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

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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.

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Authors and Affiliations

  1. DSM Food Specialties, R&D/Analysis department, P.O. Box 1, 2600 MA, Delft, The Netherlands

    Pieter Stam, Ilco Boogers & Wim Plugge

  2. DSM Pharmaceutical Products, Innovative Synthesis & Catalysis, P.O. Box 18, 6160 MD, Geleen, The Netherlands

    Alexander L. L. Duchateau

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  1. Pieter Stam
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  2. Ilco Boogers
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  3. Wim Plugge
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  4. Alexander L. L. Duchateau
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Correspondence to Alexander L. L. Duchateau.

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Stam, P., Boogers, I., Plugge, W. et al. Determination of Tryptophan in Lysozyme and Lysozyme Hydrolysate by Chiral LC Using d-Tryptophan as Internal Standard. Chroma 69, 1175–1180 (2009). https://doi.org/10.1365/s10337-009-1081-4

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