Ability to determine the desferrioxamine‐chelatable iron fractions of nontransferrin‐bound iron using HPLC

Abstract

Iron is an essential element in human development. It is imperative for oxygen and electron transport and also for DNA and neurotransmitters synthesis. On the other hand, this metal is able to participate in Fenton's reaction that in turn leads to free radical damage. The most toxic fraction of iron - nontransferrin-bound iron and its part desferrioxamine-chelatable iron - can serve as an exquisite biomarker in the identification of iron imbalance. The goal of the present study was to devise a simple, repeatable, and inexpensive method for the determination of desferrioxamine-chelatable iron in serum blood samples. The assay procedure is based on desferrioxamine complex formation with iron ions followed to ferrioxamine and its quantitative measurement using RP-HPLC method. The desferrioxamine-chelatable iron was extracted from blood by centrifugation and SPE method. Chromatographic separation was performed at 40°C by step-form gradient elution using Cadenza CD-C18 column (150 × 4.6 mm id, particle size of 3.0 μm) connected with precolumn for contaminants removal. Gradient HPLC elution has been carried out with solvent A (10 mM Tris-HCl, pH 5.5) and solvent B (ACN). The flow rate was 1.2 mL/min, and the total separation time was 5 min. The linear quantitation range was 2.5-500 μM (r = 0.9973), and the LOD and LOQ were 0.42 and 1.29 μM, respectively. Proposed HPLC method allowed for the determination of desferrioxamine-chelatable iron fraction's of nontransferrin-bound iron, both in the buffer and the serum supplemented with iron ions as well as in the patients' serum samples with good results of precision and recovery. The developed method found to be sufficiently precise and reproducible for established conditions and after validation and may be used for routine assay of desferrioxamine-chelatable iron in biological samples.

Performance of Redox Active and Chelatable Iron Assays to Determine Labile Iron Release From Intravenous Iron Formulations

“…This assay as implemented also demonstrated the greatest assay dynamic range (∼50 μM to at least 2 mM) of the assays tested in this study. The assay was not optimized in this work to minimize the LOD given that the labile iron of the agent samples in this study were in the hundreds of μM range; indeed a sub‐μM LOD has been reported for a similarly implemented assay showing further improvement in LOD may be possible . However, the LOD was routinely achievable and more than sufficient for the scope of work described.…”

Differential Pulse Polarography Procedure for the Estimation of Deferoxamine in Pharmaceuticals

“…Various techniques have been used for the determination of deferoxamine include spectrophotometry 6 , 7 HPLC 8,9 .…”

The ability to determine the non-transferrin-bound iron and total iron in the human placenta using high-performance liquid chromatography method

“…the quantitative measurement was performed using the RP-hPlc method. chromatographic separation was performed by gradient elution using a cadenza cD-c18 column with solvent 10 mM tris-hcl and solvent acetonitrile-acN on the basis of a previously reported study by Koba et al [3]. the step-form gradient elution conditions were optimised experimentally with a total run time of 15 min and with gradient changes in time of the hPlc analysis as follows: 15% during 0-1 min, 45% during 1-5 min, and 15% during 5-15 min.…”

“…the damaging effect of NtBi is associated with its ability to take part in the Fenton reaction, which may result, among others, in cellular membrane damage and induce apoptosis [3]. Previous studies have reported that NtBi is not detected in the serum of the healthy population [4,12].…”

“…Furthermore, in the human body there is also another form of iron: non-transferrin-bound iron (NtBi). the appearance of NtBi and its component -desferrioxamine-chelatable iron (Dci) -can be used for rating this element in the body [3][4].…”