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.

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.

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.

Preparation of N2-Ethyl-2'-deoxyguanosine-d4 as an Internal Standard for the Electrospray Ionization-Tandem Mass Spectrometric Determination of DNA Damage by Acetaldehyde.

The deuteration of N2-ethyl-2'-deoxyguanosine (Et-dG), which is a DNA adduct generated from acetaldehyde, was studied by the addition reaction of acetaldehyde-d4 to 2'-deoxyguanosine (dG) in deuterium oxide (D2O), with the aim to obtain an isotope internal standard for the liquid chromatography/tandem mass spectrometry (LC/MS/MS) quantitation of Et-dG. The replacement of the dG C-8 hydrogen atom by a deuteron atom took place at 50°C in D2O and afforded a mixture of Et-dG-d4 and Et-dG-d5. Et-dG-d4, which was stable in aqueous solutions, was prepared by incubating the mixture in H2O at 60°C for 48 h. The calibration curve was obtained by multiple reaction monitoring (MRM) measurements using a hydrophilic interaction chromatography-electrospray ionization-tandem mass spectrometric (HILIC/ESI-MS/MS) system between the Et-dG concentration, ranging from 1.0 × 10-10 to 4.0 × 10-9 M in the sample solutions, and the relative peak areas of Et-dG (m/z: 296.1 → 180.1) to the value of Et-dG-d4 (m/z: 300.2 → 184.2), with an internal standard showing good linearity (R2 = 0.995, n = 5).

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.

Evaluation of Type-A Endonucleases for the Quantitative Analysis of DNA Damage due to Exposure to Acetaldehyde Using Capillary Electrophoresis.

The substrate selectivities of three endonucleases were studied quantitatively using capillary zone electrophoresis to find one giving N2-ethyl(Et)-2'-deoxyguanosine-5'-monophosphate (5'-dGMP) and cyclic 1,N2-propano(CPr)-5'-dGMP from DNAs damaged by acetaldehyde (AA). Six 2'-deoxyribonucleoside-5'-monophosphates to be quantified in the hydrolysis solutions of DNAs, namely, Et-5'-dGMP, CPr-5'-dGMP, and four authentic ones, were completely separated using a 100 mM borate running buffer solution having an optimized pH of 9.67. Using the present method, nuclease reactions of nuclease S1 (NS1), nuclease P1 (NP1), and nuclease Bal 31 to 2'-deoxyribonucleoside-5'-monophosphates from damaged Calf thymus (CT-) DNAs were monitored. The CT-DNAs were prepared by treatment with AA to generate Et-guanine or CPr-guanine internally. Bal 31 hydrolyzed the damaged CT-DNAs to yield Et-5'-dGMP and CPr-5'-dGMP quantitatively. The two 5'-dGMP adducts were not detected in the hydrolysis solutions using NS1 or NP1. Bal 31 can be a suitable nuclease for analyzing DNA damages caused by AA.

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.

Progress in a selective method for the determination of the acetaldehyde-derived DNA adducts by using HILIC-ESI-MS/MS.

Acetaldehyde (AA), which is present in tobacco smoke, automobile exhaust gases and alcohol beverage, is a mutagen and carcinogen. AA reacts with 2'-deoxyguanosine (dG) in DNA to form N2-ethyl-dG (EtdG) and cyclic, 1, N2-propano-dG (CPrdG), which are considered to have a critical role in carcinogenesis induced by AA. In this study, we have developed a highly sensitive method for the quantitation of the two AA-derived DNA adducts by using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) in which hydrophilic interaction chromatography (HILIC) employing mobile phases of high organic solvent concentration was selected to improve the ionization efficiency in the ESI process. Fourteen times and 11 times larger peak areas for EtdG and CPrdG, respectively, in HILIC-ESI-MS/MS were obtained compared with those in reversed phase (RP)-LC-ESI-MS/MS. Furthermore, 6.9 times (for EtdG) and 2.4 times (for CPrdG) larger peak areas were also obtained as additional enhancement by varying additive compounds in the HILIC mobile phases from ammonium acetate to ammonium bicarbonate. In total, the enhancements in detected MS signal intensities by exchanging from the RP-LC system to the HILIC system are 97 times for EtdG and 26 times for CPrdG, respectively. Three commercially available HILIC columns with different polar functional groups were examined and sufficient separation between normal 2'-deoxynucleosides and the AA-derived DNA adducts was achieved by a carbamoyl-bonded HILIC column. Finally, we applied the established method to quantify EtdG and CPrdG in the damaged calf thymus DNA.

Simple Pretreatment and HILIC Separation for LC-ESI-MS/MS Determination of Adenosine in Human Plasma.

A simple pretreatment method and separation mode for the LC-ESI-MS/MS determination of adenosine in human plasma have been developed. Deproteinization by acetonitrile and ultrafiltration followed by chromatographic separation with a hydrophilic interaction chromatographic (HILIC) column give a highly sensitive MS/MS response without ionic suppression caused by the matrix compounds in human plasma. In addition, the presence of ammonium acetate in the mobile phase contributes to high sensitivity in MS/MS detection, facilitating the ionization of adenosine. This method seems to be amenable to the treatment of many samples in clinical practice.

Advanced Stopped-in-Loop Flow Analysis with a Reagents-Merging Zones Technique for a Catalytic Successive Determination of Vanadium and Iron.

An advanced stopped-in-loop flow analysis (SILFA) is proposed for the catalytic determinations of vanadium and iron. The chemistry relies on a vanadium- or iron-catalyzed oxidative reaction of p-anisidine by bromate or hydrogen peroxide in the presence of an activator (Tiron or 1,10-phenanthlorine) to form a red dye (510 nm). Reagents for the vanadium- or iron-catalyzed reaction are well mixed by a reagents-merging zones technique. A sample solution is loaded together with well-mixed reagents into a loop in the SILFA configuration, followed by spectrophotometric detection. The advanced SILFA system provides a selective method for the trace determination of vanadium and iron.