Chromatographia

, Volume 69, Issue 11–12, pp 1175–1180 | Cite as

Determination of Tryptophan in Lysozyme and Lysozyme Hydrolysate by Chiral LC Using d-Tryptophan as Internal Standard

  • Pieter Stam
  • Ilco Boogers
  • Wim Plugge
  • Alexander L. L. Duchateau
Original

Abstract

In the present study, a new LC method is described for the quantitation of tryptophan (Trp) in lysozyme and enzymatic lysozyme hydrolysate. To compensate for partial breakdown of Trp during hydrolysis with 4 M methanesulfonic acid, an enantiomer dilution method was developed. The method makes use of free d-Trp or a d-Trp-containing dipeptide as internal standard for the quantitation of l-tryptophan in these matrices. After acid hydrolysis in 4 M methanesulfonic acid, LC analysis is performed on a Crownpak CR chiral column in combination with fluorescence detection. Optimum time and temperature for the acid hydrolysis were investigated in order to obtain complete hydrolysis of the source materials. A comparison of the l-Trp recoveries was made for d-Trp and Gly-d-Trp as internal standards. By choosing a hydrolysis time of 150 min at 150 °C, 93% recovery of l-Trp from lysozyme was achieved. Under these conditions, no racemization occurred. When choosing d-Trp as internal standard, a direct LC method for l-Trp in lysozyme and enzymatic lysozyme hydrolysate was established without the need for pre-column derivatization and without the need to use Trp protecting agents during acid hydrolysis.

Keywords

Column liquid chromatography Fluorescence detection Enantiomers d-, l-Tryptophan 

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Copyright information

© Vieweg+Teubner | GWV Fachverlage GmbH 2009

Authors and Affiliations

  • Pieter Stam
    • 1
  • Ilco Boogers
    • 1
  • Wim Plugge
    • 1
  • Alexander L. L. Duchateau
    • 2
  1. 1.DSM Food Specialties, R&D/Analysis departmentDelftThe Netherlands
  2. 2.DSM Pharmaceutical Products, Innovative Synthesis & CatalysisGeleenThe Netherlands

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