Metallobiomolecules play a critical role in various physiological processes, such as enzymatic activity. Their distribution in biological matrices like blood serum are crucial for understanding their biochemical functions in both health and disease [1]. However, analyzing metallobiomolecules remains challenging due to biomolecule instability during separation, potential metal dissociation, and low metallobiomolecule abundance [2]. While size exclusion chromatography coupled with inductively coupled plasma mass spectrometry (SEC-ICP-MS) has been used for metallobiomolecule analysis in blood, previous methods often monitored a limited number of elements or encountered difficulties with accurate quantification [3]. To address these limitations, a robust multielement SEC-ICP-MS method was developed, which integrates multiple improvements, including total element concentration determination, instrument sensitivity monitoring, metallobiomolecule fractionation, and column recovery determination with features for enhanced column recovery, all within a single sample analysis workflow.

This method allows for size fractionation of metallobiomolecules ranging from 500 Da to 150 kDa in blood serum without prior sample preparation. It enables the simultaneous determination of total element concentrations and the element distribution in each detected chromatographic peak for 12 essential and toxic elements (Ca, Mg, Co, Zn, Cu, Fe, Mn, Se, As, Ni, Pb, and Cd) in a 35-minute analysis. By introducing strategies for sample-to-sample instrument sensitivity monitoring and minimizing metal retention on the stationary phase, recoveries are maximized while reproducible and accurate results are readily evaluated and ensured. Total concentration and column recoveries exceed 80% and 85%, respectively, for most elements, supporting the method's robustness and suitability for trace-level analysis in complex biological matrices. Moreover, the method can be easily adapted for coupling with electrospray ionization tandem mass spectrometry (ESI-MS/MS) via a simple splitting system enabling the identification of native proteins and metallobiomolecules. This SEC-ICP-MS method overcomes key challenges in metallobiomolecule analysis, offering significant advancements for both research and clinical applications.

References
[1] H. Haraguchi, J. Anal. At. Spectrom., 2004, 19, 5.
[2] J. P. Barnett, D. J. Scanlan and C. A. Blindauer, Anal. Bioanal. Chem., 2012, 402, 3311–3322.
[3] S. Mounicou, J. Szpunar and R. Lobinski, Chem. Soc. Rev., 2009, 38, 1119–1138.