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 (µSI-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 concentration range was 1.62-7.16 µmol L-1 of iron (III), with a limit of detection of 0.49 µmol L-1 and a limit of quantification of 1.62 µmol L-1. The proposed µSI-LOV-SPS method is a contribution to the development of an automatic method for the quantification of the NTBI in serum samples.

A Robust Flow-Based System for the Spectrophotometric Determination of Cr(VI) in Recreational Waters.

A flow-based method for the spectrophotometric determination of chromium (VI) in recreational waters with different salinities was developed. Chromium can occur in the environment in different oxidation states with different related physiological properties. With regard to chromium, the speciation is particularly important, as the hexavalent chromium is considered to be carcinogenic. To achieve that purpose, the use of the diphenylcarbazide (DPC) selective colored reaction with the hexavalent chromium was the chosen strategy. The main objective was to develop a direct and simple spectrophotometric method that could cope with the analysis of different types of environmental waters, within different salinity ranges (fresh to marine waters). The potential interference of metal ions, that can usually be present in environmental waters, was assessed and no significant interferences were observed (<10%). For a complete Cr(VI) determination (three replicas) cycle, the corresponding reagents consumption was 75 µg of DPC, 9 mg of ethanol and 54 mg of sulfuric acid. Each cycle takes about 5 min, including the system clean-up. The limit of detection was 6.9 and 12.2 µg L−1 for waters with low and high salt content, respectively. The method was applied for the quantification of chromium (VI) in both fresh and marine water, and the results were in agreement with the reference procedure.

Sequential injection method for bi-parametric determination of iron and manganese in soil leachates.

The aim of this work was to develop a sequential injection (SI) method for the determination of the micronutrients iron and manganese, in soil leachates, as a tool to assess potential groundwater contamination. The described sequential injection method was based on the reaction of iron with chelator MRB12, a greener alternative chromogenic reagent, and the reaction of manganese with zincon, within a single manifold. The developed SI method enabled the determination of iron in the range 0.10-1.00 mg L-1, and manganese in the range 0.25-2.5 mg L-1 with a limit of detection of 0.08 mg L-1 for iron and 0.24 mg L-1 for manganese. The determination of both parameters was made in 6 minutes, in triplicate. The application to monitor laboratory scale soil core columns (LSSCs), as a simulation of the soil leaching process, proved its efficiency to assess potential contamination of ground waters. Iron and manganese contents were effectively analysed in two different scenarios to mimic the leaching process with rainwater and fertilizer.

New microfluidic paper-based analytical device for iron determination in urine samples.

Iron is an important micronutrient involved in several mechanisms in the human body and can be an important biomarker. In this work, a simple and disposable microfluidic paper-based analytical device (µPAD) was developed for the quantification of iron in urine samples. The detection was based on the colorimetric reaction between iron(II) and bathophenanthroline and the reduction of iron(III) to iron(II) with hydroxylamine. The developed µPAD enabled iron determination in the range 0.07-1.2 mg/L, with a limit of detection of 20 µg/L and a limit of quantification of 65 µg/L, thus suitable for the expected values in human urine. Additionally, targeting urine samples, the potential interference of the samples color was overcome by incorporating a sample blank assessment for absorbance subtraction. Stability studies revealed that the device was stable for 15 days prior to usage and that the formed colored product was stable for scanning up to 3 h. The accuracy of the developed device was established by analyzing urine samples (#26) with the developed µPAD and with the atomic absorption spectrometry method; the relative deviation between the two sets of results was below 9.5%.

Greener and wide applicability range flow-based spectrophotometric method for iron determination in fresh and marine water.

A flow-based method for the spectrophotometric determination of iron in recreational waters, both fresh and marine (variable salinity content), was developed. For that purpose, 3-hydroxy-4-pyrydinone ligand functionalized with an ether function was synthetized and used as chromogenic chelator (1-(3'-methoxypropyl)-2-methyl-3-benzyloxy-4-(1H)pyridinone - MRB13) for iron quantification. This water-soluble reagent was previously reported as a greener alternative to quantify iron, due to its low toxicity and a more environmental friendly synthesis. Furthermore, it also displayed a high affinity and specificity for iron. With the main objective of quantifying iron in a variety of water types (different matrices and iron content), two strategies were developed, one of them including on-line solid-phase extraction (SPE), and the other without resorting to a SPE process. Water matrix clean-up and iron enrichment was achieved using a nitrilotriacetic acid resin column. The potential interference of metal ions usually present in water samples was assessed and no significant interference (<10%) was observed. The limits of detection were 11 and 2.9 µg L-1 without and with SPE, respectively. For one determination (three replicates), the corresponding consumption of MRB13 is 90 µg, sodium hydroxide is 1.4 mg, and boric acid is 5.6 mg. The method was applied to certified water samples and the results were in agreement with certified values. The developed method was also applied to fresh and marine water, and recovery ratios of 103 ± 4 and 101 ± 7 without and with SPE, respectively, were achieved.

Use of an ether-derived 3-hydroxy-4-pyridinone chelator as a new chromogenic reagent in the development of a microfluidic paper-based analytical device for Fe(III) determination in natural waters.

This article reports on the development and validation of a disposable microfluidic paper-based analytical device (µPAD) for on-hand, in-situ, and cheap Fe(III) determination in natural waters complying with World Health Organization guidelines. The developed µPAD used 3-hydroxy-4-pyridinone (3,4-HPO) as a colour reagent due to its considerably lower toxicity than traditionally used iron analytical reagents. It was selected among a group of hydrophilic 3,4-HPO chelators containing ether-derived chains in their structure which were prepared using green methods. The relatively high water solubility of these chelators improved the detection limit and applicability as µPAD reagents. Under optimal conditions, the µPAD is characterised by a quantification range between 0.25 and 2.0 mg/L, a detection limit of 55 µg/L and 15 min of analysis time. The signal stability extends up to 4 h and the device is stable for at least one month. The reagent consumption is below 0.2 mg per analysis and the µPAD method was validated by analysis certified reference materials and by comparison with atomic absorption results (RD < 10%). The newly developed µPAD was successfully applied to the determination of iron in river, well and tap waters with no need of any prior sample pre-treatment.

Determination of iron(III) in water samples by microsequential injection solid phase spectrometry using an hexadentate 3-hydroxy-4-pyridinone chelator as reagent.

In this work, the hexadentate 3,4-hydroxypyridinone ligand was used as reagent for the spectrophotometric quantification of iron(III) in fresh and sea waters, using a micro sequential injection lab-on-valve (µSI-LOV) system in a solid phase spectrometry (SPS) mode. To implement SPS, thus eliminating the sample matrix, a packed column in the flow cell was used; the chosen sorbent was Nitrilotriacetic Acid Superflow resin (NTA). The possibility of performing an analytical curve resorting to just one standard was also demonstrated. The consumption of the hexadentante ligand was about 30 µg per determination and the effluent production lower than 2.5 mL. The dynamic concentration range was 0.45-9.0 µmol L-1, with a limit of detection of 0.13 µmol L-1 and limit of quantification 0.43 µmol L-1. The proposed µSI-LOV-SPS methodology was successfully applied to river, ground, estuarine, tap, and sea waters.

Microsequential injection lab-on-valve system for the spectrophotometric bi-parametric determination of iron and copper in natural waters.

The determination of iron and copper exploiting a microsequential injection lab-on-valve system with online spectrophotometric detection is described. A new, environmental friendly 3-hydroxy-4-pyridinone chelator, functionalized with a polyethylene glycol chain (MRB12) to improve water solubility, was used for iron determination. For copper determination, 1-(2-pyridylazo)-2-naphthol (PAN) was used. Different parameters affecting the formation of the complexes were studied, namely sample volume, reagent concentration, and buffer composition and concentration. The optimized conditions, 150µL of sample volume and 250mgL-1 of MRB12 for iron determination, and 200µL of sample and 120mgL-1 of PAN for copper determination, enabled an LOD of 15 and 18µgL-1 for iron and copper, respectively. The robustness of the developed procedure was assessed by the calculation of the relative standard deviation (RSD), 5% for iron determination and 2% for copper determination. The accuracy of the method was assessed comparing the results with two certified samples (RD<7.5%) and calculating recovery percentages with five river water samples (average<107%).

Iron speciation in natural waters by sequential injection analysis with a hexadentate 3-hydroxy-4-pyridinone chelator as chromogenic agent.

A sequential injection method for iron speciation in various types of natural waters was developed using a synthesised hexadentate 3-hydroxy-4-pyridinone chelator (CP256). The denticity of the ligand that allow formation of the corresponding iron(III) complex in a 1:1 stoichiometry proved to be highly advantageous, in comparison with parent bidentate, hydroxy-4-piridinone chelators, with a two fold increase of reaction sensitivity and over 65% decrease of the LOD. A solid phase extraction approach was employed to attain matrix elimination, facilitating iron(III) determination and application to high salinity waters. The combination with the total iron determination obtained by the direct reaction of the ligand resulted in iron speciation. Two detection spectrophotometric cells were tested, a conventional flow cell (CFC) and a liquid waveguide capillary cell (LWCC). The dynamic concentration ranges were 0.1-2 mg/L with the CFC detection and 0.005-0.1 mg/L with the LWCC, with limit of detection of 30 µg/L and 6 µg/L, respectively. The developed method was successfully applied to a variety of natural waters.

Micro solid phase spectrophotometry in a sequential injection lab-on-valve platform for cadmium, zinc, and copper determination in freshwaters.

This work describes the development of a solid phase spectrophotometry method in a µSI-LOV system for cadmium, zinc, and copper determination in freshwaters. NTA (Nitrilotriacetic acid) beads with 60-160 µm diameter were packed in the flow cell of the LOV for a µSPE column of 1 cm length. The spectrophotometric determination is based on the colourimetric reaction between dithizone and the target metals, previously retained on NTA resin. The absorbance of the coloured product formed is measured, at 550 nm, on the surface of the NTA resin beads in a solid phase spectrophotometry approach. The developed method presented preconcentration factors in the range of 11-21 for the metal ions. A LOD of 0.23 µg L(-1) for cadmium, 2.39 µg L(-1) for zinc, and 0.11 µg L(-1) for copper and a sampling rate of 12, 13, and 15 h(-1) for cadmium, zinc, and copper were obtained, respectively. The proposed method was successfully applied to freshwater samples.

Screening of cadmium and lead in potentially contaminated waters using a spectrophotometric sequential injection lab-on-valve methodology.

The present work describes the development of a µSI-LOV method for the simultaneous screening of cadmium and lead in potentially contaminated water samples. To attain the biparametric determination, dithizone was chosen as the spectrophotometric reagent as it forms a colored complex with both metal ions, at different pH conditions. The cadmium determination was attained in strong alkaline conditions (pH≈12); the lead determination was calculated by the difference with the determination of both metals in mild alkaline conditions (pH≈8). The colored complex was measured at 550 nm and the method presented a LOD of 34 µg L(-1) for cadmium and 56 µg L(-1) for lead, with a sample consumption of 20 µL per assay and a determination rate of 55 h(-1). The results obtained were in agreement with those obtained by FAAS. The developed method was efficiently applied to the screening of cadmium and lead in marine port waters.

Iron speciation by microsequential injection solid phase spectrometry using 3-hydroxy-1(H)-2-methyl-4-pyridinone as chromogenic reagent.

The speciation of iron using the newly synthesized 3-hydroxy-1(H)-2-methyl-4-pyridinone by solid phase spectrophotometry in a microsequential injection lab-on-valve (µSI-LOV-SPS) methodology is described. Iron was retained in a reusable column, Nitrilotriacetic Acid Superflow (NTA) resin, and the ligand was used as both chromogenic and eluting reagent. This approach, analyte retention and matrix removal, enabled the assessment of iron (III) and total iron content in fresh waters and high salinity coastal waters with direct sample introduction, in the range of 20.0-100 µg/L. with a LOD of 9 µg/L. The overall effluent production was 2 mL, corresponding to the consumption of 0.48 µg of 2-metil-3-hydroxy-4-pyridinone, 0.34 mg of NaHCO3, 16 mg of HNO3, 4.4 µg H2O2 and 400µL of sample. Four reference samples were analyzed and a relative deviation<10% was obtained; furthermore, several bathing waters (♯13) were analyzed using the developed method and the results were comparable to those obtained by atomic absorption spectrophotometry (relative deviations<6%).

Exploiting the use of 3,4-HPO ligands as nontoxic reagents for the determination of iron in natural waters with a sequential injection approach.

In this paper, the use of 3-hydroxy-4-pyridinone (3,4-HPO) chelators as nontoxic chromogenic reagents for iron determination is proposed. The potential application of these compounds was studied in a sequential injection system. The 3,4-HPO ligands used in this work were specially designed to complex iron(III) at physiologic pH for clinical applications. The developed sequential injection method enabled to study the reaction conditions, such as buffering and interferences. Then, to further improve the low consumption levels, a microsequential injection method was developed and effectively applied to iron determination in bathing waters using 3,4-HPO ligands. The formed iron complex has a maximum absorbance at 460 nm. The advantage of using minimal consumption values associated with sequential injection, together with the lack of toxicity of 3,4-HPO ligands, enabled to present a greener chemistry approach for iron determination in environmental samples within the range 0.10-2.00 mg Fe/L with a LOD of 7 µg/L. The overall effluent production was 350 µL corresponding to the consumption of 0.48 mg of 3,4-HPO ligand, 0.11 mg of NaHCO3, 0.16 mg of HNO3 and 50 µL of sample. Three reference samples were assessed for accuracy studies and a relative deviation <5% was obtained. The results obtained for the assessment of iron in inland bathing waters were statistically comparable to those obtained by the reference procedure.

Gas diffusion sequential injection system for the spectrophotometric determination of free chlorine with o-dianisidine.

A gas diffusion sequential injection system for spectrophotometric determination of free chlorine is described. The detection is based in the colorimetric reaction between free chlorine and a low toxicity reagent o-dianisidine. A gas diffusion unit is used to isolate free chlorine from the sample in order to avoid possible interferences. This feature results from the conversion of free chlorine to molecular chlorine (gaseous) with sample acidification. With minor changes in the operating conditions, two different dynamic ranges were obtained enhancing the application both to water samples and bleaches. The results obtained with the developed system were compared to the reference method, iodometric titration and proved not to be statistically different. A detection limit of 0.6mg ClO(-)/L was achieved. Repeatability was evaluated from 10 consecutive determinations being the results better than 2%. The two dynamic ranges presented different determination rates: 15h(-1) for 0.6-4.8mg ClO(-)/L (water samples) and 30h(-1) for 0.047-0.188g ClO(-)/L (bleaches).