Ultra-sensitive HPLC-photochemical reaction-luminol chemiluminescence method for the measurement of secondary amines after nitrosation.

A novel method for the determination of secondary amines at the nanomolar level was developed. The method is based on the nitrosation reaction of secondary amines, with the generated N-nitrosamines being measured using an HPLC separation, photochemical reaction, and chemiluminescence detection system. The efficient nitrosation of secondary amines was performed using sodium nitrite (200 mM) and acetic acid (0.8 M) at 80 °C over 60 min. Although compounds bearing OH and SH functional groups also underwent the nitrosation reaction, the sensitivity of these compounds was 1000 times lower than that of the secondary amines. Our method was applied to the determination of low molecular weight secondary amines, including dimethylamine, morpholine, pyrrolidine, diethylamine, and piperidine, giving method detection limits of 0.7 nM, 0.2 nM, 0.4 nM, 0.7 nM, and 1.5 nM, respectively. The calibration curves were linear in the range of 5-100 nM. We then applied this method for the detection and quantification of these secondary amines in samples of tap water, river water, treated wastewater, and sea water. Dimethylamine was detected at concentrations up to 15.4 nM, <0.7 nM, and 48.5 nM in tap water, river water, and treated wastewater samples, respectively, with recoveries ranging from 94 to 103%. Other amines were also detected at nanomolar levels. These results indicate that our proposed method can be applied to the analysis of secondary amines in various environmental water samples. To the best of our knowledge, the proposed method is one of the most sensitive and selective methods for the determination of secondary amines without pre-concentration steps.

Rapid method for monitoring N-nitrosodimethylamine in drinking water at the ng/L level without pre-concentration using high-performance liquid chromatography-chemiluminescence detection.

As a contaminant in drinking water, N-nitrosodimethylamine (NDMA) is of great concern because of its carcinogenicity; it has been limited to levels of ng/L by regulatory bodies worldwide. Consequently, a rapid and sensitive method for monitoring NDMA in drinking water is urgently required. In this study, we report an improvement of our previously proposed HPLC-based system for NDMA determination. The approach consists of the HPLC separation of NDMA, followed by NDMA photolysis to form peroxynitrite and detection with a luminol chemiluminescence reaction. The detection limit for the improved HPLC method was 0.2ng/L, which is 10 times more sensitive than our previously reported system. For tap water measurements, only the addition of an ascorbic acid solution to eliminate residual chlorine and passage through an Oasis MAX solid-phase extraction cartridge are needed. The proposed NDMA determination method requires a sample volume of less than 2mL and a complete analysis time of less than 15min per sample. The method was utilized for the long-term monitoring of NDMA in tap water. The NDMA level measured in the municipal water survey was 4.9ng/L, and a seasonal change of the NDMA concentration in tap water was confirmed. The proposed method should constitute a useful NDMA monitoring method for protecting drinking water quality.

Fluorescent character of Cu(I/II) complexes with bisquinoline-based ligands and fluorometric detection of reductants.

The fluorescence ligands N,N'-bis(2-methylquinolyl)dimethylethylenediamine (BQDMEN) and N,N'-bis(2-methylquinolyl)dimethyl-1,3-propanediamine (BQDMPN) were synthesized. [Cu(II)(bqdmen)](2+) and [Cu(II)(bqdmpn)](2+) were prepared, and their reduction to [Cu(I)(bqdmen)](+) and [Cu(I)(bqdmpn)](+) was confirmed by the observed UV-Vis spectral change. Then, the fluorescence of [Cu(I/II)(bqdmen)](+/2+) and [Cu(I/II)(bqdmpn)](+/2+) in aqueous solution was characterized; [Cu(I)(bqdmen)](+) and [Cu(I)(bqdmpn)](+) were found to fluoresce in aqueous solution, whereas the fluorescence of [Cu(II)(bqdmen)](2+) and [Cu(II)(bqdmpn)](2+) was completely quenched. On the basis of these findings, fluorometric detection of reductants with a flow-injection system using a [Cu(II)(bqdmen)](2+) or [Cu(II)(bqdmpn)](2+) solution as a carrier was explored.

Chemiluminescence detection of glucose using Ce(IV) oxidation in a batch system.

A chemiluminescence method for the determination of glucose (GC) using a batch system made in our laboratory is proposed. The method is based on measures of the chemiluminesence intensity from a batch of sample GC solutions following the injection of an (NH(4))(2)[Ce(NO(3))(6)]-[Ru(bpy)(3)]Cl(2) solution. The effect of the Ru complex concentration on chemiluminescence was studied. The detection limit was 0.5 × 10(-8) M, and the linear calibration curve reached 10(-7) M. The Ru complex was selected on the basis of its desirable performance compared to those of other photosensitizers. However, flow-injection analysis (FIA) using the Ce-Ru complex reagent was not applicable for the detection of GC.

Simultaneous determination of pyridine-triphenylborane anti-fouling agent and its degradation products in paint-waste samples using capillary zone electrophoresis with field-amplified sample injection.

We proposed a capillary zone electrophoresis (CZE) procedure using field-amplified sample injection (FASI) for the simultaneous determination of pyridine-triphenylborane (PTPB) and its degradation products: diphenylborinic acid (DPB), phenylboronic acid (MPB), and phenol. The LODs for PTPB, DPB, MPB, and phenol were, respectively, 0.85, 0.88, 44, and 28 µg L(-1). The RSDs (n = 4) for the analytes listed above were in respective ranges of 6.2 - 14, 5.9 - 10, and 0.49 - 0.62% for the peak area, peak height, and migration time. The compounds were extracted from paint-waste samples collected from shipyards using a siliga-gel column. The extract was dissolved with acetonitrile containing 1% (v/v) pyridine. The samples were then analyzed using CZE, revealing respective concentrations of 0.076 - 0.53, 0.015 - 0.36, 1.7 - 22, and 1.2 - 13 µg g(-1). The proposed FASI-CZE method is a simple and promising procedure that is expected to be useful for the determination of PTPB and its degradation products in paint wastes.

Sensitive determination method for mercury ion, methyl-, ethyl-, and phenyl-mercury in water and biological samples using high-performance liquid chromatography with chemiluminescence detection.

A sensitive determination method for mercury speciation analysis was developed. Four mercury species, mercury ion, methylmercury, ethylmercury, and phenylmercury, were complexed with emetine-dithiocarbamate (emetine-CS(2)), and then injected onto a HPLC instrument coupled with a tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection system. The emetine-CS(2) complexing agent was effectively used to measure the concentration in addition to serving as a separation and detection reagent. The calibration curves for these mercury complexes were linear in the range of 0.050 - 10 µg L(-1) (as Hg). The limit of detection for (emetine-CS(2))(2)Hg, emetine-CS(2)-methylmercury, emetine-CS(2)-ethylmercury, and emetine-CS(2)-phenylmercury were 30, 17, 21, and 22 ng L(-1), respectively. The sensitivity of this method enables the determination of mercury species in water samples at sub-ppb levels. Furthermore, the method was applied to biological samples in combination with acid leaching and liquid-liquid extraction using emetine-CS(2) as an extraction reagent. The determination results were in good agreement with the values of the certified reference materials.

A novel hybrid mode of sample injection to enhance CZE sensitivity for simultaneous determination of a pyridine-triphenylborane anti-fouling agent and its degradation products.

We developed a novel hybrid sample injection mode (HSIM) that presents the combination of electrokinetic injection and vacuum injection to enhance detection sensitivity in CZE. Samples were introduced using both vacuum and electrokinetic injections simultaneously, with a water plug injected into the capillary prior to sample introduction (i.e. similarly to field-amplified sample injection, FASI). Using a sample mixture containing an anti-fouling agent applied to ship hulls, pyridine-triphenylborane and its degradation products (diphenylborinic acid, phenylboronic acid, and phenol) dissolved in ACN, the length of water plug, time, and voltage for sample introduction were optimized. The signal intensity (peak height) was found to be up to a 30-fold increased using HSIM by applying 4 kV for 4 s at the inlet end of the capillary as the cathode with supplementary vacuum in comparison with only vacuum injection for 4 s. The LODs (at a S/N of 3) for pyridine-triphenylborane, diphenylborinic acid, phenylboronic acid, and phenol were 0.88, 1.0, 21, and 23 µg/L, respectively. At the level of 0.04 mg/L, the RSDs (n=4, intra-day) for the above analytes were in the ranges of 1.9-11, 4.3-9.2, and 0.34-0.66% for peak area, peak height, and migration time, respectively. The HSIM is a simple and promising procedure useful for enhancing the sensitivity for both low-and high-mobility ions in CZE.

Rapid method for simultaneous determination of nitrite and nitrate in water samples using short-column ion-pair chromatographic separation, photochemical reaction, and chemiluminescence detection.

A rapid method has been developed for the simultaneous determination of nitrite and nitrate. The separation of nitrite and nitrate was achieved using an octadecylsilane (ODS) short column (5 µm, 20 × 4.6 mm) with 10 mM of borate buffer-methanol (99.5:0.5, v/v; pH 10.0), containing 5 mM of lauryltrimethylammonium chloride and 50 mM of NaBr. These ions were detected by luminol chemiluminescence following online UV irradiation. The calibration curves of nitrite and nitrate were linear in the range of 1.0 × 10(-7) to 2.0 × 10(-5) M and 1.0 × 10(-6) to 2.0 × 10(-4) M, respectively. The detection limits for nitrite and nitrate were 0.05 and 0.4 µM, respectively (with a signal-to-noise ratio of 3). The precisions of peak heights for 7 identical injections of a standard mixture of 0.50 µM of nitrite and 5.0 µM of nitrate were 2.7 and 2.1%, respectively. Analysis time per sample was less than 2 min, and system pressure was low (2.1 MPa). The proposed method was successfully applied to water samples from various sources.

Simultaneous determination of a pyridine-triphenylborane anti-fouling agent and its estimated degradation products using capillary zone electrophoresis.

A commercial organoborane compound, pyridine-triphenylborane (PTPB), is often applied to ship hulls as an anti-fouling agent. We developed capillary zone electrophoresis (CZE) with direct UV detection for the simultaneous determination of PTPB and its estimated degradation products: diphenylborinic acid (DPB), phenylboronic acid (MPB), and phenol. The limits of detection (LODs) for PTPB, DPB, MPB, and phenol were, respectively, 25, 30, 50, and 29 microg/l at a signal-to-noise ratio of three. At concentrations of 0.5mg/l, values of the relative standard deviation (RSD, n=6, intra-day) of peak area were obtained, respectively, for PTPB, DPB, MPB, and phenol, as 4.1, 4.1, 4.7, and 3.4% for peak heights 3.6, 3.2, 1.7, and 1.4%, and for migration times 1.1, 1.1, 1.0, and 0.73%. The analytes were detected within 14 min. Simple photodegradation experiments were conducted to verify the usefulness of the proposed method for additional PTPB degradation investigations.

Selective determination method for measurement of nitrite and nitrate in water samples using high-performance liquid chromatography with post-column photochemical reaction and chemiluminescence detection.

A simple, sensitive and selective method for the simultaneous determination of nitrite and nitrate in water samples has been developed. The method is based on ion-exchange separation, online photochemical reaction, and luminol chemiluminescence detection. The separation of nitrite and nitrate was achieved using an anion-exchange column with a 20mM borate buffer (pH 10.0). After the separation, these ions were converted to peroxynitrite by online UV irradiation using a low-pressure mercury lamp and then mixed with a luminol solution prepared with carbonate buffer (pH 10.0). The calibration graphs of the nitrite and nitrate were linear in the range from 2.0 x 10(-9) to 2.5 x 10(-6)M and 2.0 x 10(-8) to 2.5 x 10(-5)M, respectively. Since the sensitivity of nitrite was about 10 times higher than that of nitrate, the simultaneous determination of nitrite and nitrate in the water samples could be efficiently achieved. This method was successfully applied to various water samples--river water, pond water, rain water, commercial mineral water, and tap water--with only filtration and dilution steps.

Effect of a carboxyl group on the chemiluminescent reaction of tris(2,2'-bipyridine)ruthenium(III) with aliphatic amines.

The effect of a carboxyl group beside nitrogen of aliphatic amines on the tris(2,2'-bipyridine)ruthenium(III), Ru(bpy)(3)(3+), chemiluminescent reaction was examined. It has been shown that a carboxylate anion promotes the chemiluminescent reaction at a lower pH and then the aliphatic amines with this substituent can be sensitively detected compared with corresponding aliphatic amines without this substituent. Based on this finding, preliminary studies on simultaneous determination of 4-hydroxyproline, N-methylglycine, N-methylalanine, proline, and pipecolic acid in human serum have been performed using isocratic reversed-phase ion-pair high-performance liquid chromatography (HPLC) with electrogenerated Ru(bpy)(3)(3+) chemiluminescent detection. The detection limits (signal-to-noise ratio of 3) with the proposed method were 3.0, 12, 2.7, 4.6, and 10nM for 4-hydroxyproline, N-methylglycine, N-methylalanine, proline, and pipecolic acid, respectively.

Highly sensitive method for determination of N-nitrosamines using high-performance liquid chromatography with online UV irradiation and luminol chemiluminescence detection.

A new method for the measurement of N-nitrosamines in part-per-trillion concentrations from water samples without preconcentration steps has been developed. This method is based on online UV irradiation after high-performance liquid chromatographic separation and subsequent luminol chemiluminescence detection without addition of an oxidant. It was confirmed that N-nitrosamines in basic aqueous solution were transformed to peroxynitrite by UV irradiation. The detection limits for this method were 1.5 ng/L, 2.9 ng/L, 3.0 ng/L, and 2.7 ng/L for N-nitrosodimethylamine, N-nitrosomorpholine, N-nitrosomethylethylamine, and N-nitrosopyrrolidine, respectively, at a signal-to-noise ratio of 3. The calibration graphs were linear in the range of 5-1000 ng/L for these N-nitrosamines. This method was used for the determination of N-nitrosamines in tap water, river water, and industrial plant effluent samples. The recoveries of N-nitrosodimethylamine, N-nitrosomorpholine, N-nitrosomethylethylamine, and N-nitrosopyrrolidine present in tap water sample at a concentration of 10 ng/L (mean+/-standard deviation, n=4) were (94.8+/-2.7)%, (102.0+/-6.9)%, (99.3+/-3.9)%, and (102.8+/-2.5)%, respectively. These results indicate that our proposed method can be applied satisfactorily to the determination of N-nitrosamines in water samples.

Electrogenerated chemiluminescence reaction of tris(2,2'-bipyridine)ruthenium(II) with 2,5-dimethylthiophene as co-reactant in aqueous solution.

A novel effective co-reactant for electrogenerated chemiluminescence (ECL) of Ru(bpy)(3)(2+) has been found. Alpha-position-dialkylated thiophene derivatives such as 2,5-dimethylthiophene (DMT) could be used as a co-reactant for Ru(bpy)(3)(2+) ECL. The reaction mechanism of the Ru(bpy)(3)(2+)/DMT system was proposed on the basis of the identification of the reaction product, the relationship between the molecular structure and the chemiluminescent intensity, and the electrochemical study. The obtained reaction mechanism was similar to that of the Ru(bpy)(3)(2+)/aliphatic tertiary amine system. Based on these results, the preliminary studies of the Ru(bpy)(3)(2+) ECL detection system using DMT as a co-reactant were performed. Under the optimal ECL conditions, the plot of ECL intensity versus the concentration of Ru(bpy)(3)(2+) was linear over the concentration range 1.0x10(-8) to 1.5x10(-7) M (determination coefficient=0.9996).

A single-bead analysis on a disk-shaped microfluidic device using an antigen-immobilized bead.

A disk-shaped microfluidic device (lab-on-a-Disk) was developed to allow the evaluation of mental stress. As a standard sample, secretory immunoglobulin A (sIgA), which is a candidate marker of mental stress, was measured by a heterogeneous enzyme immunoassay (EIA) on the lab-on-a-Disk. Centrifugal force provided a microfluidic control on the lab-on-a-Disk. We examined the relationship between the rotational speed, the channel profile, and the position of the microfluidic chambers from the center of rotation to manipulate sample solutions into each reaction reservoir through microchannels sequentially, i.e., retain in a reservoir or flow into a subsequent reservoir. A single glass bead with immobilized sIgA on its surface was injected into a reservoir for a competitive antigen-antibody reaction, and applied to a specific surface in a heterogeneous assay. It is expected that the lab-on-a-Disk would be suitable for miniaturization and automation of the processes in EIA compared with a conventional EIA using a titer plate.

Chemiluminescence detection of five-membered heteroaromatic compounds using electrogenerated tris(2,2'-bipyridine)-ruthenium(III).

Heteroaromatic compounds, such as 2,5-dimethylthiophene, 2,5-dimethylpyrrole, and 2,5-dimethylfuran, were found to act as reducing agents for the tris(2,2'-bipyridine)ruthenium(III), Ru(bpy)3(3+), chemiluminescent reaction. In order to characterize the chemiluminescent reaction of Ru(bpy)3(3+) with heteroaromatic compounds, we have investigated various mono-, di-, and tri-heteroaromatic compounds. The pi-electron density and stability of aromatic rings influence the chemiluminescent efficiency of the reaction. Above all, 2,5-dimethylthiophene produced strong chemiluminescence under acidic conditions. In addition, we confirmed that the rate of the chemiluminescent reaction of Ru(bpy)3(3+) with 2,5-dimethylthiophene is very fast.

Highly sensitive and simple method for measurement of pipecolic acid using reverse-phase ion-pair high performance liquid chromatography with tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection.

A new, highly sensitive chemiluminescence method for measurement of pipecolic acid in various substances such as human serum, cow's milk, beer, and apple juice has been developed. The method is based on reverse-phase ion-pair high performance liquid chromatographic separation and subsequent tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection. It was confirmed that imino acids show strong chemiluminescence upon mixing with tris(2,2'-bipyridine)ruthenium(III). A calibration graph, based on a standard pipecolic acid solution, was linear over the range 5.0x10(-9)M to 2.0x10(-5)M and the detection limit was 24fmol (signal-to-noise ratio=3). This highly sensitive and selective determination method can be applied to selected samples without purification or pre-concentration procedures. Compared to the previous HPLC methods, the proposed method is easier, more sensitive, and time-saving.

High-throughput nitric oxide assay in biological fluids using microchip capillary electrophoresis.

In order to develop a high-throughput screening method for the nitrogen monoxide metabolites, nitrite and nitrate, in biological fluids, we have investigated the simultaneous determination of these metabolites using microchip capillary electrophoresis (MCE). In this study, the control of applied voltage to obtain higher sensitivity by increasing the sample injection volume was investigated. Also, the improvement of reproducibility by correcting the injection volume using the internal standard was investigated. By increasing the sample volume, the limits of detection achieved for nitrite and nitrate were 24 and 12 microM, respectively. Because we used a 10-fold diluted sample when detecting nitrite and nitrate in human serum, it was necessary to increase the sensitivity by a factor of 10-50. The run-to-run and day-to-day relative standard deviations achieved were improved to less than 10% by using an internal standard to correct the injection volume. Moreover, we obtained successful separation of nitrite and nitrate in spiked human serum within 6.5 s under optimum analytical conditions. As a result, although it is necessary to obtain greater sensitivity, it was concluded that determination of the amount of NO metabolites in biological fluids using MCE is possible.

Determination of ammonium in river water and sewage samples by capillary zone electrophoresis with direct UV detection.

We developed capillary zone electrophoresis (CZE) with direct UV detection for determination of ammonium in environmental water samples. Ammonium in the samples was partly converted into ammonia in the alkaline background electrolyte (BGE) during migration and was detected by molecular absorption of ammonia at 190 nm in approximately 7 min. The limit of detection (LOD) for ammonium was 0.24 mg/l (as nitrogen) at a signal-to-noise ratio of three. The respective values of the relative standard deviation (RSD) of peak area, peak height, and migration time for ammonium were 2.1, 1.8, and 0.46%. Major alkali and alkaline earth metal ions coexisting in the samples did not interfere with ammonium determination by the proposed method. The proposed method determined ammonium in surface water and sewage samples. The results were compared to those obtained using ion chromatography (IC).

High-performance liquid chromatography of aromatic compounds with photochemical decomposition and tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection.

A novel high-performance liquid chromatographic method for the determination of aromatic compounds based on the on-line photochemical degradation and subsequent tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection has been developed. Chemiluminescence intensity depended upon the number of aromatic rings, UV irradiation time, and variety of substituted functional groups. One of the decomposition products of aromatic compounds by UV irradiation was identified as oxalic acid. As one application of this methodology, determination of catechins in tea has been shown. Calibration graphs, based on standard (-)-epicatechin and (-)-epigallocatechin gallate solutions, were linear over the range of 0.1-50 microM. The detection limits (signal-to-noise ratio=3) were 0.8 pmol for (-)-epigallocatechin gallate and 1.2 pmol for (-)-epicatechin. The high-performance liquid chromatography-chemiluminescence (HPLC-CL) detection method with a post-column photochemical reactor can be applied to the sensitive and selective determination of catechins in tea.

Simple and sensitive method for determination of glycoalkaloids in potato tubers by high-performance liquid chromatography with chemiluminescence detection.

A novel, simple and sensitive high-performance liquid chromatographic method for the determination of the potato glycoalkaloids, alpha-solanine and alpha-chaconine, based on the chemiluminescent reaction of tris(2,2'-bipyridine)ruthenium(III) has been developed. The calibration graph was linear in the range of 5 ng/ml-10 microg/ml for both alpha-solanine and alpha-chaconine. The detection limits of alpha-solanine and alpha-chaconine were 1.2 and 1.3 ng/ml, respectively. This method was successfully applied to a potato tuber sample without cleanup, pre-concentration, and derivatization steps. The recoveries (mean +/- standard deviation, %) of alpha-solanine and alpha-chaconine spiked in tuber pith at 10 microg/g (n = 6) were 101.0 +/- 4.4% and 103.6 +/- 7.1%, respectively.