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Improved clean-up method for the enkephalins in plasma using immunoaffinity chromatography

T L Hendrickson1, G S Wilson

  • 1Department of Chemistry, University of Kansas, Lawrence 66045.

Journal of Chromatography. B, Biomedical Applications
|March 4, 1994
PubMed
Summary

A new immunoaffinity chromatography (IAC) method rapidly isolates enkephalins from plasma. This technique offers high recovery and a low detection limit, improving upon existing methods for enkephalin analysis.

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Area of Science:

  • Biochemistry
  • Analytical Chemistry

Background:

  • Enkephalins are endogenous opioid peptides with significant physiological roles.
  • Accurate quantification of enkephalins in biological samples like plasma is crucial for research.
  • Existing methods for enkephalin isolation often lack speed and selectivity.

Purpose of the Study:

  • To develop a rapid and selective immunoaffinity chromatography (IAC) method for isolating enkephalins from plasma.
  • To compare the performance of the novel IAC method with traditional solid-phase extraction (SPE).

Main Methods:

  • Enkephalin antibodies were generated using novel protein carriers and selected based on binding affinity and epitope specificity.
  • Plasma samples spiked with enkephalins were processed using the IAC column.
  • Eluted enkephalins were derivatized using naphthalene-2,3-dicarboxaldehyde and cyanide (NDA-CN).

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  • Separation and analysis were performed using reversed-phase liquid chromatography (RPLC).

    • Main Results:

    • The IAC method demonstrated high recovery rates (90%) for enkephalins from plasma with low variance (2-9%).
    • The limit of detection for the IAC method was determined to be 30 pmol.
    • The immunoaffinity column maintained performance over approximately 70 sample runs.

    Conclusions:

    • Immunoaffinity chromatography provides a rapid, selective, and robust method for enkephalin isolation from plasma.
    • The developed IAC method significantly outperforms existing solid-phase extraction techniques in terms of efficiency and sensitivity.
    • This optimized method facilitates accurate enkephalin analysis in complex biological matrices.