Abstract
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 20 mM 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 μg/L detection limit (3σ) at 13 nL injection volume. Calibrations from LOD—150 μg/L were linear [r2[Fe(II)] = 0.9999, r2[Fe(III)] = 0.9951]. At higher concentrations Fe(II) curve flattened significantly. Measurement precision was 3.5% [Fe(II) at 62 μg/L] or 2.2% [Fe(III) at 112 μg/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 μg/L or (b) +500 μg/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.
Highlights
Nowadays within neurodegeneration research it is most evident that iron-mediated oxidative stress (OS) and lipid peroxidation (LPO) plays a crucial role in multiple neurodegenerative brain disorders, such as Alzheimer’s and Parkinson’s disease (Hare et al, 2015, 2018; Ashraf et al, 2018)
We revealed that liquid chromatography-coupled-to-inductivelycoupled plasma mass spectrometry (LC-Inductively Coupled Plasma Mass Spectrometry (ICP-MS)) represents a suitable Fe(II)/Fe(III) speciation analysis method that provides good figures of merit using only 8–10 min analysis time per sample (Solovyev et al, 2017)
Based on our previous experience with LC-ICP-MS, we first started with a 50 mM ammonium citrate electrolyte containing 7 mM pyridine dicarboxylic acid (PDCA), pH 4.2 (Solovyev et al, 2017)
Summary
Nowadays within neurodegeneration research it is most evident that iron-mediated oxidative stress (OS) and lipid peroxidation (LPO) plays a crucial role in multiple neurodegenerative brain disorders, such as Alzheimer’s and Parkinson’s disease (Hare et al, 2015, 2018; Ashraf et al, 2018). We developed a CE-ICP-MS based method for the quantification of Fe(II)/Fe(III) redox species because of several advantages of CE over other techniques such as LC: (1) Capillaries have no stationary phase and depend (nearly) not on batch identity; (2) Aged or blocked CE columns can be replaced quickly without altering performance; (3) Purge steps between samples are effective and quick resulting in a shorter analysis time per sample. Our first speciation analysis approach was derived from LC eluents (Solovyev et al, 2017) serving as electrolytes, where 50 mM ammonium citrate, 7.0 mM PDCA, pH = 4.2 was the optimal condition to keep redox species stabilized and provided good separation for the ionic-Fe(II)/(III)PDCA complexes by cation exchange chromatography This electrolyte served as background electrolyte in capillary, in inlet vial and as outlet/sheath electrolyte at CE-ICP-MS interface and separation voltage was set to + 25 kV. Due to the nebulizer’s aspiration, an auxiliary flow was introduced
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