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

Biomed Chromatogr. 2009 Jun;23(6):602-6. doi: 10.1002/bmc.1158.


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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetic Acid / chemistry
  • Acetonitriles / chemistry
  • Animals
  • Biliverdine / analysis*
  • Biliverdine / isolation & purification
  • Birds / growth & development
  • Chromatography, High Pressure Liquid / methods*
  • Edetic Acid / chemistry
  • Ovum / chemistry*
  • Protoporphyrins / analysis*
  • Protoporphyrins / isolation & purification
  • Sensitivity and Specificity
  • Spectrometry, Mass, Electrospray Ionization / methods*


  • Acetonitriles
  • Protoporphyrins
  • Edetic Acid
  • Biliverdine
  • Acetic Acid
  • acetonitrile