Abstract
This paper describes an analytical method for the determination of ceruloplasmin (Cp) in human serum. The method uses immunoaffinity chromatography and size-exclusion chromatography (SEC) to “purify” the serum sample prior to analysis of 63Cu and 65Cu by inductively-coupled plasma mass spectrometry (ICPMS). By removing the six most abundant proteins from serum with immunoaffinity chromatography and by using SEC to separate Cu bound by Cp from any free Cu that might be present in the serum sample, we demonstrated that SEC-ICPMS can accurately and reproducibly measure Cp in the ERM DA470 reference serum. Cp identification is based on retention time match of the unknown in the serum sample with the Cp external standard and the presence of 63Cu and 65Cu at a ratio of 2.2±0.1. This method was used to analyze a reference serum certified for Cp, 47 serum samples from four different diseases and a set of normal controls. The reference serum and a serum sample from a patient with myocardial infarction, as well as a Cp standard, were also analyzed by electrospray mass spectrometry to confirm the presence of Cp in the SEC fraction known to contain 63Cu.
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Acknowledgements
The authors thank Toshiaki Matsuda of Agilent Tokyo Analytical Division, Tokyo, Japan, for making available an ICPMS system for performing this research; Alex Apffel of Agilent Labs, Palo Alto, CA for making available an HPLC system with fraction collection and for assistance with the immunoaffinity chromatography process; and to Darlene Solomon and Brian Peter, also of Agilent Labs, for reviewing the manuscript prior to publication.
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Lopez-Avila, V., Sharpe, O. & Robinson, W.H. Determination of ceruloplasmin in human serum by SEC-ICPMS. Anal Bioanal Chem 386, 180–187 (2006). https://doi.org/10.1007/s00216-006-0528-9
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DOI: https://doi.org/10.1007/s00216-006-0528-9