Analytical and Bioanalytical Chemistry

, Volume 394, Issue 1, pp 313–319 | Cite as

Optimization of capillary electrophoresis conditions for a glucagon competitive immunoassay using response surface methodology

  • Anna R. Lomasney
  • Christelle Guillo
  • Ashley M. Sidebottom
  • Michael G. RoperEmail author
Original Paper


The capillary electrophoresis (CE) conditions for a competitive immunoassay of glucagon were optimized for highest sensitivity of the immunoassay and resolution of the electrophoretic peaks using a Box–Behnken design. Injection time, voltage ramp time, and separation voltage were varied between three levels and two responses, bound-to-free (B/F) ratio of the immunoassay peaks and resolution between the peaks, were measured. Analysis of variance was applied to fit a predictive model, and a desirability function was used to simultaneously optimize both responses. A 10-s injection, 1.6-min ramp time, and a 22-kV separation voltage were the conditions found when high B/F was given more emphasis than high resolution. To test the model, calibration curves of a glucagon immunoassay were measured at the optimum and least optimum CE conditions. Optimal conditions increased the sensitivity of the immunoassay by 388% compared to the least optimum conditions while maintaining adequate resolution.


CE Response surface methodology Optimization Glucagon Competitive immunoassay 



This work was funded by grants from the National Institutes of Health R01 DK080714 and the American Heart Association Greater Southeast Affiliate.


  1. 1.
    Schou C, Heegaard NHH (2006) Electrophoresis 27:44–59CrossRefGoogle Scholar
  2. 2.
    Moser AC, Hage DS (2008) Electrophoresis 29:3279–3295CrossRefGoogle Scholar
  3. 3.
    Gas B, Stedry M, Kenndler E (1997) Electrophoresis 18:2123–2133CrossRefGoogle Scholar
  4. 4.
    Buchanan DD, Jameson EE, Perlette J, Malik A, Kennedy RT (2003) Electrophoresis 24:1375–1382CrossRefGoogle Scholar
  5. 5.
    Mayer BX (2001) J Chrom A 907:21–37CrossRefGoogle Scholar
  6. 6.
    Xuan XC, Hu GQ, Li DQ (2006) Electrophoresis 27:3171–3180CrossRefGoogle Scholar
  7. 7.
    Sowell J, Parihar R, Patonay J (2001) J Chromatogr B 752:1–8CrossRefGoogle Scholar
  8. 8.
    Guillo C, Roper MG (2008) Electrophoresis 29:410–416CrossRefGoogle Scholar
  9. 9.
    Sentellas S, Saurina J (2003) J Sep Sci 9–10:875–885CrossRefGoogle Scholar
  10. 10.
    Hanrahan G, Montes RE, Gomez FA (2008) Anal Bioanal Chem 390:169–179CrossRefGoogle Scholar
  11. 11.
    Montgomery DC (2005) Design and analysis of experiments. Wiley, HobokenGoogle Scholar
  12. 12.
    Ferreira SLC, Bruns RE, Ferreira HS, Matos GD, David JM, Brandao GC, da Silva EGP, Portugal LA, Reis PS, Souza AS, dos Santos WNL (2007) Anal Chim Acta 597:179–186CrossRefGoogle Scholar
  13. 13.
    Hows MEP, Perret D, Kay J (1997) J Chrom A 768:97–104CrossRefGoogle Scholar
  14. 14.
    Pfaff P, Weide F, Kuhn R (1999) Chromatographia 49:666–670CrossRefGoogle Scholar
  15. 15.
    Ragonese R, Macka M, Hughes J, Petocz P (2002) J Pharm Biomed Anal 27:995–1007CrossRefGoogle Scholar
  16. 16.
    Servais AC, Fillet M, Chiap P, Abushoff AM, Hubert P, Crommen J (2002) J Sep Sci 25:1087–1095CrossRefGoogle Scholar
  17. 17.
    Jimidar MI, Vennekens T, Van Ael W, Redlich D, De Smet M (2004) Electrophoresis 25:2876–2884CrossRefGoogle Scholar
  18. 18.
    Babu CVS, Chung BC, Yoo YS (2004) Anal Let 37:2485–2499CrossRefGoogle Scholar
  19. 19.
    Martinez-Gomez MA, Villanueva-Camanas RM, Sagrado S, Medina-Hernandez MJ (2005) Electrophoresis 26:4116–4126CrossRefGoogle Scholar
  20. 20.
    Zhang YP, Zhang YJ, Gong WJ, Wang SM, Xue HY, Lee KP (2007) J Liq Chromatogr Rel Technol 30:215–234CrossRefGoogle Scholar
  21. 21.
    Gong WJ, Zhang YP, Choi SH, Zhang YJ, Lee KP (2006) Microchim Acta 156:327–335CrossRefGoogle Scholar
  22. 22.
    Montes RE, Gomez FA, Hanrahan G (2008) Electrophoresis 29:375–380CrossRefGoogle Scholar
  23. 23.
    Schappler J, Staub A, Veuthey JL, Rudaz S (2008) J Chrom A 1204:183–190CrossRefGoogle Scholar
  24. 24.
    Hanrahan G, Montes RE, Pao A, Johnson A, Gomez FA (2007) Electrophoresis 28:2853–2860CrossRefGoogle Scholar
  25. 25.
    Babar SME, Song EJ, Hasan MN, Yoo YS (2007) J Sep Sci 30:2311–2319CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Anna R. Lomasney
    • 1
  • Christelle Guillo
    • 1
  • Ashley M. Sidebottom
    • 1
  • Michael G. Roper
    • 1
    Email author
  1. 1.Department of Chemistry and BiochemistryFlorida State UniversityTallahasseeUSA

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