Amino Acids

, Volume 30, Issue 3, pp 291–297 | Cite as

Amino acid analysis by hydrophilic interaction chromatography coupled on-line to electrospray ionization mass spectrometry

  • T. Langrock
  • P. Czihal
  • R. Hoffmann


Collagens form a common family of triple-helical proteins classified in 21 types. This unique structure is further stabilized by specific hydroxylation of distinct lysyl and prolyl residues forming 5-hydroxylysine and hydroxyproline (Hyp) isomers, mostly 4-trans and 3-trans-Hyp. The molecular distribution of the Hyp-isomers among the different collagen types is still not well investigated, even though disturbances in the hydroxylation of collagens are likely to be involved in several diseases such as osteoporosis and autoimmune diseases. Here, a new approach to analyze underivatized amino acids by hydrophilic interaction chromatography (HILIC) coupled on-line to electrospray ionization mass spectrometry (ESI-MS) is reported. This method can separate all three studied Hyp-isomers, Ile, and Leu, which are all isobaric, allowing a direct qualitative and quantitative analysis of collagen hydrolysates. The sensitivity and specificity was increased by a neutral loss scan based on the loss of formic acid (46 u).

Keywords: Collagen types – ESI-MS – HILIC – Hydroxyproline – Hyp 



electrospray ionization


hydrophilic interaction chromatography




mass spectrometry


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Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • T. Langrock
    • 1
  • P. Czihal
    • 1
  • R. Hoffmann
    • 1
  1. 1.Bioanalytics, Center for Biotechnology and Biomedicine, Faculty of Chemistry and MineralogyLeipzig UniversityLeipzigGermany

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