[A Method Based on Ru(bpy)3 3+ Electrogenerated Chemiluminescence for Assessment of Antioxidant Property of Naturally Occurring Antioxidants].

We developed a flow injection analysis (FIA) method based on tris(2,2'-bipyridine)ruthenium(III) [Ru(bpy)3 3+] electrogenerated chemiluminescence for assessment of antioxidant property. The hydroxyl radical (∙OH) were generated by H2O photolysis using an ultraviolet/H2O photoreactor. The reactor comprised a polytetrafluoroethylene tube, a quartz container, and a low-pressure mercury lamp that predominantly emitted radiation at around 185 nm. When the hydroxyl radical and Ru(bpy)3 3+ were in contact, chemiluminescence was generated as background emission. The background emission decreased when antioxidant samples were injected to the system. The antioxidant property of the naturally occurring antioxidants tested are listed herein, starting with the highest: gallic acid>ascorbic acid>quercetin. Moreover, our method allowed a sample throughput of approximately 100 samples/h. The proposed high throughput method can be used to assess the antioxidant property of the naturally occurring antioxidants.

Determination of Miglitol by Column-Switching Ion-Pair HPLC with Tris(2,2'-bipyridine)ruthenium(II)-Electrogenerated Chemiluminescence Detection.

We have developed a highly sensitive, simple method for the quantitative determination of miglitol in standard serum samples using column-switching ion-pair HPLC with tris(2,2'-bipyridine)ruthenium(II)-electrogenerated chemiluminescence detection. The serum samples were directly injected into a column-switching HPLC system with a Shim-pack MAYI-SCX precolumn to remove the serum matrix. Chromatographic separation of miglitol was achieved on a TSKgel ODS 100-V column using a mobile phase containing sodium 1-octanesulfonate as an ion-pair reagent. The detection and quantification limits of miglitol were 3 and 10 ng/mL, respectively. The calibration curve for miglitol in the serum samples showed good linearity (r(2)=0.9997) in the range of 10-2500 ng/mL. The recovery rate of miglitol from the serum samples was more than 94% as calculated from blank serum samples spiked with miglitol 50, 100, 500, 1000, and 2000 ng/mL. Therefore, this method can be applied to routine therapeutic monitoring of miglitol in serum samples.

Degradation of corticosteroids during activated sludge processing.

Laboratory tests of the decomposition of corticosteroids during activated sludge processing were investigated. Corticosteroid standards were added to activated sludge, and aliquots were regularly taken for analysis. The corticosteroids were extracted from the samples using a solid-phase extraction method and analyzed LC-MS. Ten types of corticosteroids were measured and roughly classified into three groups: 1) prednisolone, triamcinolone, betamethasone, prednisolone acetate, and hydrocortisone acetate, which decomposed within 4 h; 2) flunisolide, betamethasone valerate, and budesonide of which more than 50% remained after 4 h, but almost all of which decomposed within 24 h; and 3) triamcinolone acetonide, and fluocinolone acetonide of which more than 50% remained after 24 h. The decomposed ratio was correlated with each corticosteroid's Log P, especially groups 2) and 3).

Electrochemical detection of sugar-related compounds using boron-doped diamond electrodes.

Electrochemical detection of sugar-related compounds was conducted using a boron-doped diamond (BDD) electrode as a detector for flow-injection analysis (FIA). Sugar-related compounds oxidize at high applied potentials, for which the BDD electrode is suitable for electrochemical measurements. Conditions for an FIA system with a BDD detector were optimized, and the following detection limits were achieved for sugar-related compounds: monosaccharides, 25-100 pmol; sugar alcohols, 10 pmol; and oligosaccharides, 10 pmol. The detection limit for monosaccharide D-glucose (Glu) was 105 pmol (S/N = 3). A linear range was acquired from the detection limit to 50 nmol, and the relative standard deviation was 0.65% (20 nmol, n = 6). A high-performance liquid chromatography (HPLC) column was added to the system between the sample injector and the detector and detection limits to the picomole level were achieved, which is the same for the HPLC system and the FIA system. The electrochemical oxidation reaction of Glu was examined using cyclic voltammetry with the BDD detector. The reaction proved to be irreversible, and proceeded according to the following two-step mechanism: (1) application of a high potential (2.00 V vs. Ag/AgCl) to the electrode causes water to electrolyze on the electrode surface with the simultaneous generation of a hydroxyl radical on the surface, and (2) the hydroxyl radical indirectly oxidizes Glu. Thus, Glu can be detected by an increase in the oxidation current caused by reactions with hydroxy radicals.

Development and application of a method to investigate drug-metabolizing enzyme inhibitors using sparteine for probe of cytochrome P450 2D6 and tris(2,2'-bipyridine)ruthenium(II)-electrogenerated chemiluminescence detection.

We studied the detection of drug-metabolizing enzyme inhibitiors using column-switching high performance liquid chromatography with tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)(3)(2+))-electrogenerated chemiluminescence detection. This can be applied to evaluate the genetic diversity concerning the ability of cytochrome P450 (CYP) 2D6 to metabolize drug in vitro. We demonstrated the ability of CYP2D6 to enable us to examine drugs metabolizing enzyme inhibition with high performance and sensitivity. This method can be applied to investigate metabolite inhibitors of CYP2D6 in vitro and in vivo. Thus, Metixene was found to be a potential CYP2D6 inhibitor.

Column-switching HPLC determination of mexiletine using tris(bipyridine)ruthenium(III) electrogenerated chemiluminescence and precolumn derivatization with divinylsulfone.

A sensitive HPLC method combined with a column-switching system and tris(bipyridine)ruthenium(III) electrogenerated chemiluminescence (ECL) detection has been developed for the quantitative determination of mexiletine (MEX). MEX was derivatized by divinylsulfone (DVS) and then measured. The optimum conditions for the derivatization reaction were 10 µL of sample solutions, 40 mM DVS (pH 8.0), a reaction temperature of 50°C, and a reaction time of 15 min. The derivatized samples were cleaned up by an on-line pretreatment column. Also, after column-switching to the analytical column, the derivatized MEX was separated and detected. The calibration curves of MEX in human control serum showed good linear regression (r = 0.9996) from 0.008 to 6.56 µg ml(-1). The detection limit of MEX was 0.008 µg ml(-1) (S/N = 3). At a concentration of 2.0 µg ml(-1) MEX, the relative standard deviation (n = 5) was 0.98%. In this method the concentration of MEX in human control serum was readily measured, and this method was successfully applied to the time courses of the concentration of MEX in rabbit plasma after intravenous administration. The proposed method involved a simple and minimum sample-preparation procedure and a short run time (<20 min). Therefore it can be applied to routine therapeutic monitoring and pharmacokinetic studies of MEX.

Selective detection of hydroxyl radical scavenging capacity based on electrogenerated chemiluminescence detection using tris(2,2'-bipyridine)ruthenium(III) by flow injection analysis.

We describe a new method for detecting hydroxyl radical scavenging capacity based on tris(2,2'-bipyridine)ruthenium(III) [Ru(bpy)(3)(3+)] chemiluminescence and flow injection analysis. Hydroxyl radicals were generated by the Fenton reaction. The scavenging capacity of the six antioxidants tested decreased in the following order: edaravone>L-tryptophan>gallic acid>Trolox>N-acetyl-L-cysteine>ascorbic acid. The proposed method allowed a sample throughput of about 80 samples/h. The six antioxidants were found to inhibit chemiluminescence intensity of Ru(bpy)(3)(2+). The proposed method is a rapid, selective, and accurate procedure for the study of hydroxyl radical scavenging capacity by Ru(bpy)(3)(3+) chemiluminescence.

Application of an improved proteomics method, fluorogenic derivatization-liquid chromatography-tandem mass spectrometry, to differential analysis of proteins in small regions of mouse brain.

To identify age-related proteins in small regions of mouse brain, we improved a proteomics approach, fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS), and applied the method to the differential proteome analysis of aging in cerebral cortex, hippocampus and brainstem. The method showed good accuracy with RSDs <10% for between-day protein peak heights, and much better sensitivity for the detection of proteins compared to other proteomics approaches. The existence of 28 regionally specific age-related proteins in mouse brain was demonstrated. These results verified that the small brain regions could be the targets for proteome analysis by the FD-LC-MS/MS method.

Existence of low-molecular-weight thiols in Caenorhabditis elegans demonstrated by HPLC-fluorescene detection utilizing 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide.

A highly sensitive and simple method using HPLC-fluorescence detection with 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-Cl) as a fluorogenic reagent demonstrated the existence of the low-molecular-weight thiols in the extract of Caenorhabditis elegans (C. elegans). The method includes derivatization of the thiols with DAABD-Cl at 40 degrees C for 10 min in borate buffer (pH 9.0) containing TCEP, CHAPS and EDTA, separation of the derivatives on an ODS column and fluorometric determination of the derivatives at 510 +/- 15 nm with excitation at 400 +/- 15 nm. The identification of the thiols was made by HPLC-electrospray ionization mass spectrometry (LC-MS) following isolation of the derivatives using HPLC-fluorescence detection. Low-molecular-weight thiols were found to exist in the extract of C. elegans, such as cysteine, cysteinylglycine, gamma-glutamylcysteine, reduced glutathione and two other unidentified thiol compounds, confirming the existence of the 'glutathione cycle' in C. elegans similar to the mammalian body.

Triterpene acids from Poria cocos and their anti-tumor-promoting effects.

The structures of six new lanostane-type triterpene acids isolated from the epidermis of the sclerotia of Poria cocos were established to be 15alpha-hydroxydehydrotumulosic acid (5), 16alpha,25-dihydroxydehydroeburicoic acid (9), 5alpha,8alpha-peroxydehydrotumulosic acid (10), 25-hydroxyporicoic acid H (11), 16-deoxyporicoic acid B (12), and poricoic acid CM (16) on the basis of spectroscopic methods. On evaluation of these six and 11 other known triterpene acids isolated from the sclerotium, 1-4, 6-8, 13-15, and 17, against the Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells, all of the compounds except for 1, 3, 4, and 8 exhibited inhibitory effects with IC50 values of 195-340 mol ratio/32 pmol TPA. Compound 12 and poricoic acid C (13) exhibited inhibitory effects on skin tumor promotion in an in vivo two-stage carcinogenesis test using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter.

Determination of aromatic and branched-chain amino acids in plasma by HPLC with electrogenerated Ru(bpy)3(3+) chemiluminescence detection.

An HPLC method is described for the electrochemiluminescence (ECL) detection of amino acids, following cycloaddition reaction of their amino groups with divinyl sulfone (DVS), using electrogenerated tris(bipyridine)ruthenium(III). The derivatization reaction conditions were examined, with the optimum conditions found to be 40 mM DVS (pH 8.0) at 50 degrees C for 15 min. Detection limits for the 15 amino acids examined varied greatly (0.04-8.0 pmol) using a standard solution by flow injection analysis (FIA). These optimized conditions were used for HPLC determination of the amino acids in human plasma. A linear relationship was obtained up to 100 pmol on a column for aromatic and branched-chain amino acids. Recoveries of Tyr, Met, Val, Leu, Ile, Phe and Trp when added to human plasma (1 micromol/10 ml plasma, n=5) were 101.5+/-1.1, 99.0+/-1.2, 98.0+/-1.4, 101.1+/-1.6, 95.1+/-1.6, 99.2+/-1.5 and 97.7+/-1.3 % (mean+/-S.D.) respectively. The concentrations of the amino acids in the plasma are in good agreement with other published data.