This iron redox speciation method is based on capillary electrophoresis-inductively coupled plasma mass spectrometry with sample stacking combined with short analysis in one run. The method quickly analyzes and provides low limits of quantification for iron redox species across a diverse range of tissues and biofluid samples.
Excessive iron results in free redox-active iron two-plus that can cause devastating effects within the cell, such as oxidative stress and cell deaths by lipid peroxidation known as ferroptosis. Quantitative measurements of ferrous and ferric iron is the key for a closer insight into these detrimental processes. This CE-ICP-Ms technique provides both:fast analysis and low quantification limits, which are both necessary for quantifying iron-redox species.
Begin by selecting a nebulizer with a low self aspirating volume and preparing the appropriate parts to mount the homemade interface. Connect two three-way female luer connectors with a male cone luer connector by connecting the left end of the lower three-way luer bar to the male connector and the male connector to the middle connection of the upper three-way luer. Next, put a one centimeter tube over the platinum wire and use a one centimeter silicone tube to connect the first tube and the nozzle of a male luer connector.
Fix the assembly to the middle connection of the lower three-way luer connector by screw rotation. Push a one centimeter tube over the outlet of the CE capillary and position at eight to nine centimeters from the end, then connect it to the nozzle of the male luer connector with a silicone tube. Put the whole assembly through the bar of the upper three-way T connector and fix the male luer connector and the left end of the female three-way luer connector by screw rotation.
Then fix the 25 centimeter silicone tube at the nozzle of a male luer connector and fix the whole assembly at the bottom of the bar from the lower three-way lure connector. Tightly push the one centimeter silicone tube, five millimeters over the end of the nebulizer. While the second male luer cone connector is plugged into the protruding part of the tube.
Move the interface part with the protruding CE capillary to the male cone at the nebulizer, then carefully insert the capillary through the male cone and the wider part of the nebulizer capillary. If necessary, adjust the length of the protruding capillary by unscrewing the left-hand male luer, moving the luer with attached tubes into the required position and reinstalling it. The upper female three-way luer connectors should fit tightly to the male cone.
When finished with the setup proceed with ICP-Ms. Calibration curves were created for iron three and iron two redox species. The limits of detection were 3.1 micrograms per liter for iron two and 3.2 micrograms per liter for iron three and linearity was demonstrated up to 150 micrograms per liter.
The analysis of the SH-SY5Y Cell Lysate showed a slightly slower migration for iron redox species due to the somewhat higher conductivity. The measured concentrations of iron three and iron two were 330 micrograms per liter and 84 micrograms per liter, respectively. Resulting in an iron two to three ratio of 0.25.
The most important step of this protocol is the careful insertion of the protruding CE capillary through the mounted interface and its suitable positioning related to the nebulizer capillary. The here presented methods may become a promising tool to deepen the insight into iron dependent molecular processes. Carefully designed studies are needed to validate if iron two-plus, iron three-plus quantification can serve as a potential biomarker to pinpoint tissue at risk for iron-mediated lethal damage in the context of neurodegenerative and cancer diseases.
