Iron Redox Speciation Analysis Using Capillary Electrophoresis Coupled to Inductively Coupled Plasma Mass Spectrometry (CE-ICP-MS)

Neuronal iron dyshomeostasis occurs in multiple neurodegenerative diseases. Changes in the Fe(II)/Fe(III) ratio toward Fe(II) is closely related to oxidative stress, lipid peroxidation, and represents a hallmark feature of ferroptosis. In particular for body fluids, like cerebrospinal fluid (CSF), reliable quantitative methods for Fe(II)/(III) redox-speciation analysis are needed to better assess the risk of Fe(II)-mediated damage in brain tissue. Currently in the field of metallomics, the most direct method to analyze both iron species is via LC-ICP-MS. However, this Fe(II)/(III) speciation analysis method suffers from several limitations. Here, we describe a unique method using capillary electrophoresis (CE)-ICP-MS for quantitative Fe(II)/(III) speciation analysis that can be applied for cell lysates and biofluid samples. Compared to LC, CE offers various advantages:(1) Capillaries have no stationary phase and do not depend on batch identity of stationary phases; (2) Replacement of aged or blocked capillaries is quick with no performance change; (3) Purge steps are effective and short; (4) Short sample analysis time. The final method employed 20mM HCl as background electrolyte and a separation voltage of +25 kV. In contrary to the LC-method, no complexation of Fe-species with pyridine dicarboxylic acid (PDCA) was applied, since it hampered separation. Peak shapes and concentration detection limits were improved by combined conductivity-pH-stacking achieving 3 mu g/L detection limit (3 s) at 13 nL injection volume. Calibrations from LOD-150 mu g/L were linear [r(2) [Fe(II)] = 0.9999, r(2) [Fe(III)] = 0.9951]. At higher concentrations Fe(II) curve flattened significantly. Measurement precision was 3.5% [Fe(II) at 62 mu g/L] or 2.2% [Fe(III) at 112 mu g/L] and migration time precision was 2% for Fe(III) and 3% for Fe(II), each determined in 1:2 diluted lysates of human neuroblastoma cells. Concentration determination accuracy was checked by parallel measurements of SH-SY5Y cell lysates with validated LC-ICP-MS method and by recovery experiments after standard addition. Accuracy (n = 6) was 97.6 +/- 3.7% Fe(III) and 105 +/- 6.6% Fe(II). Recovery [(a) + 33 mu g/L or (b) + 500 mu g/L, addition per species] was (a):97.2 +/- 13% [Fe(II)], 108 +/- 15% [Fe(III)], 102.5 +/- 7% (sum of species), and (b) 99 +/- 4% [Fe(II)], 101 +/- 6% [Fe(III)], 100 +/- 5% (sum of species). Migration time shifts in CSF samples were due to high salinity, but both Fe-species were identified by standard addition.