Extraction and analysis of colourful eggshell pigments using HPLC and HPLC/electrospray ionization tandem mass spectrometry

Authors

  • A. Gorchein,

    Corresponding author
    1. MRC Bioanalytical Science Group, School of Biological and Chemical Sciences, Birkbeck, University of London, Malet Street, London WC1E 7HX, UK
    • MRC Bioanalytical Science Group, School of Biological and Chemical Sciences, Birkbeck, University of London, Malet Street, London WC1E 7HX, UK.
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  • C. K. Lim,

    1. MRC Bioanalytical Science Group, School of Biological and Chemical Sciences, Birkbeck, University of London, Malet Street, London WC1E 7HX, UK
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  • P. Cassey

    1. Centre for Ornithology, School of Biosciences, University of Birmingham, Edgbaston B15 2TT, UK
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Abstract

The literature on the pigments of avian eggshells is critically reviewed. Methods using methanolic sulfuric acid or hydrochloric acid to extract eggshell pigments are unsuitable to detect the occurrence of zinc protoporphyrin or zinc biliverdin because they demetallate these compounds. Extraction methods are described here using EDTA and acetonitrile–acetic acid or acetonitrile–dimethyl sulfoxide, which do not demetallate zinc protoporphyrin. Such extracts were prepared from eggshell of the common nighthawk, Chordeiles minor, and from another six bird species. Protoporphyrin and biliverdin were identified and fully characterized by HPLC/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) in all samples, but none contained zinc protoporphyrin. The zinc complex of biliverdin, claimed to be an additional pigment responsible for eggshell background colours, was labile to EDTA and acid pH and if occurring naturally could not be extracted intact by the published or the modified protocols. An explanation is advanced for the exceptional report that all porphyrins from uroporphyrin to protoporphyrin were found in eggshells of the fowl Gallus domesticus. Copyright © 2009 John Wiley & Sons, Ltd.

Abbreviations used
TFA

trifluoroacetic acid

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