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A doxorubicin (Dox) and curcumin (Cur) combination treatment regimen has been widely studied in pre-clinical research. However, the nanoparticles developed for this combination therapy require a consecutive drug loading process because of the different water-solubility of these drugs. This study provides a strategy for the “one-step” formation of nanoparticles encapsulating both Dox and Cur. We took advantage of polyacrylic acid (PAA) and calcium carbonate (CaCO3) to realise a high drug entrapment efficiency (EE) and pH-sensitive drug release using a simplified preparation method. Optimisation of lipid ratios and concentrations of CaCO3 was conducted. Under optimal conditions, the mean diameter of PEGylated lipid/PAA/CaCO3 nanoparticles with encapsulated Cur and Dox (LPCCD) was less than 100?nm. An obvious pH-sensitive release of both drugs was observed, with different Dox and Cur release rates. Successful co-delivery of Cur and Dox was achieved via LPCCD on HepG2 cells. LPCCD altered the bio-distribution of Dox and Cur in vivo and decreased Dox-induced cardiotoxicity. The current investigation has developed an efficient ternary system for co-delivery of Dox and Cur to tumours, using a “one-step” formation resulting in nanoparticles possessing remarkable pH-sensitive drug release behaviour, which may be valuable for further clinical studies and eventual clinical application., Journal of Drug Targeting, 25(8), pp.704-714; 2017

The luminol chemiluminescence (CL) profile of an oil-in-water (O/W) emulsion during thermal oxidation (60°C) was assessed using the luminol-K3[Fe(CN)6] assay, in which the oxidation species produced by the autoxidation of an O/W emulsion generated CL emission. Increased CL intensity was observed for O/W emulsions prepared using either linseed or corn oil, which was increased by the addition of Fe2+ to the O/W emulsion. The relationship between the CL profile and results obtained by conventional approaches, such as the peroxide value (PV) and thiobarbituric acid (TBA) methods, were compared. Owing to good correlation between the CL intensity and results obtained by the methods, the CL method might be applicable for estimating the oil oxidizing of an emulsion in thermal oxidation.

The drain current model of AlGaN/GaN Ridge HEMTs has been studied for switching power supply circuits of flexible devices. The physically derived model is developed, and then implemented in a circuit simulator with Verilog-A source codes. To apply the models to simulate power supply circuits for flexible devices, the weak inversion to linear characteristics and the maximum drain current are important. The model is verified with measured data of transistor TEGs that we fabricated with a source field plate technology. The results show reasonable agreements between measurements and simulations.

Switching time characterization has been presented to verify our AlN/GaN based metal-insulator-semiconductor (MIS) high-electron-mobility-transistor (HEMT) model. To apply the GaN MIS-HEMT model to any fast power switching applications, drain current scalability of the number of gate fingers and the number of transistor units, bias dependent capacitances, and the small signal AC equivalent circuit are implemented in the model. All model parameters are accurately extracted with measurements of dedicated test structures. The timing measurement result shows good agreement with the simulation.

Hydrogen sulfide (H2S) is a colorless toxic gas which can be found as HS− in rivers and waste waters especially in the occupational susceptible environment. Herein we synthesized a lophine analogue, 2-(4-hydroxyphenyl)-4,5-di(2-pyridyl)imidazole (HPI), which fluoresces at 410 nm after excitation at 280 nm. HPI has an imidazole ring and a pyridine ring which are capable of forming coordinate bonds with copper (Cu(II)) that cause quenching of HPI fluorescence. We found that HS− can selectively liberate HPI from the complex via formation of CuS, thus, HPI regains its fluorescence properties. Interestingly, the probe was proved to be regenerable. This reaction was used for the development of a fluorescence microplate assay for the determination of HS− in environmental samples. The method was applied to river water samples and was able to detect HS− in concentrations down to 5 ppb with acceptable accuracy (90.3–103.0%) and good precision (%RSD ≤ 4.1). The method showed many advantages over the previously reported ones including instantaneous reaction, simple probe synthesis, high-throughput, high selectivity toward hydrogen sulfide over other ions and sulfur or thiol containing compounds and at last, it complies with the green chemistry rules through using a regenerable probe, aqueous solvents, and miniaturized measurement system.

Quinones are ubiquitous compounds that have two-sided nature; a beneficial one, especially in the electron transport chain, and a toxic one through the generation of reactive oxygen species in vivo. Herein we developed a new dual-function chemiluminescence probe for their determination namely N-(4-aminobutyl)-N-ethylisoluminol-lipoic acid (ABEI-LA). This probe has two moieties, a reductant one, lipoic acid (LA), which upon its redox cycle reaction with quinones, generate superoxide anion, and the other moiety is the luminophore, ABEI, which generates intense chemiluminescence upon reaction with the generated superoxide anion. ABEI-LA is the first single reagent that could generate CL with quinones in a one-step operation. A simple rapid chemiluminescence assay for the anticancer drug, doxorubicin, was developed using ABEI-LA as a probe. The method showed good linearity towards doxorubicin in the range of 1−200 nM and excellent sensitivity with a detection limit of 0.17 nM (25 pg/assay). The proposed probe, ABEI-LA, was successfully applied for the quantification of doxorubicin, in lyophilized powder for injections and human serum samples and it showed very good recoveries of 84–91 % and 98–101 %, respectively. These results demonstrate the ability of ABEI-LA for the determination of quinones in different matrices enabling the in-depth study of quinones two-sided nature.

Cancer immunotherapies such as antibodies targeting T cell checkpoints, or adaptive tumor-infiltrating lymphocyte (TIL) transfer, have been developed to boost the endogenous immune response against human malignancies. However, activation of T cells by such antibodies can lead to the risk of autoimmune diseases. Also, the selection of tumor-reactive T cells for TIL relies on information regarding mutated antigens in tumors and does not reflect other factors involved in protein antigenicity. It is therefore essential to engineer therapeutic interventions by which T cell reactivity against tumor cells is selectively enhanced (i.e., “focused cancer immunotherapy”) based on tumor antigens that are specifically expressed in the tumor of a certain cancer and in many patients with this cancer. Immune complexes (ICs) are the direct and stable products of immunological recognition by humoral immunity. Here, we searched for tumor-specific IC antigens in each of five cancers (lung (n=28), colon (n=20), bladder (n=20), renal cell (n=15) and malignant lymphoma (n=9)), by using immune complexome analysis that comprehensively identifies and profiles the constituent antigens in ICs. This analysis indicated that gelsolin and inter-alpha-trypsin inhibitor heavy chains were specifically and frequently detected (at a frequency higher than 80%), and that phosphoproteins (VENTX, VCIP135) were also specifically present in the ICs of lung cancer patients. Immune complexome analysis successfully identified several tumor-specific IC antigens with high detection frequency in lung cancer patients. These specific antigens are required to validate the clinical benefit by further analysis using a large number of patients. This article is protected by copyright. All rights reserved.

9,10-Phenanthrenequinone (PQ) was successfully used as a new mass-tagging reagent for sensitive labeling of aliphatic aldehydes (C3-C10) prior liquid chromatography-electrospray ionization?tandem mass spectrometry (LC/ESI?MS/MS). This reagent could overcome the drawbacks of previous amine or hydrazine-based reagents, such as lower sensitivity, formation of two stereoisomeric reaction products for each single analyte, need for longer derivatization time, and poor reactivity with aliphatic aldehydes. The PQ-aldehyde derivatives exhibited intense [M+H]+ and a common product ion with ESI in the positive-ion mode. The derivatives were monitored at the transition of [M+H]+ → m/z 231.9 with detection limits from 4.0 to 100 pM (signal to noise ratio = 3). 3-Phenylpropanal was used as an internal standard (IS) and the separation of the eight aldehydes and IS was achieved in less than 10 min employing gradient elution with methanol and ammonium formate buffer (20 mM, pH 4.0). The method employed salting out liquid?liquid extraction for aliphatic aldehydes form serum for the first time with excellent recoveries (92.6?110.8%). The developed method was validated and applied for quantification of the target aldehydes in serum of healthy volunteers (n = 14)., Journal of Chromatography A, 1462, pp.80-89; 2016

High-functionality and -quality (HFQ) lipids have a discrete molecular weight and good water dispersibility and can be produced by solid-phase peptide synthesis. Therefore, HFQ lipids are a promising material for the preparation of ligand-grafted PEGylated liposomes. Recently, we have reported serine–glycine repeated peptides ((SG)n) as a spacer of HFQ lipids and to substitute a conventional PEG spacer. We demonstrated the advantage of using (SG)n spacers for peptide ligand presentation on the liposomal surface in vitro; however, the use of (SG)n spacers in ligand-grafted PEGylated liposomes in vivo has not been validated. The aim of this study was to validate the in vivo targeting ability of HFQ lipid-grafted PEGylated liposomes. We synthesized lipids containing GRGDS (RGD–(SG)n–lipid) to target integrin αvβ3 and prepared RGD–(SG)n/PEGylated liposomes. Subsequently, their cellular uptake characteristics in murine colon carcinoma (Colon-26) cells were evaluated. Two-color imaging of liposomes and tumor bl...

A compact model of AlN/GaN metal-insulator-semiconductor (MIS)-HEMTs which supports DC and small-signal AC with self-heating and frequency dispersion effects is developed. The model is implemented in Verilog-A source codes. The model parameters are extracted from measured data of the G-S-G transistor test structures that we fabricated. Both of self-heating and frequency dispersion characteristics are successfully handled in the model. The results show good agreements between device measurements and simulations.

A small signal AC equivalent circuit with scalable drain current equations of AlN/GaN metal-insulator-semiconductor (MIS)-HEMTs has been developed for embedded source field-plate structures. The scalable source field-plate capacitance is included in addition to the dispersion, gate, and drain capacitances. The models are implemented in Verilog-A source codes. The model parameters are extracted from measured data of the G-S-G transistor test structures that we fabricated. The results show good agreements between device measurements and simulations.

Background: Immune complexes (ICs) may clearly reflect immunological abnormalities caused by disease, especially for autoimmune diseases. Although ICs have been detected in cerebrospinal fluid (CSF) from patients with CNS autoimmune diseases, identities of antigens in such ICs have not been comprehensively determined. Methods: We used immune complexome analysis, in which nano-liquid chromatography-tandem mass spectrometry is employed to comprehensively identify antigens incorporated into ICs in biological fluids, to characterize ICs in CSF samples from patients with CNS autoimmune diseases, and to find disease-specific IC antigen to a certain CNS autoimmune disease. Also, we compared the IC antigens we identified with the reported CSF proteome or with the published plasma proteome to examine if the method is distinguished from the conventional CSF proteome analysis. Results: We identified 176 antigens in 78 CSF samples. We then assessed the overlaps among these antigens, the CSF proteome, and the plasma proteome; 140 of the 176 antigens were found to be exclusively detected by our method. Notably, IC-associated suprabasin in CSF was 100% specific to neuropsychiatric systemic lupus erythematosus (NPSLE). Conclusions: This report is the first to comprehensively identify the antigens incorporated into ICs in CSF. There was limited overlap between the antigens we identified and the CSF proteome or the plasma proteome; therefore, our method can be distinguished from the conventional CSF proteome analysis. Although the sensitivity of disease-specific IC-antigens detected in immune complexome analysis screening, the sensitivity may be improved by developing an ELISA method specifically for detecting the ICs. Immune complexome analysis of CSF may be a new and promising path to biomarker discovery for diagnosis and study for CNS autoimmune diseases., Clinica Chimica Acta, 484, pp.26-31; 2018

A scalable drain current model of AlN/GaN MIS-HEMTs has been developed for embedded source field-plate structures. The physically derived model is scalable to the length, width, and the number of fingers, and then implemented in a circuit simulator with Verilog-A source codes. To apply the models to simulate power switching applications including DC-DC converters, the weak inversion to linear characteristics and the maximum drain current are important. The model is verified with measured data of three dimensions and two sets of multi-finger transistor TEGs that we fabricated with an embedded source field plate technology. The results show good agreements between measurements and simulations.

Comprehensive identification and profiling of antigens in immune complexes (ICs) in biological fluids, such as serum and cerebrospinal fluid, is useful for developing early diagnostic markers and specific treatments for many diseases. We have developed a method, designated "immune complexome analysis", to comprehensively identify the antigens in ICs. In this method, we first purify ICs from biological fluid by using Protein G- or Protein A-coated beads, then these ICs are subjected to tryptic digestion on the beads and subsequent analysis using nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). We previously used this method to find specific antigens in circulating ICs (CIC-antigens) in serum for autoimmune diseases, infectious disease and cancers. However, this method detects not only CIC-antigens but also antibodies and proteins bound non-specifically to the beads, which restricts the detection of minor peptides released by the digestion of CIC-antigens whose amounts are generally much less than antibodies and the proteins. To selectively detect CIC-antigens with enhanced sensitivity, in this study we compared three methods (Method A, direct tryptic digestion on the beads; Method B, low-pH elution and tryptic digestion; Method C, papain-digestion, elution, and tryptic digestion) and examined which method selectively elutes CIC-antigens from CICs bound to the beads and selectively detects CIC-antigens using nano-LC-MS/MS. We also compared three types of CIC-capturing beads (Protein G-coated magnetic beads, Protein A-coated magnetic beads and Proceptor™-sepharose beads) to examine if parallel use of these beads aids the comprehensive detection of CIC-antigens in immune complexome analysis. Comparison showed that Method C provided the most selective and sensitive detection of CIC-antigens, without interference by antibodies and proteins non-specifically bound to the beads. In addition, using three types of beads allowed the examination of a wide range of CIC-antigens in immune complexome analysis. Therefore, combining Method C with three types of beads should allow the selective and sensitive identification of IC-antigens present in biological fluids from patients with a variety of diseases. The identification of IC-antigens may lead to the development of diagnostic methods and protocols for specific treatments for these diseases.

Trolox, a water-soluble vitamin E analogue has been used as a positive control in Trolox equivalent antioxidant capacity and oxygen radical antioxidant capacity assays due to its high antioxidative effect. In this study, the ex vivo antioxidative effects of Trolox and its concentration in blood and brain microdialysates from rat after administration were evaluated by newly established semi-microflow injection analysis, chemiluminescence detection and HPLC-UV. In the administration test, the antioxidative effect of Trolox in blood and brain microdialysates after a single administration of 200 mg/kg of Trolox to rats could be monitored. The antioxidative effects in blood (12.0 ± 2.1) and brain (8.4 ± 2.1, × 103 antioxidative effect % × min) also increased. Additionally, the areas under the curve (AUC)s0–360 (n = 3) for blood and brain calculated with quantitative data were 10.5 ± 1.2 and 9.7 ± 2.5 mg/mL × min, respectively. This result indicates that Trolox transferability through the blood–brain barrier is high. The increase in the antioxidative effects caused by Trolox in the blood and brain could be confirmed because good correlations between concentration and antioxidative effects (r ≥ 0.702) were obtained. The fact that Trolox can produce an antioxidative effect in rat brain was clarified. Copyright © 2015 John Wiley & Sons, Ltd.

A fast, simple, and sensitive flow injection analysis method was developed for the measurement of semicarbazide-sensitive amine oxidase (SSAO) activity in human serum. Benzaldehyde, generated by the action of SSAO after incubation of serum with benzylamine, was derivatized with a novel aromatic aldehyde-specific reagent (1,2-diaminoanthraquinone) and the fluorescent product was measured by fluorescence detection at excitation and emission wavelengths of 390 and 570nm, respectively. Serum SSAO activity was defined as benzaldehyde (nmol) formed per milliliter serum per hour. The method was linear over SSAO activity of 0.2-150.0nmolmL-1h-1 with a detection limit of 0.06nmolmL-1h-1. The %RSD of intra-day and inter-day precision did not exceed 9.4% and the accuracy ranged from -6.5 to -0.6%. The method was applied for the determination of the serum SSAO activity in healthy controls (C, n=24) and diabetes mellitus patients (DM, n=18). It was demonstrated that the activity (mean±SE) of SSAO in diabetics sera was significantly higher than that in healthy subjects' ones (DM; 73.3±1.8nmolmL-1h-1 vs C; 58.9±2.2nmolmL-1h-1, P, Analytica Chimica Acta, 881, pp.139-147; 2015

The ketamine analogue, 2-(3-methoxyphenyl)-2-(ethylamino)cyclohexanone (methoxetamine) has emerged as a drug of abuse. Both methoxetamine and ketamine are antagonists of glutamate N-methyl-d-asparate receptors, and several case reports show that methoxetamine produces similar schizophrenia-like symptoms and hallucinations to ketamine. Although methoxetamine is believed to change levels of dopamine, glutamate, and serotonin in the brain, few studies thus far have examined these effects. We investigated the influence of methoxetamine on dopamine and serotonin concentrations using microdialysis and high performance liquid chromatography with electrochemical detection. To reveal the effects of methoxetamine, we monitored dopamine and serotonin concentrations in several brain areas [striatum, nucleus accumbens, and prefrontal cortex (mPFC)] after an administration of 20 mg/kg of methoxetamine. We compared the effects of methoxetamine with those of ketamine using two ketamine doses. Methoxetamine increased dopamine and serotonin concentrations most robustly in the mPFC. In addition, its effects were stronger than those of ketamine at the same molar dose, suggesting that methoxetamine causes schizophrenia-like symptoms and hallucinations by increasing the dopamine and serotonin concentrations. We conclude that consumption of methoxetamine may be more dangerous than consumption of ketamine.

Acrolein is a major unsaturated aldehyde that is generated during the lipid peroxidation process. The measurement of acrolein in biological samples should be useful to estimate the degree of lipid peroxidation and to evaluate the effect of hazardous properties of acrolein on human health. In this study, a highly sensitive and selective high-performance liquid chromatography with fluorescence detection method was developed for the determination of acrolein in human serum. The proposed method involves the pre-column fluorogenic derivatization of acrolein with 1,2-diamino-4,5-dimethoxybenzene (DDB) as a reagent. The fluorescent derivative of acrolein could be detected clearly without any interfering reagent blank peaks because DDB does not have intrinsic fluorescence itself, and the detection limit was 10 nM (signal-to-noise ratio = 3). The proposed method could selectively detect acrolein in human serum with a simple protein precipitation treatment. Copyright © 2015 John Wiley & Sons, Ltd.

Neuropsychiatric systemic lupus erythematosus (NPSLE) is often difficult to diagnose and distinguish from other diseases, because no NPSLE-specific antibodies have been identified. We developed a novel proteomic strategy for identifying and profiling antigens in immune complexes in the cerebrospinal fluid (CSF), and applied this strategy to 26 NPSLE patients. As controls, we also included 25 SLE patients without neuropsychiatric manifestations (SLE), 15 with relapsing remitting multiple sclerosis (MS) and 10 with normal pressure hydrocephalus (NPH). We identified immune complexes of suprabasin (SBSN) in the CSF of the NPSLE group. The titer of anti-SBSN antibodies was significantly higher in the CSF of the NPSLE group compared to those of the SLE, MS and NPH groups. Microarray data showed that the senescence and autophagy pathways were significantly changed in astrocytes exposed to anti-SBSN antibodies. Our findings indicate that SBSN could be a novel autoantibody for the evaluation of suspected NPSLE.

Tanshinones are a major class of bioactive ingredients in the traditional herbal medicines, Danshen (Salvia miltiorrhiza). A sensitive and reliable determination method for tanshinones is useful to ensure the quality of Danshen.To develop a sensitive and selective analytical method for tanshinones by high-performance liquid chromatography (HPLC) with fluorescence detection after pre-column derivatisation.The proposed method depends on derivatisation reaction of tanshinones with 4-carbomethoxybenzaldehyde and ammonium acetate forming intensely fluorescent imidazole derivative.The proposed method provided excellent sensitivity with the detection limits of 3.3 nM (66 fmol/injection), 3.2 nM (64 fmol/injection) and 2.0 nM (40 fmol/injection) for cryptotanshinone, tanshinone I and tanshinone IIA, respectively, without the necessity of complicated instrumentations. The developed method is successfully applied to quantify the contents of tanshinones in Danshen.The developed method is the first analytical method for tanshinones by fluorescence detection. Since the derivatisation reaction is selective for the o-quinone structure of tanshinone, the developed method will become a suitable mean for the discovering of tanshinone type diterpenoids from herbal samples. Copyright © 2017 John WileySons, Ltd.

A fast, accurate, and ultrasensitive high-performance liquid chromatography method with chemiluminescence detection (HPLC-CL) was optimized and validated for the determination of pyrroloquinoline quinone (PQQ) concentration in human plasma following solid-phase extraction (SPE). This method is based on the redox cycle of the reaction between PQQ and dithiothreitol, which generates reactive oxygen species that can be detected using luminol as a CL probe. The isocratic HPLC system comprised an ODS column and 4.0 mM tetra-n-butylammonium bromide in Tris-HNO3 buffer (pH 8.8; 50 mM)-acetonitrile (7:3, v/v) as mobile phase. A novel, rapid, and simple SPE method was also developed providing excellent %recovery (?95.2%) for PQQ from human plasma samples. The proposed method was linear over the range of 4.0?400 nmol/L plasma of PQQ with a lower detection limit (S/N=3) of 1.08 nmol/L plasma (0.27 nM). The method was successfully implemented to determine PQQ concentration in the plasma of healthy individuals after administration of PQQ supplements., Journal of Pharmaceutical and Biomedical Analysis, 145, pp.814-820; 2017

A validated, simple and sensitive HPLC method was developed for the simultaneous determination of lipoperoxidation relevant reactive aldehydes: glyoxal (GO), acrolein (ACR), malondialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) in human serum. The studied aldehydes were reacted with 2,2′-furil to form fluorescent difurylimidazole derivatives that were separated on a C 18 column using gradient elution and fluorescence detection at excitation and emission wavelengths of 250 and 355 nm, respectively. The method showed good linearity over the concentration ranges of 0.100-5.00, 0.200-10.0, 0.200-40.0, and 0.400-10.0 nmol/mL for GO, ACR, HNE, and MDA, respectively, with detection limits ranging from 0.030 to 0.11 nmol/mL. The percentage RSD of intraday and interday precision did not exceed 5.0 and 6.2%, respectively, and the accuracy (%found) ranged from 95.5 to 103%. The proposed method was applied for monitoring the four aldehydes in sera of healthy, diabetic, and rheumatic human subjects with simple pretreatment steps and without interference from endogenous components. By virtue of its high sensitivity and accuracy, our method enabled detection of differences between analytes concentrations in sera of human subjects under different clinical conditions., Analytical Biochemistry, 464, pp.36-42; 2014

Sulfur-containing amino acids (SAAs), homocysteine (Hcy), methionine (Met) and cysteine (Cys) in blood are related to homocystinuria, an inborn error of metabolism. In this study, an assay method with HPLC-fluorescence detection to quantify the SAAs in a dried blood spot was established and applied to samples from newborn babies (n=200). Sample pretreatment involving reduction, derivatization with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole, and liquid-liquid extraction with ethyl acetate gave the separation of the derivatives with retention times within 12 min. The method was enough sensitive to determine the SAAs in a dried blood spot with 0.04-0.14 µm as the limit of detection at a signal-to-noise ratio of 3. However, the absolute recoveries were very low (5.7% for Hcy, 4.6% for Cys) except for Met (105.4%) owing to inefficient recovery of Hcy and Cys from the blood matrix. Other validation parameters such as accuracy (93.5-106.2%) and intra- (≤ 9.0%) and inter-day precisions (≤ 8.7%) were acceptable. The reliability of a dried blood spot as an analytical sample was estimated. Furthermore, the proposed method was successfully applied to dried blood spots prepared from newborn babies.

A highly sensitive, selective and reproducible chromatographic method is described for determination of low-molecular mass unsaturated aliphatic aldehydes in human serum. The method combines fluorescent labeling using 4-(N,N-Dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole with peroxyoxalate chemiluminescence. The derivatives were separated on a reversed-phase column C8 isocratically using a mixture of acetonitrile and 90mM imidazole-HNO3 buffer (pH 6.4, 1:1, % v/v). The calibration ranges were: 20-420nM for methylglyoxal, 16-320nM for acrolein, 15-360nM for crotonaldehyde and 20-320nM for trans-2-hexenal. The detection limits were ranged from 4.4 to 6.5nM (88-130fmol/injection), the recovery results were within the range of 87.4-103.8% and the intra and inter-day precision results were lower than 5.5%. The proposed validated method has been successfully applied to healthy, diabetic and rheumatic arthritis patients' sera with simple pretreatment method. In conclusion, this new method is suitable for routine analysis of large numbers of clinical samples for assessment of the oxidative stress state in patients., Journal of Chromatography B, 953-954; pp.147-152; 2014

Quinones are compounds that have various characteristics such as biological electron transporter, therapeutic agent and harmful environmental pollutant. Therefore, an effective analytical method for quinones is useful in many fields including biomedical, clinical and toxicological studies. This review describes the principle and feature of analytical techniques for quinones including high-performance liquid chromatography with ultraviolet, fluorescence, chemiluminescence, electrochemical detection and mass spectrometry, gas chromatography with mass spectrometry and capillary electrophoresis. Furthermore, the sensitivity and the sample preparation method for the determination of several quinones such as vitamin K, ubiquinone, doxorubicin and polycyclic aromatic hydrocarbon quinone in biological and environmental samples are summarized., Journal of Pharmaceutical and Biomedical Analysis, 87, pp.261-270; 2014

In vitro screening of a Fe 2+ -chelating effect using a Fenton's reaction-luminol chemiluminescence (CL) system is described.TheluminescencebetweenthereactiveoxygenspeciesgeneratedbytheFenton'sreactionandluminolwasdecreased on capturing Fe 2+ using a chelator. The proposed method can prevent the consumption of expensive seed compounds (drug discovery candidates) owing to the high sensitivity of CL detection. Therefore, the assay could be performed using small volumes of sample solution (150 μL) at micromolar concentrations. After optimization of the screening conditions, the effica- cies of conventional chelators such as ethylenediaminetetraacetic acid (EDTA), diethylentriaminepentaacetic acid (DETAPAC), deferoxamine, deferiprone and 1,10-phenanthroline were examined. EC50 values for these compounds (except 1,10- phenanthroline) were in the range 3.20±0.87 to 9.57±0.64 μ M( n=3). Rapid measurement of the Fe 2+ -chelating effect with an assay run time of a few minutes could be achieved using the proposed method. In addition, the specificity of the method was discussed. Copyright © 2014 John Wiley & Sons, Ltd.

A chemiluminescence (CL) reaction of folic acid (FA) with ruthenium (II) and cerium (IV) was applied to quantify FA-related compounds such as FA, dihydrofolic acid, tetrahydrofolic acid, 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid and methotrexate (MTX). Among the FAs, 5-methyltetrahydrofolic acid provided the highest CL intensity. HPLC-CL detection of FA was applied to quantify FA in pharmaceutical preparations and supplements. Analytical samples were separated on a semi-micro ODS column with a mixture of 20 mM phosphate buffer (pH 5.7) and acetonitrile (94 : 6, v/v %). The separated samples were mixed with a post-column CL reagent consisting of 1.5 mM Ru(bipy)32+ and 1.0 mM Ce(SO4)2, then the generated CL was monitored. The calibration range for FA was 10–100 μM and the limit of detection was 1.34 μM (signal-to-noise ratio of 3). Repeatabilities were 4.2, 4.6 and 5.0 RSD% (10, 25, 50 μM), and the recoveries for FA supplement, vitamin B complex supplement and FA-containing medication (tablet) were 102.4 ± 10.5, 103.3 ± 13.3 and 100.3 ± 8.5%, respectively. The described method is robust against changes in the chromatographic parameters of ± 3.3 or ± 1.5%. The measured FA content corresponded well to the labeled content of FA-containing products (100.6–104.9%), demonstrating the precision and accuracy of this method for the evaluation of FA pharmaceutical preparations. Copyright © 2014 John Wiley & Sons, Ltd.

A simple and selective HPLC-fluorescence (FL) method with FL probe, 4-[4-(4-dimethylaminophenyl)-5-phenyl-1. H-imidazol-2-yl]benzoic acid methyl ester (DAPIM), for simultaneous determination of mercaptalbumin (HMA) and nonmercaptalbumin (HNA1) was developed. After HMA and HNA1 were separated on an ion-exchange column, they were on-line and post-column mixed with DAPIM. The DAPIM-albumin complex produces FL (λex 370. nm and λem 510. nm); however, DAPIM solution never gives the FL. Based on this mechanism, selective determination of HMA and HNA1 were achieved without any pretreatment and interfering peak. The proposed method was applied to the measurement of HMA and HNA1 in human serum of healthy volunteers and diabetes mellitus patients., Journal of Pharmaceutical and Biomedical Analysis, 88, pp.170-173; 2014

A simple, rapid, and sensitive microplate assay method for determination of ubiquinone in cosmetic products has been developed and validated. This method is based on measuring reactive oxygen species (ROS) generated from the redox cycle of quinone using redox colorimetric reagent, 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT). The reaction with ROS converts INT to a formazan dye having a strong absorbance at 510 nm that is increased with increasing concentration of ubiquinone. On the basis of this reaction, we developed a microplate assay method that allows high-throughput measurement of ubiquinone. This method uses microplate reader for the measurement of absorbance and it can determine many samples at one time. Under the optimized conditions, the proposed method was linear in the range of 0.02–4 μM and the limit of detection (blank +3SD) was 14.8 nM. We applied the proposed method to determine ubiquinone contents in cosmetic products, and the measurement values were in good agreement with the indicated value. The proposed colorimetric method should be useful as a high-throughput analysis for quality management of ubiquinone containing products.

Aldehydes are very reactive carbonyl compounds that are widespread naturally. Aldehydes can be produced in vivo by oxidative reactions and are able to disturb biological functions through binding and modifying biomolecules. Thus, it is necessary to determine their levels in biological samples to estimate their possible adverse effect on human health in addition to their consideration as a biomarker for oxidative stress-related diseases. Furthermore, aldehydes have been found in foodstuffs as by-products of food processing or deterioration and their amounts in food samples were used as an indicator of its quality. Aldehydes also are widely distributed in the environment sourced from the industrial and motor vehicular exhausts and human exposure to them could bring many adverse health effects. From these viewpoints, an effective analytical method for the determination of aldehydes should be essential in various fields including biological, clinical, environmental, and food sciences. Among analytical methodologies, chromatographic determination methods should be suitable tools for the simultaneous determination of wide variety of aldehydes. Derivatization reactions are frequently applied for aldehydes to improve their detection sensitivity as most of them do not possess detectable moiety. Also, derivatization can control their retention behavior on HPLC columns. Moreover, sample pretreatment procedures for aldehydes to modify their high volatility, reactivity, polarity, and inherent instability is vital for their pre-concentration and avoiding matrix effects. In this review, chromatographic determination methods for aldehydes are summarized mainly according to the recent reports with the analytical techniques including the effective extraction and chemical derivatization. Besides, the applications for the chromatographic determination of aldehydes are summarized and significant findings obtained by the application studies are described., Journal of Pharmaceutical and Biomedical Analysis, 175, art.no.112782; 2019

Immune complexome analysis is a method for identifying and profiling of antigens in circulating immune complexes (CICs); it involves separation of immune complexes from serum, direct tryptic digestion of these complexes, and protein analysis via nano-liquid chromatography–tandem mass spectrometry (nano-LC–MS/MS). To improve this method, we initially investigated the effects of two factors—the gradient elution program and nano-LC column type (C18-packed, C8-packed, or packed spray capillary column)—on the numbers of peptides and proteins identified. Longer gradient elution times resulted in higher identification capability throughout the range of 25–400 min. Moreover, the packed spray capillary column supported identification of more peptides and proteins than did any other column. In addition, microwave-assisted digestion was compared with conventional digestion, which involved incubation overnight at 37 °C. Microwave-assisted digestion produced more partially digested peptides than did conventional digestion. However, the percentages of miscleaved peptides in all of the identified peptides in microwave-assisted digestion of immune complexes (a protein mixture) were lower than those in the physical stimulation-assisted digestion of a model protein. Microwave-assisted digestion is slightly inferior to, or as effective as, conventional digestion, but it drastically reduces the digestion time., Analytical Biochemistry, 443(2), pp.181-186; 2013

IgG4-related disease (IgG4-RD) is characterized by various serological abnormalities. Some patients with IgG4-RD present with hypergammaglobulinemia, hypocomplementemia, and autoantibodies recognizing rheumatoid factors and nuclear factors. However, whether IgG4-RD is an autoimmune disease remains unclear.Here, we used immune complexome analysis to comprehensively identify constituent antigens of circulating immune complexes isolated from 10 patients with IgG4-related dacryoadenitis and/or sialadenitis (IgG4-RDS) which is one condition associated with IgG4-RD.We detected each of 125 distinct antigens in independent samples from two or more patients with IgG4-RDS. Of them, 17 antigens were found to be specific to patients with IgG4-RDS by comparing 125 antigens with all the antigens found in other connective tissue diseases (antineutrophil cytoplasmic antibody-associated vasculitis, Takayasu's arteritis, mixed connective tissue disease, dermatomyositis, Sjögren's syndrome, systemic scleroderma, and systemic lupus erythematosus) or healthy donors. The number of disease-specific antigens associated with IgG4-RDS was comparable to those autoimmune diseases.Studies of the 17 IgG4-RDS-specific antigens might lead to a better understanding of the pathogenesis of IgG4-RD, but further study of the prevalence of these CIC-associated antigens that involves a selective and sensitive assay will be needed to determine their potential as diagnostic or pathogenic biomarkers.

The present study aims to investigate the possibility of interaction of donepezil (DP) and galantamine (GAL) as acetylcholinestrase inhibitors, on memantine (MT) hydrochloride in rat plasma by HPLC-fluorescence detection. The separation of MT was achieved within 12 min without interference of DP and GAL on the chromatogram. MT levels in rat plasma with a single administration of MT (2.5 mg/kg, i.p.) and those with a co-administration of DP (5.0 mg/kg, i.p.) and GAL (3 mg/kg, i.p.) were monitored. MT concentrations determined in rat plasma ranged from 10.0 to 245.6 ng/mL. Significant difference was observed in the behavior of MT with a co-administration of DP, while no significant difference was observed with a co-administration of GAL.

Paraquat (N,N′-dimethyl-4,4′-bipyridinium dichloride) is one of the most widely used herbicides owing to its high efficacy and low environmental persistence. However, because paraquat has significant acute toxicity, fatalities are often caused by accidental or voluntary ingestion of paraquat. In consideration of the strong toxicity and fast-Acting property of paraquat, on-site analysis at accident scenes should be effective in facilitating immediate medical treatment. In this study, a simple and rapid chemiluminescence assay using a portable luminometer was developed for on-site analysis of paraquat. The proposed assay is based on luminol chemiluminescence detection of superoxide anion radical resulting from the redox reaction between paraquat and dithiothreitol. Intense chemiluminescence was observed after mixing of paraquat and dithiothreitol in the presence of luminol. Because the chemiluminescence intensity was proportional to the concentration of paraquat, a quantitative measurement of paraquat was possible. The calibration curve for standard paraquat solution was linear from 0.025 to 2.5 μM with the correlation coefficient of 0.992; the detection limit (blank + 3SD) was 22 nM. The proposed assay was applied to determine paraquat in beverage samples after a cation exchange clean-up procedure. Given that the portable luminometer used in this study is small and lightweight, the proposed assay should be useful for on-site analysis of paraquat., Forensic Toxicology, 31(2), pp.301-306; 2013

A fluorogenic derivatization method for the determination of chlorpropamide in human serum was developed by means of high-performance liquid chromatography (HPLC) with fluorescence detection. The Suzuki coupling reaction with a non-fluorescent reagent, phenylboronic acid (PBA), was employed to convert chlorpropamide into highly fluorescent biphenyl derivative. Chlorpropamide was extracted from human serum by liquid-liquid extraction with toluene after addition of hydrochloric acid, and subsequently reacted with PBA. Because the fluorogenic derivatization was highly selective for aryl halide, the proposed method allowed sensitive and selective detection of chlorpropamide with a detection limit (at a signal to noise ratio of 3) of 0.5 ng mL-1. The sensitivity of our method was from 4 to 100 times better than HPLC-UV, gas chromatography, and LC-mass spectrometry., Chromatographia, 76(11-12), pp.703-706; 2013

The binding of a lophine-based fluorescence probe, 4-[4-(4-dimethylaminophenyl)-5-phenyl-1H-imidazol-2-yl]benzoic acid methyl ester (DAPIM) with human serum albumin (HSA) was investigated by fluorescence spectroscopy under physiological conditions. While DAPIM shows extreme low fluorescence in aqueous solution, DAPIM binding with HSA emits strong fluorescence at 510nm. The binding constant and binding number determined by Scatchard plot was 3.65×106M-1 and 1.07, respectively. Competitive binding between DAPIM and other ligands such as warfarin, valproic acid, diazepam and oleic acid, were also studied fluorometrically. The results indicated that the primary binding site of DAPIM to HSA is site II at subdomain IIIA. DAPIM can be a useful fluorescence probe for the characterization of drug-binding sites. In addition to the interaction study, because the fluorescence intensity of DAPIM increased in proportion to HSA concentration, its potential in HSA assay for serum sample was also evaluated., Analytica Chimica Acta, 780, pp.1-6; 2013

Butyl methacrylate (BMA)-ethylene dimethacrylate (EDMA)-methacrylic acid (MAA) and BMA-EDMA-2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) monolithic columns were prepared by varying the percentage of ionic monomers for capillary electrochromatography. Monolithic columns with a higher content of ionic monomers provided better column efficiency, and the performance of BMA-EDMA-MAA monoliths was better than BMA-EDMA-AMPS. To characterize and optimize BMA-EDMA-MAA monoliths, the effects of the content of cross-linker and the total monomer in the polymerization mixture on column performance were also studied. Plate heights of 8.2μm for the unretained solute (thiourea) and 12.6μm for the retained solute (naphthalene) were achieved with a monolithic column using 2.5% MAA (Column I)., Journal of Chromatographic Science, 51(5), pp.425-429; 2013

8-Amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione (L-012) was recently synthesized as a new chemiluminescence (CL) probe; the light intensity and the sensitivity of L-012 are higher than those of other CL probes such as luminol. Previously, our group developed four lophine-based CL enhancers of the horseradish peroxidase (HRP)-catalyzed CL oxidation of luminol, namely 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI), 2-(4-hydroxyphenyl)-4,5-di(2-pyridyl)imidazole (HPI), 4-(4,5-diphenyl-1H-imidazol-2-yl)phenylboronic acid (DPA), and 4-[4,5-di(2-pyridyl)-1H-imidazol-2-yl]phenylboronic acid (DPPA), and showed that DPPA was suitable for the photographic detection of HRP. In this study, we replaced luminol with L-012 and evaluated these as L-012-dependent CL enhancers. In addition, to detect HRP and/or H2O2 with higher sensitivity, each detection condition for the L-012-HRP-H2O2 enhanced CL was optimized. All the derivatives enhanced the L-012-dependent CL as well as luminol CL; HPI generated the highest enhanced luminescence. Under optimized conditions for HRP detection, the detection limit of HRP was 0.08 fmol. By contrast, the detection limit of HRP with the enhanced L-012-dependent CL using 4-iodophenol, which is a common enhancer of luminol CL, was 1.1 fmol. With regard to H2O2 detection, the detection limits for enhanced CL with HPI and 4-iodophenol were 0.29 and 1.5 pmol, respectively. Therefore, it is demonstrated that HPI is the most superior L-012-dependent CL enhancer.

Isoamyl nitrite is used as a therapeutic reagent for cardiac angina and as an antidote for cyanide poisoning, but it is abused because of its euphoric properties. Therefore, a method to determine isoamyl nitrite is required in many fields, including pharmaceutical and forensic studies. In this study, a simple, rapid and sensitive method for the determination of isoamyl nitrite was developed using a flow injection analysis system equipped with a chemiluminescence detector and on-line photoreactor. This method is based on on-line ultraviolet irradiation of isoamyl nitrite and subsequent luminol chemiluminescence detection without the addition of an oxidant. A linear standard curve was obtained up to 1.0 μM of isoamyl nitrite with a detection limit (blank + 3SD) of 0.03 μM. The method was successfully applied to determine isoamyl nitrite content in pharmaceutical preparations. Copyright © 2013 John Wiley & Sons, Ltd.

An HPLC-fluorescence (FL) method for determination of sulfur-containing amino acids such as homocysteine (Hcy), methionine (Met) and cysteine (Cys) in human plasma was developed. The sulfur-containing amino acids were labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F). Calibration curves in the range of 1–100 µm (Hcy and Met) and 5–500 µm (Cys) indicated good linearities (r ≥ 0.998). The limits of detection at a signal-to-noise ratio of 3 were 0.13 (Hcy), 0.02 (Met) and 0.11 µm (Cys), respectively. Acceptable results for accuracy and precision of intra- and inter-day measurements were obtained. The results of Hcy and Cys obtained by the proposed method indicated good correlations with the conventional method (r > 0.911, n = 20). Furthermore, the method was applied to determination of the sulfur-containing amino acids in maternal plasma (n = 200) after delivery. The concentrations of Hcy, Met and Cys as a median (inter quartile range, Q1 and Q3) were 5.37 (3.32–7.79) μm, 25.20 (20.10–31.06) μm and 147.25 (102.81–189.31) μm, respectively. Copyright © 2012 John Wiley & Sons, Ltd.

The biological importance of aminothiols is well recognized with the concentration of these compounds within biological fluids such as plasma and urine functioning as valuable biomarkers in a number of clinical circumstances and a wide variety of diseases. Herein, for the first time, chromatographic coupled chemiluminescent assay was used for simultaneous determination of aminothiols in human urine. The method exploits nucleophilic nature of aminothiols to form adducts in the existence of quinones. The released adducts retain the redox-cycling capability of parent quinones and able to liberate reactive oxygen species (ROS) when come in contact with dithiothreitol (DTT). Strong glow is released upon reaction of ROS with luminol. The method succeeded to determine aminothiols in human urine after solid phase extraction achieving good linearity and high sensitivity shown by low limit of detection (LOD) ranged from 3.8 to 16 (fmol per injection).

Electron mobility has been characterized for drain current simulations of AlN/GaN MIS-HEMTs. We especially focus on the embedded source field-plate structures (ESFP) for high power applications. To apply the models to simulate power switching applications including DC-DC converters, electron mobility equations are the key to characterize DC bias conditions under dynamic operations. The model is implemented in MIT Virtual Source model with modifications of Verilog-A source codes. The model parameters are extracted from measured data of the transistor test structures that we fabricated with an ESFP technology. The results show excellent agreements between measurements and simulations.

Electron mobility and self-heating models for drain current simulations of AlN/GaN MIS-HEMTs have been derived for embedded source field-plate structures. They are scalable physical models. To apply the models to simulate power switching applications including DC-DC converters, the weak inversion to linear characteristics and the maximum drain current are important. The models are implemented in MIT Virtual Source model with modifications of Verilog-A source codes. The model parameters are extracted from measured data of the transistor test structures that we fabricated with an embedded source field-plate technology. The results show excellent agreements between measurements and simulations.

Immune complexes (ICs) are the direct and real-time products of humoral immune responses. The identification of constituent foreign or autoantigens within ICs might bring new insights into the pathology of infectious diseases. We applied immune complexome analysis of plasma to the study of Chagas disease caused by Trypanosoma cruzi. Twenty seropositive plasma samples including cardiac and/or megacolon determinate patients (n = 11) and indeterminate (n = 9) were analysed along with 10 seronegative individuals to characterize the antigens bound to circulating ICs. We identified 39 T. cruzi antigens and 114 human autoantigens specific to patients with Chagas. Among those antigens, two T. cruzi antigens (surface protease GP63, glucose-6-isomerase) and six human autoantigens (CD180 antigen, ceruloplasmin, fibrinogen beta chain, fibrinogen beta chain isoform 2 preprotein, isoform gamma-A of fibrinogen γ-chain, serum paraoxonase) were detected in more than 50% of the patients tested. Human isoform short of complement factor H-related protein 2 and trans-sialidase of T. cruzi were more frequently found in the indeterminate (5/9 for both) compared with in the determinate Chagas (0/11, P = 0・046 for human, 1/11, P = 0・0498 for T. cruzi). The immune complexome could illustrate the difference of immune status between clinical forms of chronic Chagas disease., Parasite Immunology, 38(10), pp.609-617; 2016

Quinones are widely distributed in nature and have various bioactivities. Besides, quinones are also considered as toxicological intermediates which cause severe dangerous effects. Hereby, a sensitive, simple, and rapid method is reported for quinones determination. The proposed method employed time resolved fluorescence (TRF) microplate reader based chemiluminescent (CL) detection for the first time as a novel approach for measurement. Under pulse photo-irradiation, the unique photochemical characteristic of quinones is exploited to liberate reactive oxygen species (ROS) which reacted with photosensitized CL reagent. L-012, luminol analogue, was selected for its high sensitivity. Under our investigation, para-quinones showed high CL response when compared to ortho-quinones. A linear response was obtained for studied quinone concentrations in the range of 0.05-50 μM for 1,4-naphthquinone and of 0.05-150 μM for 2-methyl-1,4-naphthoquinone (menadione) and 9,10-anthraquinone with detection limit (blank + 3SD) of 0.01 μM. The proposed method allowed the rapid determination of large number of samples in very short time (96 sample/125 s). The proposed method was successfully applied for determination of menadione in spiked human serum., Analyst, 137(20), pp.4802-4808; 2012

Simultaneous determination of amphetamine-type stimulants (ATSs), such as methamphetamine (MA), amphetamine (AP), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), and p-methoxymethamphetamine (PMMA), has been made for hair of 32 female abusers from different ethnic backgrounds. One milligram of abuser's hair spiked with 1-methyl-3-phenylpropylamine as internal standard was used for each determination of the ATSs. After digestion with 1 M NaOH, the samples were extracted with n-heptane; the organic layer was evaporated to dryness, dissolved in 75 mM of borate buffer solution (pH 8.5), and labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole. The determination of the ATSs in hair was performed by the semi-micro high-performance liquid chromatography-chemiluminescence method. Thirty-two subjects who had self-report histories of ATS use for more than 1 week prior to hair sample collection participated in this study. Concentration ranges of ATSs in abusers’ hair were 0.07–6.73 ng/mg for MA (n = 24), 0.18–2.47 ng/mg for AP (n = 13), 0.05–0.86 ng/mg for MDMA (n = 12), 0.52–1.38 ng/mg for MDA (n = 3), and 0.09–1.88 ng/mg for PMMA (n = 6). Several discrepancies between detected drugs and information obtained from the interviewed abusers were found. Most of the drugs determined in this study could not be detected by the urine test performed at hospitalization. Correlation between ATS concentration and psychiatric symptoms on the basis of Brief Psychiatric Rating Scale (BPRS) was also studied. Simultaneous determination of ATSs in hair of outpatients and inpatients at psychiatric clinics and hospitals should be effective in providing proper diagnosis and treatment. This is the first report to demonstrate the presence of PMMA in hair of human subjects.