Analytical and Bioanalytical Chemistry

, Volume 407, Issue 15, pp 4351–4362 | Cite as

Use of LC–MS–MS as an alternative to currently available immunoassay methods to quantitate corticosterone in egg yolk and albumen

  • Siegrid De BaereEmail author
  • Tom Rosendahl Larsen
  • Mathias Devreese
  • Patrick De Backer
  • Liesbeth De Neve
  • Graham Fairhurst
  • Luc Lens
  • Siska Croubels
Paper in Forefront
Part of the following topical collections:
  1. Hormone and Veterinary Drug Residue Analysis


Corticosterone (CORT) is the dominant plasma glucocorticoid in birds. There has been increasing interest in the function of CORT in avian egg yolk and in the potential to use CORT concentrations in eggs to quantify stress and to assess the effect of maternal stress on offspring. The concentration of CORT in egg yolk is most frequently assessed using enzyme or radioimmunoassays, alone or in combination with high-performance liquid chromatography. However, the quantification of CORT is frequently hampered by the presence of high concentrations of other steroid hormones which cross-react with the CORT antibody. As an alternative, we developed a sensitive and specific LC–MS–MS method. The sample-preparation procedure consisted of a protein–lipid precipitation step, followed by defatting and clean-up using a C18 SPE column. Chromatography was performed on an Acquity C18 BEH column (50 mm × 2.1 mm i.d., dp: 1.7 μm, run-time: 6 min), using 0.1 % formic acid in both water (A) and acetonitrile (B) as mobile phases. The MS–MS instrument was operated in the positive-electrospray-ionization mode. The method was validated in-house according to European Guidelines (linearity, accuracy and precision, limits of quantification and detection, specificity, stability) and the results fell within the accepted ranges. The method was successfully used for the analysis of CORT in yolk and albumen of eggs collected from eight breeding lesser black-backed gulls at a Flemish coastal colony. CORT concentrations were in the range 42.4–166.3 pg g−1 in albumen and < LOQ (75 pg g−1)–762.5 pg g−1 in yolk.

Graphical Abstract


Corticosterone LC–MS–MS Eggs Yolk Albumen Steroid hormones 







Slope calibration curve






Atmospheric-pressure chemical ionization




Deuterated corticosterone






Enzyme immunoassay


Electrospray ionization




European Union


Food and Drug Administration


Goodness-of-fit coefficient


High-performance liquid chromatography


Internal standard


Liquid chromatography tandem mass spectrometry


Limit of detection


Limit of quantification


Lipid–protein precipitation


Multiple reaction monitoring


Mass-to-charge ratio




Phosphate-buffered saline






Quality control


Correlation coefficient


Apparent recovery


Extraction recovery




Rotations per minute


Relative standard deviation


Relative retention time


Signal-to-noise ratio


Solid-phase extraction


Signal suppression and/or enhancement




Retention time



The authors wish to thank Mrs J. Lambrecht for her kind assistance during sample analysis, Hans Matheve for his help with data collection, and Marjut Paljakka and Cátia Santos for their help with sample preparation. This study was financially supported by FWO project G0E1614N to LL and LDN.

Conflicts of interest

The authors report no conflicts of interest.

Supplementary material

216_2014_8269_MOESM1_ESM.pdf (28 kb)
ESM 1 (PDF 28.2 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Siegrid De Baere
    • 1
    Email author
  • Tom Rosendahl Larsen
    • 2
  • Mathias Devreese
    • 1
  • Patrick De Backer
    • 1
  • Liesbeth De Neve
    • 2
  • Graham Fairhurst
    • 3
  • Luc Lens
    • 2
  • Siska Croubels
    • 1
  1. 1.Department of Pharmacology, Toxicology and Biochemistry, Laboratory of Pharmacology and Toxicology, Faculty of Veterinary MedicineGhent UniversityMerelbekeBelgium
  2. 2.Department of Biology, Terrestrial Ecology Unit, Faculty of ScienceGhent UniversityGhentBelgium
  3. 3.Department of BiologyUniversity of SaskatchewanSaskatoonCanada

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