Phyto-Facilitated Bimetallic ZnFe2O4 Nanoparticles via Boswellia carteri: Synthesis, Characterization, and Anti-Cancer Activity.

BACKGROUND: The growing dissatisfaction with the available traditional chemotherapeutic agents has enhanced the need to develop new methods for obtaining materials with more effective and safe anti-cancer properties. Over the past few years, the usage of metallic nanoparticles has been a target for researchers of different scientific and commercial fields due to their tiny sizes, environment-friendly properties, and a wide range of applications. To overcome the obstacles of traditional physical and chemical methods for the synthesis of such nanoparticles, a new, less expensive, and eco-friendly method has been adopted using natural existing organisms as a reducing agent to mediate the synthesis of the desired metallic nanoparticles from their precursors, a process called green biosynthesis of nanoparticles. OBJECTIVE: In the present study, zinc-iron bimetallic nanoparticles (ZnFe2O4) were synthesized via an aqueous extract of Boswellia carteri resin mixed with zinc acetate and iron chloride precursors, and they were tested for their anticancer activity. METHODS: Various analytic methods were applied for the characterization of the phyto synthesized ZnFe2O4, and they were tested for their anticancer activity against MDA-MB-231, K562, MCF-7 cancer cell lines, and normal fibroblasts. RESULTS: Our results demonstrate the synthesis of cubic structured bimetallic nanoparticles ZnFe2O4 with an average diameter of 10.54 nm. MTT cytotoxicity assay demonstrates that our phyto-synthesized ZnFe2O4 nanoparticles exhibited a selective and potent anticancer activity against K562 and MDA-MB-231 cell lines with IC50 values 4.53 µM and 4.19 µM, respectively. CONCLUSION: In conclusion, our biosynthesized ZnFe2O4 nanoparticles show a promising, environmentally friendly, and low coast chemotherapeutic approach against selective cancers with a predicted low adverse side effect toward normal cells. Further, in vivo, advanced animal research should be done to execute their applicability in living organisms.

Deep Eutectic Solvent-Based Microextraction of Lead(II) Traces from Water and Aqueous Extracts before FAAS Measurements.

Microextraction procedures for the separation of Pb(II) from water and food samples extracts were developed. A deep eutectic solvent composed of α-benzoin oxime and iron(III) chloride dissolved in phenol was applied as a phase separator support. In addition, this deep eutectic mixture worked as an efficient extractor of Pb(II). The developed microextraction process showed a high ability to tolerate the common coexisting ions in the real samples. The optimum conditions for quantitative recoveries of Pb(II) from aqueous extracts were at pH 2.0, conducted by adding 150 µL from the deep eutectic solvent. The quantitative recoveries were obtained with various initial sample volumes up to 30 mL. Limits of detection and limits of quantification of 0.008 and 0.025 µg L-1 were achieved with a relative standard deviation (RSD%) of 2.9, which indicates the accuracy and sensitivity of the developed procedure. Recoveries from the reference materials, including TMDA 64.2, TMDA 53.3, and NCSDC-73349, were 100%, 97%, and 102%, respectively. Real samples, such as tap, lake, and river water, as well as food samples, including salted peanuts, chickpeas, roasted yellow corn, pistachios, and almonds, were successfully applied for Pb(II) analysis by atomic absorption spectroscopy (AAS) after applying the developed deep eutectic solvent-based microextraction procedures.

Siderophores in plant root tissue: Tagetes patula nana colonized by the arbuscular mycorrhizal fungus Gigaspora margarita.

More than 70% of vascular plant species live in symbiosis with arbuscular mycorrhizal (AM) fungi. In addition to other effects this symbiosis is known for its significance for plant nutrition including iron. Fungal iron mobilization from soil is commonly dependent on siderophores. This study reports on a search for such iron-chelators in root tissue of Tagetes patula nana var. plena colonized by Gigaspora margarita. The AM colonized plants and uninoculated controls were grown under strictly axenic conditions. HPLC analyses of aqueous extracts from plant roots have provided clear evidence for the presence of a rhizoferrin type siderophore, named glomuferrin, in root tissue of mycorrhizal seedlings. Results from HPLC analytical work are seconded by molecular biological data: A BLASTp search revealed that the AM fungal species Gigaspora rosea, Rhizophagus irregularis (formerly Glomus intraradices), Glomus cerebriformis and Diversispora epigea encode a non-ribosomal peptide synthetase (NRPS)-independent siderophore synthase (NIS), which is homologous to the rhizoferrin synthetase of Rhizopus delemar. Thus this study indicates that the biosynthesis of rhizoferrin type siderophores such as glomuferrin (= bis-imidorhizoferrin) may be widespread in the AM symbiosis.

Catalytic degradation of p-nitrophenol by magnetically recoverable Fe3O4 as a persulfate activator under microwave irradiation.

In this study, Fe3O4 and microwave (MW) were combined to activate persulfate (PS) for the removal of organic matter, resulting in the enhanced degradation of p-nitrophenol (PNP) in solution. During the preparation of Fe3O4, the effect of sodium acetate was examined, and the results showed that the concentration of sodium acetate had little effect on the catalytic activity of the Fe3O4/PS/MW system but did have an effect on the Fe3O4 yield. In addition, with regards to the representative environmental factors, the degradation experiment showed that humic acid and the co-existing anions of chloride, sulfate, nitrate, and phosphate had little effects on p-nitrophenol removal; however, carbonate had a negative effect. In addition, the Fe3O4/PS/MW system performed well in the initial pH range of 3.0-9.0. According to the quenching experiment and electron paramagnetic resonance (EPR) detection, sulfate radicals and a minority of hydroxyl radicals play dominant roles in the degradation process. In addition, the role of Fe3O4 was confirmed to take part in the degradation process by X-ray photoelectron spectroscopy (XPS) analysis. Because of the good performance observed in the water matrices of tap water and the Songhua River, these results demonstrate the potential application of the Fe3O4/PS/MW system for wastewater treatment.

Pesticide-derived bright chlorine-doped carbon dots for selective determination and intracellular imaging of Fe(III).

Inspired by the conversion from organics or biomass to fluorescent carbon dots (C-dots), the use of pesticide 4-chlorophenol (4-CP) as a precursor to prepare C-dots has been reported. The as-prepared chlorine-doped C-dots display a brightly blue emission at ∼445 nm with ∼22.8% quantum yield. Also, the surface of C-dots enriches functional groups, such as phenolic hydroxyl and carboxylic acid, etc., which can capture ferric ion (Fe(III)), resulting in the quenching of blue fluorescence of C-dots through an inner filter effect. The quantitative assay for Fe(III) was therefore realized by this probe with a 0.36 µM detection limit in the 0.6-25 µM concentration range. Most significantly, the cytotoxicity on Hela cells indicates the 4-CP-derived C-dots have a negligible cytotoxicity. The C-dots were applied in detection in environmental samples and imaging in Hela cells of Fe(III), demonstrating their good applicability, low toxicity and good biocompatibility, and providing an alterative approach to totally eliminate the harm of chlorophenols (CPs).

A novel step-wise indirect bioleaching using biogenic ferric agent for enhancement recovery of valuable metals from waste light emitting diode (WLED).

Waste light-emitting diodes (WLED) are of major interest as they are a considered secondary source of valuable metals with a high potential for polluting the environment. To recover the valuable metals from WLEDs, various methods have been applied such as direct and indirect bioleaching. A novel step-wise indirect bioleaching process has been developed in this study for recycling valuable metals from WLEDs using adapted Acidithiobacillus ferrooxidans. The ferric ion concentration was controlled at 4-5 g/L with step-wise addition of biogenic ferric for faster bioleaching rate. The results indicated the negative effect of bacterial attachment in bioleaching of WLEDs. A direct bioleaching offers low copper, nickel, and gallium leach yields, while all metals' recovery improved with step-wise indirect bioleaching. At a pulp density of 20 g/L, the copper, nickel, and gallium recovery efficiency was 83%, 97%, 84%, respectively. In addition, leaching time was reduced to 15 days from 30 days. From a technological perspective, the study proved that step-wise indirect bioleaching by biogenic ferric resulted in maximum valuable metal recovery from WLEDs at a low cost and via a short, simple and environmentally-friendly process.

Analysis of Microbial Siderophores by Mass Spectrometry.

Siderophores represent important microbial virulence factors and infection biomarkers. Their monitoring in fermentation broths, bodily fluids, and tissues should be reproducible. Similar isolation, characterization, and quantitation studies can often have conflicting results, and without proper documentation of sample collection, data processing, and analysis methods, it is difficult to reexamine the data and reconcile these differences. In this Springer Nature Protocol, we present the procedure optimized for ferricrocin/triacetylfusarinine C extraction from biological material as well as for tissue fixation and cryosectioning for optical microscopy and for both elemental and molecular mass spectrometry imaging. Special attention is paid to siderophore data mining from conventional and product ion mass spectra, liquid chromatography, and mass spectrometry imaging datasets, performed here by our free software called CycloBranch.

Magnetically separable BiOBr/Fe3O4@SiO2 for visible-light-driven photocatalytic degradation of ibuprofen: Mechanistic investigation and prototype development.

The increasingly ubiquitous release of emerging refractory pollutants into water is a serious concern due to associated risks. In this study, mesoporous hierarchical BiOBr/Fe3O4@SiO2-a solvothermally synthesized visible-light-driven magnetic photocatalyst-not only exhibited fast kinetics (t1/2 = 8.7 min) in the photocatalytic degradation of ibuprofen in water but also achieved almost complete mineralization over a prolonged irradiation of 6 h. Various reactive species, including O2¯, OH, and H2O2, were detected, while the scavenging experiments revealed that eCB--mediated reactions and direct-hole oxidation are the major degradation routes. The magnetically recycled BiOBr/Fe3O4@SiO2 maintained ∼80% of its initial photocatalytic activity even after five consecutive cycles. The typically copresent wastewater constituents, including NOM and anions, inhibited the photocatalytic performance to varying extents, and hence necessitated an increase in the photocatalyst dosage to achieve complete ibuprofen degradation in real sewage. Based on the findings of batch experiments, the process was scaled up by developing a 5 L prototype photocatalytic reactor integrated with an electromagnetic separation unit. The results of prototype photocatalytic experiments were comparable to those of batch experiments, and an electromagnetic separation efficiency of ∼99% was achievable in 5 min.

A search for glomuferrin: a potential siderophore of arbuscular mycorrhizal fungi of the genus Glomus.

Most fungi are known to synthesize siderophores under iron limitation. However, arbuscular mycorrhizal fungi (AM fungi) have so far not been reported to produce siderophores, although their metabolism is iron-dependent. In an approach to isolate siderophores from AM fungi, we have grown plants of Tagetes patula nana in the presence of spores from AM fungi of the genus Glomus (G. etunicatum, G. mossae & unidentified Glomus sp.) symbiotically under iron limitation and sterile conditions. A siderophore was isolated from infected roots after 2-3 weeks of growth in pots containing low-iron sand with Hoagland solution. HPLC analysis of the root cell lysate revealed a peak at a retention time of 6.7 min which showed iron-binding properties in a chrome azurol S test. The compound was isolated by preparative HPLC and the structure was determined by high resolution electrospray FTICR-MS and GC/MS analysis of the hydrolysis products. From an observed absolute mass to charge ratio (m/z) of 401.11925 [M+H]+ with a relative mass error of ∆ = 0.47 ppm an elemental composition of C16H21N2O10 [M+H]+ was derived, suggesting a molecular weight of 400 Da for glomuferrin. Corresponnding ion masses of m/z 423.10 and m/z 439.06 were asigned to the Na-adduct and K-adduct respectively. A mass of 455.03836 confirmed an Fe- complex with an elemental composition of C16H19N2O10Fe (∆ = 0.15 ppm). GC/MS analysis of the HCl lysate (6 N HCL, 12 h) revealed 1,4 butanediamine. Thus the proposed structure of the isolated siderophore from Glomus species consisted of 1,4 butanediamine amidically linked to two dehydrated citrate residues, similar to the previously identified bis-amidorhizoferrin. Thus, the isolated siderophore (glomuferrin) is a member of the rhizoferrin family previously isolated from fungi of the Mucorales (Zygomycetes).

Optimization of an endovascular magnetic filter for maximized capture of magnetic nanoparticles.

To computationally optimize the design of an endovascular magnetic filtration device that binds iron oxide nanoparticles and to validate simulations with experimental results of prototype devices in physiologic flow testing. Three-dimensional computational models of different endovascular magnetic filter devices assessed magnetic particle capture. We simulated a series of cylindrical neodymium N52 magnets and capture of 1500 iron oxide nanoparticles infused in a simulated 14 mm-diameter vessel. Device parameters varied included: magnetization orientation (across the diameter, "D", along the length, "L", of the filter), magnet outer diameter (3, 4, 5 mm), magnet length (5, 10 mm), and spacing between magnets (1, 3 mm). Top designs were tested in vitro using 89Zr-radiolabeled iron oxide nanoparticles and gamma counting both in continuous and multiple pass flow model. Computationally, "D" magnetized devices had greater capture than "L" magnetized devices. Increasing outer diameter of magnets increased particle capture as follows: "D" designs, 3 mm: 12.8-13.6 %, 4 mm: 16.6-17.6 %, 5 mm: 21.8-24.6 %; "L" designs, 3 mm: 5.6-10 %, 4 mm: 9.4-15.8 %, 5 mm: 14.8-21.2 %. In vitro, while there was significant capture by all device designs, with most capturing 87-93 % within the first two minutes, compared to control non-magnetic devices, there was no significant difference in particle capture with the parameters varied. The computational study predicts that endovascular magnetic filters demonstrate maximum particle capture with "D" magnetization. In vitro flow testing demonstrated no difference in capture with varied parameters. Clinically, "D" magnetized devices would be most practical, sized as large as possible without causing intravascular flow obstruction.

Novel regeneration method for phosphate loaded granular ferric (hydr)oxide--a contribution to phosphorus recycling.

At a progressive rate, small wastewater treatment plants in rural areas need to be equipped with an additional phosphorus removal stage in order to achieve a good chemical status in the receiving natural water bodies. A conventional regeneration method for ferric (hydr)oxides such as phosphate specific adsorbents, which can be applied to remove and recover phosphorus in fixed bed filters, was investigated and improved. It was shown that a loss of up to 85% of the initial capacity can be observed when regeneration with 1 M NaOH is implemented. The losses are caused by surface blocking with different calcium-containing compounds as revealed by an EDX analysis. These blocking compounds could be removed completely with an additional acidic regeneration step at pH = 2.5. During the alkaline desorption that followed, complete phosphorus removal and a full recovery of the adsorption capacity were achieved for goethite-rich Bayoxide(®) E 33 HC (E33HC) and akaganéite-rich GEH(®) 104 (GEH). The regeneration procedure was repeated up to eight times without any signs of further decline in the phosphate adsorption capacity or any changes in the specific surface area or pore size distribution of the adsorbent. In contrast to GEH and E33HC, ferric hydroxide- and calcite-rich FerroSorp(®) Plus (FSP) was partly dissolved during acid treatment.

Facile preparation of a pH-sensitive nano-magnetic targeted system to deliver doxorubicin to tumor tissues.

We have developed a drug-loaded, pH-sensitive, nano-magnetic targeted system (DPNTS) for delivering doxorubicin (DOX) to tumor tissues through a facile route. Iron oxide (Fe3O4) nanoparticles were used as magnetically-responsive carriers, polyethyleneglycol (PEG) as the surface-modifying agent, and polyethyleneimine (PEI) as the drug-loading site whose primary amine reacts with the 13-carbonyl of DOX. The prepared DPNTS was within 20 nm and had good stability in dispersion and superparamagnetic properties. DOX was grafted to PEG/PEI@Fe3O4 at up to 85%. During in vitro release studies, nearly 81% DOX was released from DPNTS within 72 h at pH 4.5, compared with only 28% at pH 7.4.

Metachelins, mannosylated and N-oxidized coprogen-type siderophores from Metarhizium robertsii.

Under iron-depleted culture conditions, the entomopathogenic fungus Metarhizium robertsii (Bischoff, Humber, and Rehner) (= M. anisopliae) produces a complex of extracellular siderophores including novel O-glycosylated and N-oxidized coprogen-type compounds as well as the known fungal siderophores N(α)-dimethylcoprogen (NADC) and dimerumic acid (DA). Metachelin A (1), the most abundant component in the M. robertsii siderophore mixture, was characterized as a 1094 Da analogue of NADC that is O-glycosylated by ß-mannose at both terminal hydroxyl groups and N-oxidized at the dimethylated α-nitrogen. The mixture also contained a 1078 Da analogue, metachelin B (2), which lacks the N-oxide modification. Also characterized were the aglycone of 1, i.e., the N-oxide of NADC (3), and the monomannoside of DA (6). N-Oxide and O-glycosyl substituents are unprecedented among microbial siderophores. At high ESIMS source energy and at room temperature in DMSO, 1 underwent Cope elimination, resulting in loss of the N(α)-dimethyl group and dehydration of the α-ß bond. High-resolution ESIMS data confirmed that all tri- and dihydroxamate siderophores (1-6) complex with trivalent Fe, Al, and Ga. In a chrome azurol S assay, all of the M. robertsii siderophores showed iron-binding activity roughly equivalent to that of desferrioxamine B.

Novel DFO-functionalized mesoporous silica for iron sensing. Part 2. Experimental detection of free iron concentration (pFe) in urine samples.

Successful in vivo chelation treatment of iron(iii) overload pathologies requires that a significant fraction of the administered drug actually chelates the toxic metal. Increased mobilization of the iron(iii) in experiments on animals or humans, most often evaluated from urinary output, is usually used as an assessment tool for chelation therapy. Alternatively, the efficiency of a drug is estimated by calculating the complexing ability of a chelating agent towards Fe(iii). The latter is calculated by the pFe value, defined as the negative logarithm of the concentration of the free metal ion in a solution containing 10 µM total ligand and 1 µM total metal at a physiological pH of 7.4. In theory, pFe has to be calculated taking into account all the complexation equilibria involving the metal and the possible ligands. Nevertheless, complexation reactions in complex systems such as serum and urine may hardly be accurately modelled by computer software. The experimental determination of the bioavailable fraction of iron(iii) in biological fluids would therefore be of the utmost relevance in the clinical practice. The efficiency of the therapy could be more easily estimated as well as the course of overload pathologies. In this context, the aim of the present work was the development of a sensor to assess the free iron directly in biological fluids (urine) of patients under treatment with chelating agents. In the proposed device (DFO-MS), the strong iron chelator deferoxamine (DFO) is immobilized on the MCM-41 mesoporous silica. The characterization of the iron(iii) sorption on DFO-MS was undertaken, firstly in 0.1 M KNO3, then directly in urine samples, in order to identify the sorption mechanism. The stoichiometry of the reaction in the solid phase was found to be: with an exchange constant (average value) of log ßex = 40(1). The application of DFO-MS to assess pFe in SPU (Simulating Pathology Urine) samples was also considered. The results obtained were very promising for a future validation and subsequent application of the sensor in samples of patients undergoing chelation therapy.

Novel DFO-SAM on mesoporous silica for iron sensing. Part I. Synthesis optimization and characterization of the material.

The synthesis and the physico-chemical characterisation of a novel solid phase, designed for iron(iii) sorption, are presented. The solid (indicated in the following as DFO-SAMMS) is made with a hydroxamate siderophore, the deferoxamine (DFO), covalently bound on a self-assembled monolayer on mesoporous silica (SAMMS). The data demonstrate that the DFO molecules are bound to the solid material, grafted on the surface and do not enter the silica pores. A new one-pot synthesis is presented in which DFO is dissolved in DMSO, and left to react with GPTMS with stirring overnight. In the same mixture, SAMMS is added to get the final product. The optimisation of experimental conditions of this novel one-pot synthesis is presented, with results indicating that a temperature of 90 °C, for the reaction between DFO and GPTMS, and the use of MCM-41 silica are the most convenient conditions. The kinetics of sorption reveals that the iron uptake is relatively fast, around 100 min at pH = 2.5, and from the sorption profile of iron(iii), the estimated capacity of the product obtained under optimized conditions was higher than 0.3 mmol g(-1). The results found in the present research are very promising for application in real biological samples.

Magnetophoresis of iron oxide nanoparticles at low field gradient: the role of shape anisotropy.

Magnetophoresis of iron oxide magnetic nanoparticle (IOMNP) under low magnetic field gradient (<100 T/m) is significantly enhanced by particle shape anisotropy. This unique feature of magnetophoresis is influenced by the particle concentration and applied magnetic field gradient. By comparing the nanosphere and nanorod magnetophoresis at different concentration, we revealed the ability for these two species of particles to achieve the same separation rate by adjusting the field gradient. Under cooperative magnetophoresis, the nanorods would first go through self- and magnetic field induced aggregation followed by the alignment of the particle clusters formed with magnetic field. Time scale associated to these two processes is investigated to understand the kinetic behavior of nanorod separation under low field gradient. Surface functionalization of nanoparticles can be employed as an effective strategy to vary the temporal evolution of these two aggregation processes which subsequently influence the magnetophoretic separation time and rate.

A novel electrochemical process for the recovery and recycling of ferric chloride from precipitation sludge.

During wastewater treatment and drinking water production, significant amounts of ferric sludge (comprising ferric oxy-hydroxides and FePO4) are generated that require disposal. This practice has a major impact on the overall treatment cost as a result of both chemical addition and the disposal of the generated chemical sludge. Iron sulfide (FeS) precipitation via sulfide addition to ferric phosphate (FePO4) sludge has been proven as an effective process for phosphate recovery. In turn, iron and sulfide could potentially be recovered from the FeS sludge, and recycled back to the process. In this work, a novel process was investigated at lab scale for the recovery of soluble iron and sulfide from FeS sludge. Soluble iron is regenerated electrochemically at a graphite anode, while sulfide is recovered at the cathode of the same electrochemical cell. Up to 60 ± 18% soluble Fe and 46 ± 11% sulfide were recovered on graphite granules for up-stream reuse. Peak current densities of 9.5 ± 4.2 A m(-2) and minimum power requirements of 2.4 ± 0.5 kWh kg Fe(-1) were reached with real full strength FeS suspensions. Multiple consecutive runs of the electrochemical process were performed, leading to the successful demonstration of an integrated process, comprising FeS formation/separation and ferric/sulfide electrochemical regeneration.

Linear fusigen as the major hydroxamate siderophore of the ectomycorrhizal Basidiomycota Laccaria laccata and Laccaria bicolor.

A screening for siderophores produced by the ectomycorrhizal fungi Laccaria laccata and Laccaria bicolor in synthetic low iron medium revealed the release of several different hydroxamate siderophores of which four major siderophores could be identified by high resolution mass spectrometry. While ferricrocin, coprogen and triacetylfusarinine C were assigned as well as other known fungal siderophores, a major peak of the siderophore mixture revealed an average molecular mass of 797 for the iron-loaded compound. High resolution mass spectrometry indicated an absolute mass of m/z = 798.30973 ([M + H](+)). With a relative error of Δ = 0.56 ppm this corresponds to linear fusigen (C33H52N6O13Fe; MW = 797.3). The production of large amounts of linear fusigen by these basidiomycetous mycorrhizal fungi may possibly explain the observed suppression of plant pathogenic Fusarium species. For comparative purposes Fusarium roseum was included in this study as a well known producer of cyclic and linear fusigen.

Decontamination of spent iron-oxide coated sand from filters used in arsenic removal.

Sand filters devised with iron-rich adsorbents are extensively promoted and deployed in the arsenic-prone south and south-east Asian countries (e.g., Bangladesh). The approach offers superior performance in removing arsenic while the spent sludge from the sand filters is an issue of concern due to the possibility of toxic releases after being discarded. In this work, a new technique is proposed for the treatment of spent iron-oxide coated sand (IOCS) from filters used in arsenic removal. Chelant-washing of the arsenic-loaded IOCS is combined with the solid phase extraction treatment to accomplish the objective. The unique point of the proposed process is the cost-effective scheme, which includes the option of recycling of the washing solvent beside the decontamination of the spent arsenic-rich sludge.

Development and validation of a LC method for the separation and determination of the anticancer-active Fe(III) (4-methoxy-salophene) using the new second-generation monolith.

LC method with the newly introduced second-generation monolithic silica RP-18e column has been developed for the separation of Fe(III) (salophene) and four methoxy-substituted Fe(III) (salophene) complexes. The method has been validated for the quantitation of Fe(III) (4-OMe-salophene), a highly active anticancer substance in vitro, bound to serum albumin. Our routinely used high-resolution continuum-source atomic absorption spectroscopy method based on the determination of the central iron atom was unsuitable in this case because serum originally contains significant amounts of iron as revealed by a blank sample of serum albumin. The developed LC method depends on detecting the whole complex rather than the bound iron. Two morphologically different first- and second-generation HPLC monolithic columns have been compared for this purpose. The newly introduced second-generation monolithic silica column Chromolith(®) HighResolution RP-18e column (100 × 4.6 mm, Merck) separated the mixture successful within 13 min. A mobile phase consisting of 25 mM phosphate buffer pH 3/methanol (60:40, v/v) was used at a flow rate of 1 mL/min. The dynamic linear working range of the calibration curve for Fe(III) (4-OMe-salophene) was found to be between 1 and 200 µg/mL. Detection and quantitation limits were 0.3 and 1 µg/mL, respectively.