Cobalt speciation assay for human serum, Part I. Method for measuring large and small molecular cobalt and protein-binding capacity using size exclusion chromatography with inductively coupled plasma-mass spectroscopy detection

Abstract

A method utilizing size exclusion liquid chromatography (SEC) was developed to separate and quantify large molecular cobalt (Co) (e.g., albumin-Co) from cyanocobalamin (vitamin B₁₂) and small molecular Co (e.g., glutathione-Co and free Co) in human serum. Highly selective and sensitive detection using inductively coupled plasma–mass spectrometry was coupled with SEC to provide a method with reliable accuracy, precision, recoveries, stability, and a detection limit of 0.037 μg/L in undiluted serum. Other divalent metal cations known to compete with Co(II) for serum albumin-binding sites (such as iron, zinc, manganese, cadmium, copper, nickel, and lead) did not significantly alter Co(II) quantification. Co–protein binding capacity determination of individual serum samples indicated that addition of 2500 μg Co/L to undiluted human serum resulted in approximately 90% distribution to the large molecular Co peak, consistent with Co binding to high-affinity divalent metal binding sites on albumin. Since serum albumin binding partially sequesters biologically active Co(II) ions, this method provides an important tool for better understanding the kinetics and toxicology of Co compounds. Thus, the proposed method might play an important role in establishing Co dose–response relationships that affect the equilibrium concentrations of free ionic Co(II).

Keywords:

• cobalt binding capacity
• cobalt supplement
• cobalt-chromium alloy implants
• occupational cobalt exposure

Acknowledgements

The authors would like to acknowledge Ken Unice and Brooke Tvermoes for their helpful input and peer review of the manuscript.

Funding/conflict of interest

Two of the authors (RG, HG) are employed by Applied Speciation and Consulting, an analytical laboratory and metals chemistry consulting firm. Four of the authors (BK, BF, KT, DP) are employed by ChemRisk, a consulting firm that provides scientific advice to the government, corporations, law firms, and various scientific/professional organizations. ChemRisk has been engaged by DePuy Orthopedics, Inc., a manufacturer of prosthetic devices, some of which contain cobalt. This paper was prepared and written exclusively by the authors without review or comment by DePuy employees or counsel. It is likely that this work will be relied upon in medical research, nutrition research, and litigation. Some of the authors may be called upon to serve as expert witnesses. Funding for this paper and for the analytical work and method development was provided by DePuy, with additional funding provided by the two firms (ChemRisk and Applied Speciation and Consulting) that employ the authors.