Advertisement

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 Baere
  • 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

Abstract

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

Keywords

Corticosterone LC–MS–MS Eggs Yolk Albumen Steroid hormones 

Abbreviations

11-deoxy-CRTL

11-Deoxycortisol

17-α-OH-PROG

17-α-Hydroxyprogesterone

a

Slope calibration curve

ALD

Aldosterone

ANDR

Androstenedione

APCI

Atmospheric-pressure chemical ionization

CORT

Corticosterone

CORT-d8

Deuterated corticosterone

CRTL

Cortisol

DHEA

Dehydroepiandrosterone

EIA

Enzyme immunoassay

ESI

Electrospray ionization

ESTR

Estradiol

EU

European Union

FDA

Food and Drug Administration

g

Goodness-of-fit coefficient

HPLC

High-performance liquid chromatography

IS

Internal standard

LC–MS–MS

Liquid chromatography tandem mass spectrometry

LOD

Limit of detection

LOQ

Limit of quantification

L–PPT

Lipid–protein precipitation

MRM

Multiple reaction monitoring

m/z

Mass-to-charge ratio

N2

Nitrogen

PBS

Phosphate-buffered saline

PREG

Pregnenolone

PROG

Progesterone

QC

Quality control

r

Correlation coefficient

RA

Apparent recovery

RE

Extraction recovery

RIA

Radioimmunoassay

rpm

Rotations per minute

RSD

Relative standard deviation

RRT

Relative retention time

S/N

Signal-to-noise ratio

SPE

Solid-phase extraction

SSE

Signal suppression and/or enhancement

TEST

Testosterone

Tr

Retention time

Notes

Acknowledgments

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)

References

  1. 1.
    Lipar JL, Ketterson ED, Nolan V, Casto JM (1999) Egg Yolk Layers Vary in the Concentration of Steroid Hormones in Two Avian Species. Gen Comp Endocrinol 115:220–227CrossRefGoogle Scholar
  2. 2.
    Rettenbacher S, Möstl E, Groothuis TGG (2009) Gestagens and glucocorticoids in chicken eggs. Gen Comp Endocrinol 164:125–129CrossRefGoogle Scholar
  3. 3.
    von Engelhardt N, Henriksen R, Groothuis TGG (2009) Steroids in chicken egg yolk: Metabolism and uptake during early embryonic development. Gen Comp Endocrinol 163:175–183CrossRefGoogle Scholar
  4. 4.
    Rettenbacher S, Groothuis TG, Henriksen R, Möstl E (2013) Corticosterone in bird eggs: The importance of analytical validation. Wien Tierärztl Monat 100:283–290Google Scholar
  5. 5.
    Groothuis TGG, Schwabl H (2008) Hormone-mediated maternal effects in birds: mechanisms matter but what do we know of them. Phil Trans R Soc B 363:1647–1661CrossRefGoogle Scholar
  6. 6.
    Almasi B, Rettenbacher S, Müller C, Brill S, Wagner H, Jenni L (2012) Maternal corticosterone is transferred into the egg yolk. Gen Comp Endocrinol 178:139–144CrossRefGoogle Scholar
  7. 7.
    Navara KJ, Pinson SE (2010) Yolk and albumen corticosterone concentrations in eggs laid by white versus brown caged laying hens. Poult Sci 89:1509–1513CrossRefGoogle Scholar
  8. 8.
    Saino N, Romano M, Ferrari RP, Martinelli R, Møller AP (2005) J Exp Zool 303A:998–1006CrossRefGoogle Scholar
  9. 9.
    Kozlowski CP, Bauman JE, Hahn DC (2009) A Simplified Method for Extracting Androgens From Avian Egg Yolks. Zool Biol 28:137–143CrossRefGoogle Scholar
  10. 10.
    Quillfeldt P, Poisbleau M, Parenteau C, Trouvé C, Demongin L, van Noordwijk HJ, Möstl E (2011) Measuring corticosterone in seabird egg yolk and the presence of high yolk gestagen concentrations. Gen Comp Endocrinol 173:11–14CrossRefGoogle Scholar
  11. 11.
    Möstl E, Spendier H, Kotrschal K (2001) Concentration of immunoreactive progesterone and androgens in the yolk of hens’ eggs (Gallus domesticus). Wien Tierärztl Monat 88(3):62–65Google Scholar
  12. 12.
    Bertin A, Richard-Yris M-A, Houdelier C, Lumineau S, Möstl E, Kuchar A, Hirschenhauser K, Kotrschal K (2008) Habituation to humans affects yolk steroid levels and offspring phenotype in quail. Horm Behav 54:396–402CrossRefGoogle Scholar
  13. 13.
    Sas B, Domány G, Gyimóthy I, Gaál Kovácsné K, Süth M (2006) Influence of the type of management system on corticosterone transfer into eggs in laying hens. Acta Vet Hung 54(3):343–352CrossRefGoogle Scholar
  14. 14.
    Koren L, Ng ESM, Soma KK, Wynne-Edwards KE (2012) Sample Preparation and Liquid Chromatography-Tandem Mass Spectrometry for Multiple Steroids in Mammalian and Avian Circulation. PLoS ONE 7:e32496CrossRefGoogle Scholar
  15. 15.
    Samtani MN, Jusko WJ (2007) Quantification of dexamethasone and corticosterone in rat biofluids and fetal tissue using highly sensitive analytical methods: assay validation and application to a pharmacokinetic study. Biomed Chromatogr 21:585–597CrossRefGoogle Scholar
  16. 16.
    Fanelli F, Belluomo I, Di Lallo VD, Cuomo G, De Iasio R, Baccini M, Casadio E, Casetta B, Vicennati V, Gambineri A, Grossi G, Pasquali R, Pagotto U (2011) Serum steroid profiling by isotopic dilution-liquid chromatography-mass spectrometry: Comparison with current immunoassays and reference intervals in healthy adults. Steroids 76:244–253CrossRefGoogle Scholar
  17. 17.
    Rönquist-Nii Y, Edlund PO (2005) Determination of corticosteroids in tissue samples by liquid chromatography-tandem mass spectrometry. J Pharm Biomed 37:341–350CrossRefGoogle Scholar
  18. 18.
    Commision Decision 2002/657/EC implementing Council Directive 96/23/EC concerning the performances of analytical methods and the interpretation of results, Official Journal of the European Communities, N° L221, 17/08/2002, Decision of 12 August 2002, European Commision, Directorate General for Public Health and Consumers ProtectionGoogle Scholar
  19. 19.
    VICH GL 49 (2012-Final), Guidance for Industry, Studies to evaluate the metabolism and residue kinetics of veterinary drugs in food producing animals: validation of analytical methods used in residue depletion studies, February 2011Google Scholar
  20. 20.
    Knecht J, Stork G (1974) Prozentuales und Logarithmisches Verfahren zur Berechnung von Eichkurven. Fresen’ Z Anal Chem 270:97–99CrossRefGoogle Scholar
  21. 21.
    R.J. Heitzman, Ed. Veterinary Drug Residues, Report Eur. 14126-EN, Commision of the EC, Brussels-Luxembourg, 1994Google Scholar
  22. 22.
    Matuszewski BK, Constanzer ML, Chavez-Eng CM (2003) Strategies for the Assessment of Matrix Effect in Quantitative Bioanalytical Methods Based on HPLC-MS/MS. Anal Chem 75:3019–3030CrossRefGoogle Scholar
  23. 23.
    Hewavitharana AK (2011) Matrix matching in liquid chromatography-mass spectrometry with stable isotope labelled internal standards – is it necessary? J Chromatogr A 1218:359–361CrossRefGoogle Scholar
  24. 24.
    Abdel-Khalik J, Björklund E, Hansen M (2013) Development of a solid phase extraction method for the simultaneous determination of steroid hormones in H295R cell line using liquid chromatography-tandem mass spectrometry. J Chromatogr B 935:61–69CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Siegrid De Baere
    • 1
  • 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

Personalised recommendations