ionization-MS/MS with multiple reaction monitoring. Anal Chem 82: 548–558PubMedCrossRefGoogle Scholar
  10. 10.
    Bosch L, Alegria A, and Farre R (2006) Application of the 6-aminoquinolyl-N-hydroxysccinimidyl carbamate (AQC) reagent to the RP-HPLC determination of amino acids in infant foods. J Chromatogr B Analyt Technol Biomed Life Sci 831: 176–183PubMedCrossRefGoogle Scholar
  11. 11.
    Pappa-Louisi A et al (2007) Optimization of separation and detection of 6-aminoquinolyl derivatives of amino acids by using reversed-phase liquid chromatography with on line UV, fluorescence and electrochemical detection, Anal Chim Acta 593: 92–97PubMedCrossRefGoogle Scholar
  12. 12.
    Martínez-Girón AB et al (2009) Development of an in-capillary derivatization method by CE for the determination of chiral amino acids in dietary supplements and wines. Electrophoresis 30: 696–704PubMedCrossRefGoogle Scholar
  13. 13.
    Cohen SA, and Michaud DP (1993) Synthesis of a fluorescent derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino acids via high-performance liquid chromatography. Anal Biochem 211: 279–287PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Carolina Salazar
    • 1
  • Jenny M. Armenta
    • 2
  • Diego F. Cortés
    • 3
  • Vladimir Shulaev
    • 1
    Email author
  1. 1.Department of Biological SciencesUniversity of North Texas, College of Arts and SciencesDentonUSA
  2. 2.Waters CorporationBeverlyUSA
  3. 3.Virginia Bioinformatics InstituteVirginia Polytechnic Institute and State UniversityBlacksburgUSA

Personalised recommendations