## Reference  
1 Mounicou, S.; Szpunar, J.; Lobinski, R. Metallomics: The concept and methodology. _Chem. Soc. Rev._**2009**, _38_, 1119-1138. Reviewing Determination Methods for \"Free Cu\" and Fe(II)/Fe(III) Ratio and Development of New and Fast Speciation Techniques for Reliable Measurements of \"Free Cu\" and Fe(II)Fe(III) Ratio in Various Samples  
B. Michalke  
Helmholtz Center Munich--German Research Center for Environmental Health, Research Unit Analytical BioGeoChemistry, Ingolstadter Landstr. 1, 85764 Neuherberg, Germany; bernhard.michalke@helmholtz-muenchen.de  
Trace elements (or trace metals) are minerals present in living tissues in small amounts. Some of them are known to be essential, others may be essential aside from the remaining elements, which are considered to be nonessential. Trace elements function primarily as catalysts in enzyme systems; some metallic ions, such as iron and copper, participate in oxidation-reduction reactions in energy metabolism. Iron, as a constituent of hemoglobin and myoglobin, also plays a vital role in the transport of oxygen. However, as copper and iron can show different oxidation states--i.e., they can be redox-active--both elements are probably causative in pathologic disease mechanisms of an extended set of severe illnesses. Such diseases include (for iron) forms of cancers, such as prostate cancer and melanoma, as well as ferrotosis, which is a newly discoveredpeculiar form of non-apoptotic cell death, and (for copper) Menkes and Wilson's disease. Menkes disease is a congenital metabolic disorder based on a copper metabolic disfunction. Wilson's disease is a rare inherited disorder that causes copper to accumulate in your liver, brain, and other vital organs. For understanding pathogenesis mechanism and diagnosis purposes, it is of vital importance to know the quantitative level either of free copper or quantitatively the Fe(II) and Fe(III) concentrations to quantify the cellular Fe(II)Fe(III) ratio. Several methods have been published providing different sensitivity, selectivity, and time-spending/saving procedures, for both free copper determination and iron redox state. This contribution summarizes these methods, highlighting the strengths and limitations of each method. For Cu, the \"difference\" method is the common clinical method for determining extractable Cu levels in serum, but also an IP-SCX-ICP-sf-MS method is reviewed, which is providing differentiation of Cu(I) and Cu(II), but also (Fe(II) vs. Fe(III). Molecular sieves and measurement of filtrate by ICP-MS or a newly published simple LP-SCX-ICP-MS method will be shortly evaluated. Finally, this lecture introduces a new SCX-ICP-MS or ICP-OES based determination method for free Cu determination, avoiding identified limitations from previous methods. This new approach also allows parallel monitoring of sulfur-compounds (mainly proteins), in this way proving the free Cu signal is not co-eluting with (not bound to) S-ligands (proteins). For iron redox speciation, only semi-quantitative dyes are in use for Fe(II). The Fe(II)/(III) ratio thus cannot be estimated from that approach. To overcome this lack, we developed an innovative CE-ICP-MS technique that allows the simultaneous quantification of redox-active Fe(II) and redox-inactive Fe(III) ions, aside from total iron. Via this method, we found that amyloid precursor protein suppression results in a robust accumulation of Fe(II)-driven lipid peroxidation, sensitizing cancer cells to cell death by ferprotosis. This method was further modified to quantitatively distinguish ferritin-bound iron from other iron species, enabling us to calculate the Fe-load of ferritin in the respective sample. Sequentially, in a second analytical run, the cellular GSH:GSSG ratio in the same cell lysate is assessed. As a proof of principle, the application to several different cell lysate types is shown, as well as the respective changes in Fe(II)/(III) ratio due to cellular knock-downs or iron regulators.