Colorimetric and fluorometric determination of uric acid by a suspension-based assay using enzyme-immobilized micro-sized particles.

Daily monitoring of serum uric acid levels is very important to provide appropriate treatment according to the constitution and lifestyle of individual hyperuricemic patients. We have developed a suspension-based assay to measure uric acid by adding a sample solution to the suspension containing micro-sized particles immobilized on uricase and horseradish peroxidase (HRP). In the proposed method, the mediator reaction of uricase, HRP, and uric acid produces resorufin from Amplex red. This resorufin is adsorbed onto enzyme-immobilized micro-sized particles simultaneously with its production, resulting in the red color of the micro-sized particles. The concentration of resorufin on the small surface area of the microscopic particles achieves a colorimetric analysis of uric acid with superior visibility. In addition, ethanol-induced desorption of resorufin allowed quantitative measurement of uric acid using a 96-well fluorescent microplate reader. The limit of detection (3σ) and RSD (n = 3) were estimated to be 2.2 × 10-2 µg/mL and ≤ 12.1%, respectively. This approach could also be applied to a portable fluorometer.

A sensitive paper-based vapor-test kit for instant formalin detection in food products.

A colorimetric sensing method based on a paper-based vapor-test kit was successfully developed for the selective and sensitive real-time monitoring of formalin in food samples. The device was specifically designed to efficiently extract and detect formalin simultaneously. A microcentrifuge tube was used as the sample solution container, with the inner cap serving as the reaction and detection zone. Formalin was converted into gaseous formaldehyde through controlled heating, which was then extracted and collected on a filter paper coated with Nash's reagent. The color change on paper was used for formalin quantification using a smartphone for detection and image analysis. Under optimal conditions, our method provided a linear range of 0.5-75 mg L-1 with a detection limit of 0.11 mg L-1. This method effectively determined formalin in fresh food and vegetable samples, with recoveries ranging from 92 to 111%, demonstrating comparable accuracy to the standard method for practical food quality control and safety.

Trace-Level Determination of Triazole Fungicides Using Effervescence-Assisted Liquid-Liquid Microextraction Based on Ternary Deep Eutectic Solvent Prior to High-Performance Liquid Chromatography.

A simple and sensitive preconcentration method, namely, effervescence-assisted liquid-liquid microextraction based on the ternary deep eutectic solvent method, was developed for enrichment of triazole fungicide residues prior to their determination by high-performance liquid chromatography coupled with UV detection. In this method, a ternary deep eutectic solvent (as extractant) was prepared by combination of octanoic acid, decanoic acid, and dodecanoic acid. The solution was well dispersed with sodium bicarbonate (as effervescence powder) without using auxiliary devices. In order to obtain relatively high extraction efficiency, analytical parameters were investigated and optimized. Under optimum conditions, the proposed method showed good linearity within the range of 1-1000 µg L-1 with a coefficient for determination (R2) greater than 0.997. The low limits of detection (LODs) were in the range of 0.3-1.0 µg L-1. The precisions were assessed from the relative standard deviations (RSDs) of retention time and peak area obtained from intra- (n = 3) and inter-day (n = 5 × 5) experiments, which were greater than 1.21 and 4.79%, respectively. Moreover, the proposed method provided high enrichment factors ranging from 112 to 142 folds. A matrix-match calibration method was used for analysis of real samples. Finally, the developed method was successfully applied for determination of the triazole fungicide in environmental water (near agricultural area), honey, and bean samples, and it represents a promising alternative method for analysis of triazoles. The recoveries of the studied triazoles were obtained in the range of 82-106% with an RSD less than 4.89.

Hydrophilic interaction chromatography-type sorbent prepared by the modification of methacrylate-base resin with polyethyleneimine for solid-phase extraction of polar compounds.

Hydrophilic interaction chromatography (HILIC)-type sorbents were newly developed for the solid-phase extraction (SPE) of polar compounds. Two methacrylate-base resins with different cross-linking monomers and pore properties were synthesized, and three polyethyleneimines (PEIs) with different molecular weights were modified onto each base resin. In both cases, PEIs with a molecular weight of 10,000 (PEI-10,000) exhibited the highest adsorption properties for polar compounds (uracil, uridine, adenosine, cytidine, and guanosine). To control the water-enriched layer at the surface of the PEI-10,000-modified sorbents, the additive amount of PEI-10,000 in the modified reaction was also optimized. When 10 times the amount of PEI-10,000 to each base resin was added, an improvement in adsorption property was observed. Moreover, the use of a nonaqueous sample solution (100% acetonitrile) during the sample loading process drastically improved adsorption, especially for uracil (about 80%) and adenosine (100%). These results indicate that the formation of a strong water-enriched layer at the surface of sorbents with an effective expression of hydrophilic interaction was an important factor in the adsorption properties of polar compounds in HILIC mode-SPE.

Novel chemically cross-linked self-molding particulate sorbents as solid-phase extraction media.

Here, we describe novel, chemically cross-linked, self-molding particulate polymer sorbents that are utilized as a molding-type solid-phase extraction medium (M-SPEM), which exhibits high permeability and rigidness. To fabricate such M-SPEM, first, polyethyleneimine (PEI)-modified reversed-phase (RP)-type particulate sorbents were synthesized, thereafter, they were chemically cross-linked by a polymer having many epoxy groups together with additional PEI. By optimizing the binding conditions of the particulate sorbents, the resultant M-SPEM has almost the same adsorption properties as the corresponding unmolded particulate sorbent for some polar (e.g., uracil and adenine) compounds. The binding technique proposed here is expected to facilitate the fabrication of molding-type sorbents and improve the performance of the SPE procedure.

Electrochemical detection of methyl parathion using calix[6]arene/bismuth ferrite/multiwall carbon nanotube-modified fluorine-doped tin oxide electrode.

A highly sensitive electrochemical sensor using a calix[6]arene/bismuth ferrite/multiwall carbon nanotube-modified fluorine-doped tin oxide electrode (CA6/BFO/MWCNTs/FTO) was fabricated for the detection of methyl parathion. The MWCNTs, BFO, and CA6 were consecutively cast onto the FTO electrode surface to enhance the surface area, electron transfer, and selectivity of sensors. The electrochemical behavior of CA6/BFO/MWCNTs/FTO was studied via cyclic voltammetry and electrochemical impedance spectroscopy. MP was detected via cyclic voltammetry in a phosphate buffer solution at pH 7.0. The working principle of the sensor involves a linear decrease in the anodic peak current of BFO with increasing MP concentration. The linear working ranges are 0.005-0.05 nM and 0.07-1.5 nM. The CA6/BFO/MWCNTs/FTO sensor provides a low detection limit (S/N = 3) of 5 pM and a high electrochemical sensitivity of 1.23 A µM-1 cm-2. The fabricated sensor was successfully applied to assess the presence and amount of MP in vegetables and fruits (recoveries of 82.0-106.8%), with results comparable to high-performance liquid chromatography.

Simultaneous Preconcentration of Triazole Fungicide Residues Using In-Situ Coacervative Extraction Based on a Double-Solvent Supramolecular System Prior to High Performance Liquid Chromatographic Analysis.

An in situ coacervative extraction (IS-CAE) based on a double-solvent supramolecular system coupled to liquid-liquid microextraction is investigated for extraction and enrichment of triazole fungicides. The formation of a double-solvent supramolecular system was generated by in situ formation and used as an extraction solvent for the coacervative extraction method. No disperser solvent was required. This new double-solvent supramolecular system has a higher extraction ability than any of its components alone. The different factors that could affect the extraction capability were studied and optimized, including the type of double extractant and its volume, salt addition, vortex time, and centrifugation time. Under optimum extraction conditions, this method provides high enrichment factors (EFs) of 73-318 with low limits of detection (LODs) of 0.3-1 µg L-1 and limits of quantitation (LOQs) of 1-3 µg L-1. In addition, the proposed method was prosperously applied for the determination of triazole fungicides in water, fruit juice, and soy milk samples.

Dual determination of nitrite and iron by a single greener sequential injection spectrophotometric system employing a simple single aqueous extract from Areca catechu Linn. serving as a natural reagent.

Dual determination of nitrite and iron was proposed by using a single greener sequential injection (SI) spectrophotometric system employing a simple single aqueous extract from Areca catechu Linn. The extract served as a natural reagent to replace N-(1-naphthyl)ethylenediamine (NED) of the Griess reagent with nitrite and 1,10-phenanthroline with iron. The color products possessed analytical wavelengths at 430 and 560 nm, respectively. Conditions for the SI procedure were optimized using a univariate experimental design. Calibration ranges were up to 5.0 mg L-1 and 10.0 mg L-1 with limits of detection (LODs) of 0.04 mg L-1 and 0.05 mg L-1 for nitrite and iron(iii), respectively, and relative standard deviations (RSDs) being less than 3%. Recoveries of spiked standard nitrite and iron(iii) at 0.3 mg L-1 and 0.5 mg L-1 in water samples were 88 to 104% and 84 to 109%, respectively. The developed method successfully achieved dual determination of nitrite and total iron agreeing at a 95% confidence level with the reference methods of the conventional Griess assay and flame atomic absorption spectrometry (FAAS), respectively. The proposed method utilized locally available material from plants and serves the UN-SDGs.

Preparation and evaluation of molding-type solid-phase extraction media binding with commercially available adhesives.

A fabrication method of molding-type solid-phase extraction media (M-SPEM) bound with commercially available adhesive is presented. Six pieces of M-SPEM were prepared by heating each kneaded product of a particulate sorbent and an adhesive inserted into a six-hole cylindrical mold for hardening under an open system and normal pressure. The particulate sorbent contained in M-SPEM was divinylbenzene-based reversed-phase mode solid-phase extractants that we have reported. An examination of several adhesives showed that the moldability of M-SPEM depended on the composition and properties of the adhesive. The optimized procedure can be used to prepare an M-SPEM containing an 85 wt% particulate sorbent (particulate sorbent/adhesive, 100 mg/17 mg; particle diameter, 90-150 µm), and the M-SPEM has a specific surface area of about 500 m2/g. The established procedure in this study can bind particulate sorbents together, which showed almost no reductions in the adsorption property and liquid permeability compared with those of the particulate sorbent.

Development of a fluorescence microplate reader using an organic photodiode array with a large light receiving area.

We developed a small fluorescence microplate reader with an organic photodiode (OPD) array. The OPD array has nine OPDs that have a large light receiving area (9.62 mm2 per one OPD). Since the OPD array is fabricated on a flat glass plate, it can be placed just below microwells and can detect fluorescence emitted through the entire surface of the microwell bottom. The analytical performance of the developed plate reader was evaluated by measuring an aqueous solution of resorufin. The limit of detection (LOD) for resorufin (0.01-0.05 µM) was lower than that obtained with a plate reader equipped with nine inorganic photodiodes developed in a previous study (0.30 µM) and a commercially available microplate reader (0.16 µM). These results indicate that the large light receiving area improves the detection performance of the system. In addition, the developed reader was successfully used to quantify immunoglobulin A (IgA) in human saliva. The LOD for IgA was estimated to be 1.2 ng/mL, which is low enough to objectively evaluate human stress.

Lead Assays with Smartphone Detection Using a Monolithic Rod with 4-(2-Pyridylazo) Resorcinol.

A monolithic rod of polyurethane foam-[4-(2-pyridylazo) resorcinol] (PUF-PAR) as a simple chemical sensor for lead assays with smartphone detection and image processing was developed. With readily available simple apparatus such as a plastic cup and a stirrer rod, the monolithic PUF rod was synthesized in a glass tube. The monolithic PUF-PAR rod could be directly loaded by standard/sample solution without sample preparation. A one-shot image in G/B value from a profile plot in ImageJ for a sample with triplicate results via a single standard calibration approach was obtained. A linear single standard calibration was: [G/B value] = -0.038[µg Pb2+] + 2.827, R2 = 0.95 for 10-30 µg Pb2+ with a limit of quantitation (LOQ) of 33 µg L-1. The precision was lower than 15% RSD. The proposed method was tested by an assay for Pb2+ contents in drinking water samples from Bangkok. The results obtained by the proposed method agree with those of ICP-OES and with 100-120% recovery, demonstrating that the method is useful for screening on-site water monitoring.

Determination of Lead Employing Simple Flow Injection AAS with Monolithic Alginate-Polyurethane Composite Packed In-Valve Column.

A simple flow injection FlameAAS for lead determination with an alginate-polyurethane composite (ALG-PUC) monolithic in-valve column has been developed. The ALG-PUC monolithic rod was prepared by mixing methylene diphenyl diisocyanate with polyol and sodium alginate with the ratio of 2:1:1 by weight for a 5 min polymerization reaction. It was then put into a column (0.8 cm i.d × 11 cm length) situated in a switching valve for the FI set up. A single standard calibration could be obtained by plotting the loaded µg Pb2+ vs. FI response (absorbances). The loaded µg Pb2+ is calculated: µg Pb2+ = FRload × LT × CPb2+, where the FR load is the flow rate of the loading analyte solution (mL min-1), LT is the loading time (min), and CPb2+ is the Pb2+ concentration (µg mL-1). A linear calibration equation was obtained: FI response (absorbances) = 0.0018 [µg Pb2+] + 0.0032, R2 = 0.9927 for 1-150 µg Pb2+, and RSD of less than 20% was also obtained. Application of the developed procedure has been demonstrated in real samples.

Development and Evaluation of HILIC-type Sorbents Modified with Hydrophilic Copolymers for Solid-phase Extraction.

Hydrophilic interaction chromatography (HILIC) has attractive attention for the separation of water-soluble compounds via HPLC. There are, however, few studies on the pretreatment of the HILIC-type solid-phase extraction (SPE) due to the difficulty of obtaining the HILIC-type sorbent. Therefore, the development of HILIC-type sorbents for SPE is essential. In this study, four different hydrophilic copolymers, namely diallylamine-maleic acid copolymer (DAM), diallylamine-acrylamide copolymer (DAA), allylamine-maleic acid copolymer (MAM), and partly methylcarbonylated allylamine acetate copolymer (MAC), were immobilized on glycidyl methacrylate (GMA)-base resin, and their adsorptive properties were evaluated. The results of the physical and adsorptive properties indicated that a balance between the water content of the water-enriched layer on sorbent and the amount of hydrophilic copolymer immobilized on the GMA-base resin was vital for the adsorption in HILIC-type sorbent for SPE.

Molding-type Solid-phase Extraction Media Glued with Commercially Available Adhesives.

The handling of a particulate sorbent for solid-phase extraction is often troublesome because it causes static clinging and scattering. To overcome this problem, a production method for a simple molding-type solid-phase extraction medium (M-SPEM) was developed in this study by using commercially available adhesives. The content of a particulate sorbent can increase to as much as 85 wt% in the M-SPEM. Because of the high content, the proposed M-SPEMs have a higher specific surface area than previous monolithic media.

Evaluation of the adsorption properties of nucleobase-modified sorbents for a solid-phase extraction of water-soluble compounds.

We developed hydrophilic interaction chromatography (HILIC)-type sorbents modified with nucleobases for solid phase extraction (SPE). The synthesized hydrophilic base resins were modified by each nucleobase (adenine, guanine, and cytosine). The measurement of the amount of water content indicated that each nucleobase-modified sorbent had a water layer. To evaluate the adsorption properties in the HILIC mode, we chose two nucleobases (uracil and adenine) and four nucleosides (uridine, adenosine, cytidine, guanosine) as water-soluble analytes, which were loaded into an SPE cartridge packed with the nucleobase-modified sorbent. Firstly, 95% acetonitrile (ACN) solutions were used in the process of conditioning and sample loading of the above polar analytes. High recoveries of the analytes were observed in each nucleobase-modified sorbent, and the Diol-type sorbent (no modification with any of the nucleobases) did not adsorb each water-soluble analyte. On the basis of this result, a 98% ACN solution was used during the process of conditioning and sample loading to decrease the concentration of water in the sample, which potentially inhibited the formation of hydrogen bonding between each analyte and the modified nucleobase. Considerable improvements of recoveries were observed in Adenine- and Cytosine-modified sorbents. These results were possibly attributed to the effective expression of hydrogen bonding by decreasing water concentration in the sample solution. Although a non-aqueous (100% ACN) sample solution can be expected to obtain higher recoveries compared with the 98% ACN solution, a decrease in recoveries was observed in Adenine-modified sorbent. From these results, the highest adsorption property was observed in Adenine-modified sorbent using 98% ACN as a sample condition, and the combination of this sample condition and sorbent is effective for high adsorption under HILIC condition. Moreover, we also revealed that a balance between the thickness of water layer and the modification amount of nucleobase is important for retention in the HILIC-type sorbent.

Determination of Albumin, Glucose, and Creatinine Employing a Single Sequential Injection Lab-at-Valve with Mono-Segmented Flow System Enabling In-Line Dilution, In-Line Single-Standard Calibration, and In-Line Standard Addition.

A mono-segmented sequential injection lab-at-valve (SI-LAV) system for the determination of albumin, glucose, and creatinine, three key biomarkers in diabetes screening and diagnosis, was developed as a single system for multi-analyte analysis. The mono-segmentation technique was employed for in-line dilution, in-line single-standard calibration, and in-line standard addition. This made adjustments to the sample preparation step easy unlike the batch-wise method. The results showed that the system could be used for both fast reaction (albumin) and slow reaction (glucose with enzymatic reaction and creatinine). In the case of slow reaction, the analysis time could be shortened by using the reaction rate obtained with the SI-LAV system. This proposed system is for cost-effective and downscaling analysis, which would be applicable for small hospitals and clinics in remote places with a small number of samples but relatively fast screening would be needed.

Preparation of Cyclic-1,N2-propano-2'-deoxyguanosine-d7 as an Internal Standard for ESI-MS/MS Determination of DNA Damage from Acetaldehyde.

Cyclic-1,N2-propano-2'-deoxyguanosine-d7 (CPr-dG-d7) was prepared as an isotopic internal standard (IS) for electrospray ionization tandem mass spectrometry (ESI-MS/MS) quantification of CPr-dG in DNA as a candidate cancer risk marker of acetaldehyde intake, mainly from drinking. The deuterated compound was reasonably synthesized from acetaldehyde-d4 and 2'-deoxyguanosine in deuterium oxide (D2O), preventing the deuterium atoms of acetaldehyde-d4 from being substituted by hydrogen atoms, which occurred seriously in aqueous synthesis media via keto-enol tautomerism. Furthermore, another deuterium atom was added from D2O to form CPr-dG-d7. After four weeks of storage in H2O at 10°C, CPr-dG-d7 was found to be sufficiently stable for practical use. The calibration curve of CPr-dG by using a hydrophilic interaction chromatography-ESI-MS/MS system with CPr-dG-d7 as the IS showed sufficient linearity from 1.0 × 10-10 to 4.0 × 10-9 M with r2 = 0.998.

Effects of pendant-like hydrophilic monomers on the adsorption properties of reversed-phase-type sorbents for solid-phase extraction.

Solid-phase extraction (SPE) has been extensively employed as a pretreatment method. In SPE, reversed-phase-type sorbents have been widely applied for the pretreatment of environmental or biological samples. Hydrophilic-lipophilic balance (HLB)-type sorbents, constituting the copolymers used as reversed-phase-type sorbents, have been applied for various sample pretreatment methods. In HLB-type sorbents, the hydrophilic monomer contributes to the improved wettability of sorbents and increase of polar interactions. In this study, three pendant-like hydrophilic monomers, viz. N-vinylpyrrolidone (NVP), 4-acryloylmorpholine (AMO), and 4-vinyl-1,3-dioxolan-2-one (VDO), respectively, exhibiting different Log P values and possibly causing different polar interactions, were selected to improve the adsorption properties of polar compounds, and divinylbenzene (DVB)-based HLB-type sorbents containing each hydrophilic monomer were synthesized and examined. By the optimization of the molar ratio of DVB and the hydrophilic monomer (i.e. HLB), the inert diluent, and the degree of cross-linking, the developed sorbents exhibited higher recoveries for various polar compounds (viz. cytosine, uracil, cytidine, uridine, 2'-deoxycytidine, 2'-deoxyguanosine, adenine, thymidine, adenosine, and 2'-deoxyadenosine) compared to commercially available HLB-type sorbents.

Optimization of background electrolyte composition for simultaneous contactless conductivity and fluorescence detection in capillary electrophoresis of biological samples.

In this article, optimization of BGE for simultaneous separation of inorganic ions, organic acids, and glutathione using dual C4 D-LIF detection in capillary electrophoresis is presented. The optimized BGE consisted of 30 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, 15 mM 2-amino-2-hydroxymethyl-propane-1,3-diol, and 2 mM 18-crown-6 at pH 7.2 and allowed simultaneous separation of ten inorganic anions and cations, three organic acids and glutathione in 20 min. The samples were injected hydrodynamically from both capillary ends using the double-opposite end injection principle. Sensitive detection of anions, cations, and organic acids with micromolar LODs using C4 D and simultaneously glutathione with nanomolar LODs using LIF was achieved in a single run. The developed BGE may be useful in analyses of biological samples containing analytes with differing concentrations of several orders of magnitude that is not possible with single detection mode.

Effects of hydrophilic monomers on sorptive properties of divinylbenzene-based reversed phase sorbents.

Solid phase extraction (SPE) has been extensively used as a pretreatment method. In SPE methods, commercially available reversed phase type sorbents, which consist of macroporus styrene-divinylbenzene or copolymers including divinylbenzene (DVB) and hydrophilic monomers, have been applied to a variety of samples. The later sorbents are called hydrophilic lipophilic balanced (HLB) type sorbents. Hydrophilic monomers in hydrophilic lipophilic balanced type sorbents contribute to the increase in retention of polar compounds, because hydrophilic monomers improve the wettability and increase the interaction with polar compounds as analytes. In this study, three different methacrylate monomers (ethylene glycol dimethacrylate (EGDMA), glycerol dimethacrylate (GDMA) and trimethylolpropane trimethacrylate (TMPTMA)), which are expected to improve the retention of polar compounds, were chosen, and DVB-based copolymetric sorbents including the three monomers were newly synthesized. Among them, the sorbents including GDMA or TMPTMA gave higher recoveries to polar compounds such as uridine and adenine than that including EGDMA. The optimization studies of hydrophilic lipophilic balance, inert diluent and the purity of DVB improved the sorptive abilities of the sorbents. The developed sorbents have higher recoveries for variety of polar compounds (cytosine, uracil, cytidine, uridine, 2'-deoxycytidine, 2'-deoxyguanosine, adenine, thymidine, adenosine and 2'-deoxyadenosine) than commercially available hydrophilic lipophilic balanced type sorbents, while the recoveries for theophylline were comparable between the proposed sorbents and the commercial sorbents.