Non-transferrin-bound iron determination in blood serum using microsequential injection solid phase spectrometry- proof of concept

Non-transferrin-bound iron (NTBI) is a group of circulating toxic iron forms, which occur in iron overload or health conditions with dysregulation of iron metabolism. NTBI is responsible for increased oxidative stress and tissue iron loading. Despite its relevance as a biochemical marker in several diseases, a standardized assay is still lacking. Several methods were developed to quantify NTBI, but results show high inter-method and even inter -laboratory variability. Thus, the development of a consistent NTBI assay is a major goal in the management of iron overload and related clinical conditions. In this work, a micro sequential injection lab-on-valve (mu SI-LOV) method in a solid phase spectrophotometry (SPS) mode was developed for the quantification of NTBI, using a bidentate 3,4-hydroxypyridinone (3,4-HPO) ligand anchored to sepharose beads as a chromogenic reagent. To attain SPS, the functionalized beads were packed into a column in the flow cell, and the analyte, NTBI retained as iron (III), formed a colored complex at the beads while eliminating the sample matrix. The dynamic concen-tration range was 1.62-7.16 mu mol L-1 of iron (III), with a limit of detection of 0.49 mu mol L-1 and a limit of quantification of 1.62 mu mol L-1. The proposed mu SI-LOV-SPS method is a contribution to the development of an automatic method for the quantification of the NTBI in serum samples.

Automated summary

Non-transferrin-bound iron (NTBI) is a group of toxic iron forms that circulate in conditions with iron overload or dysregulation of iron metabolism. The quantification of NTBI is still lacking a standardized assay due to high variability among methods and laboratories. This study developed a micro sequential injection lab-on-valve (mu SI-LOV) method using solid phase spectrophotometry (SPS) to quantify NTBI in serum samples. The proposed method achieved a dynamic concentration range of 1.62-7.16 μmol L^-1 iron (III), with a limit of detection of 0.49 μmol L^-1 and a limit of quantification of 1.62 μmol L^-1.