Online acid barrage stacking anti-salt injection for capillary electrophoresis of 9-fluorenylmethylchloroformate-derivatized amino acids in high ionic strength solutions by UV detection

An acid barrage stacking (ABS) method has been shown to be feasible for online anti-salt injection in CE of 9-fluorenylmethyl chloroformate (FMOC)-labeled amino acids (AAs) detected by common UV absorption. The operation was performed on normal polar CE by sucking in an extra plug of acid following a sample zone, serving as a selective acid barrage to block the backward migration of weak anionic analytes due to a sudden mobility reduction via acid-base reaction which does not affect strong co-ions such as Cl(-) to penetrate the barrage freely. By CE-UV of FMOC-AAs in various NaCl solutions, the effectiveness of ABS was firmly validated, able to stand up to 500 mM NaCl and to stack analytes by 10(3)-fold calculated from the UV detection limits, that is 0.01 microM for ABS and 10 microM for non-stacking injection. The method was also validated by determining trace Glu and Asp in real samples of rat brain microdialysate, rat serum and human saliva. The intraday RSDs were 0.33-4.9% for migration time and 1.8-9.6% for peak area. The recoveries measured by spiking technique were 82-115% for Glu and 86-116% for Asp. Working equations were obtained by plotting peak height vs. concentration at 0.1-50 microM, with correlation coefficients of >0.999. The contents of Glu and Asp were thus found at 0.26-0.83 microM and 0.24-0.64 microM respectively, in rat brain microdialyste; 37-40 microM and 8.4-10 microM, respectively, in rat serum; and 3.5-5.8 microM and 1.0-4.1 microM, respectively in human saliva. They were consistent with the data from other methods.     

Simultaneous determination of retinol, alpha-tocopherol and beta-carotene in serum by isocratic high-performance liquid chromatography.

A simultaneous determination of retinol, alpha-tocopherol and beta-carotene in serum by high-performance liquid chromatography is described. Total analysis time is 13 min. A reversed-phase (Ultrasphere ODS, 5 microns) column is used with a mobile phase of acetonitrile-methanol-dichloromethane (70:10:20, v/v/v) and a flow-rate of 1.2 ml/min. Retinol is monitored at 325 nm, alpha-tocopherol at 292 nm and beta-carotene at 450 nm. Serum is deproteinized with ethanol containing the internal standard (alpha-tocopherol acetate), then extracted with hexane. The evaporated organic layer is reconstituted with the mobile phase and injected. The choice of the eluent is discussed, as well as the choice of an internal standard and the need for an antioxidant during the extraction step. Sixteen different eluents are compared in terms of analysis time and selectivity. The linear concentration ranges (retinol 0.016-13.7 microM, alpha-tocopherol 0.18-91.8 microM, beta-carotene 0.05-5.75 microM), within-run coefficients of variation (retinol less than 7%; alpha-tocopherol less than 8%, beta-carotene less than 7%), between-run coefficients of variation (retinol less than 13%, alpha-tocopherol less than 9%, beta-carotene less than 8%) and recoveries (retinol greater than 95%, alpha-tocopherol greater than 91%, beta-carotene greater than 80%) are suitable for clinical investigations. Serum reference values were found to be 2.47 +/- 0.61 microM (retinol), 30.5 +/- 6.8 microM (alpha-tocopherol) and 0.91 +/- 0.55 microM (beta-carotene). A significant difference (p less than 0.001) between males and females was found for retinol.     

Green fluorescent protein in inertially injected aqueous nanodroplets

We inertially inject and study the contents of optically trappable aqueous nanodroplets (hydrosomes) emulsified in a perfluorinated matrix. A new piezoelectric actuated device for production of single hydrosomes on demand is introduced. Hydrosomes containing enhanced green fluorescent protein (EGFP) were injected, optically trapped, and held at the focus of an excitation laser in a confocal microscope, and single-molecule photobleaching events were observed. The rotational diffusion time of EGFP in trapped hydrosomes was measured using time-resolved fluorescence anisotropy. In free solution, the mean rotational diffusion time was determined to be 13.8 +/- 0.1 ns at 3 microM and 14.0 +/- 0.2 ns at 10 microM. In hydrosomes, the mean rotational diffusion time was similar and determined to be 12.6 +/- 1.0 ns at 3 microM and 15.5 +/- 1.6 ns at 10 microM. We conclude that the rotational motion inside the nanodroplets is consistent with rotation in free solution and that the protein therefore does not aggregate at the water-oil interface. Protein can be confined in hydrosomes with high efficiency using this technique, which provides an alternative to surface attachment or lipid encapsulation and opens up new avenues of research using single molecules contained in fluid nanovolumes.     

Determination of chlorpheniramine and its binding with human serum albumin by capillary electrophoresis with tris(2,2'-bipyridyl)ruthenium(II) electrochemiluminescence detection.

Tris(2,2'-bipyridyl)ruthenium(II) electrochemiluminescence (ECL) detection in a capillary electrophoresis separation system was used for the determination of chlorpheniramine (CPM). The experimental conditions, such as the applied potential, separation voltage, injection voltage, injection time and the pH of the separation buffer were considered in detail. The ECL intensity showed two linear responses to CPM, i.e., from 15 microM to 1 mM and from 0.8 microM to 15 microM with a detection limit of 0.5 microM. The binding of CPM with human serum albumin was also monitored using this method and the binding constant was estimated to be 4.1 x 103 M(-1).     

Selective detection of extracellular glutamate in brain tissue using microelectrode arrays coated with over-oxidized polypyrrole

We describe the selective measurement of extracellular glutamate concentration in rodent brain using ceramic-based platinum microelectrode arrays (MEAs) coated with electropolymerized, over-oxidized polypyrrole (OPP) as a permselective barrier to interference from dopamine and ascorbate. OPP-coated MEAs displayed similar sensitivity (97 +/- 9 nA microM(-1) cm(-2)) and response time (ca. 1 s) to glutamate as previously described Nafion-coated MEAs but, unlike Nafion-coated MEAs, were unresponsive to 5 microM dopamine. Such OPP-coated MEAs afforded selective detection of evoked glutamate release in the dopamine-rich striatum of the mouse brain.     

Method development for the analysis of particle phase substituted methoxy phenols and aromatic acids from biomass burning using capillary electrophoresis/electrospray ionization mass spectrometry (CE/ESI-MS)

A method is developed for the determination of substituted methoxy phenols and aromatic acids in biomass burning aerosol using capillary electrophoresis (CE) coupled to an electrospray ionization mass spectrometer. Background electrolytes (BGEs) containing ammonium acetate, ammonium hydroxide and 10% (v/v) methanol at pH 9.1 and ammonium hydroxide at pH 11 are investigated for their suitability. A good linearity is found for all analytes in the range of 1-50 microM for the ammonium acetate based BGE and 1-40 microM for the ammonium hydroxide BGE. The detection limit ranged from 0.1 to 1.0 microM for the ammonium acetate based BGE and 0.3 to 0.7 microM for the ammonium hydroxide BGE. The relative standard deviation (R.S.D.) is typically less than 0.5% (ammonium acetate based BGE) and 4.2% (ammonium hydroxide BGE) for the migration time and 3-9% (ammonium acetate based BGE) and 2.5-8% (ammonium hydroxide BGE) for the peak area (n = 5). The analytical time was less than 10 min for both methods. The proposed methods are fast, sensitive and quantitative and can be applied to the analysis of complex biomass burning aerosol samples without complex pre-treatment. The results from the analysis of real biomass burning samples demonstrate the suitability of the proposed methods to the analysis of low concentration water soluble organic carbon (WSOC) in biomass burning samples. The fast analytical time and high sensitivity of the proposed methods enables the analysis of a large number of size segregated impactor samples from biomass burning aerosols.     

Inhibition on HIV-1 integrase activity and nitric oxide production of compounds from Ficus glomerata

An ethanol Ficus glomerata wood extract and its purified components were investigated for their HIV-1 integrase (IN) and nitric oxide (NO) inhibitory activities. From bioassay-guided isolation, five compounds: beta-sitosterol-D-glucoside (1), aloe-emodin (2), genistein (3), 1,3,6-trihydroxy-8-methyl-anthraquinone (4) and 3-(1-C-beta-D-glucopyranosyl)-2,6-dihydroxy-5-methoxybenzoic acid (5) were isolated. Among the tested samples, at concentrations of 100 microM; compound 2 showed 31.9% inhibition of HIV-1 IN, followed by 4 (19.5%), whereas other compounds were inactive. With regard to the inhibitory effect on NO production, 3 possessed the highest activity with an IC50 value of 27.5 microM, followed by 4 (IC50 = 34.7 microM) and 2 (IC50 = 41.8 microM), respectively. This is the first time that compounds 2-5 have been isolated from Ficus glomerata.     

A new labdane diterpene from the flowers of Solidago canadensis

A new labdane diterpene, 9alpha,16xi-dihydroxy-6-oxo-7,13-labdadien-15,16-olide (solicanolide, 1) and six known compounds identified as quercetin (2), 3-O-caffeoylquinic acid (3, neochlorogenic acid), 5-O-caffeoylquinic acid (4, chlorogenic acid), 4,5-di-O-caffeoylquinic acid (5), 3,5-di-O-caffeoylquinic acid (6) and 3,4-di-O-caffeoylquinic acid (7) were isolated from the flowers of Solidago canadensis. To our knowledge, compound 7 was isolated for the first time in S. canadensis. This work describes the isolation of compounds 1-7 and the structure elucidation of a new compound identified as compound 1. Solicanolide (1) showed cytotoxic activity against A549 (IC(50): 13+/-2 microM), DLD-1 (IC(50): 26+/-2 microM) and WS1 (IC(50): 17+/-1 microM) cell lines.     

A new fatty ester and a new triterpene from Skimmia laureola

Aerial parts of Skimmia laureola yielded a new fatty ester, (+)-skimmilaureol (1), and a new triterpene 16-29-dihydroxy, 20-ene cyclolaudenol (2). Five known compounds, namely, O-methyl-cyclolaudenol (3), (R)-7-methoxy-6-(3'-hydroxy-2'R-methoxy-3'-methyl butyl)coumarin (4), (+)-(S)-psi-ribalinine (5), (R)-(+)-ribalinine (6) and methyl isoplatydesmine (7), previously isolated from this plant were subjected to enzymatic bioassays for the first time. Compounds 3 and 4 were found to be prolyl endopeptidase inhibitors with IC(50) 8.21 +/- 0.407 and 39.63 +/- 1.502 microM, respectively, while compounds 5-7 were found to be acetyl-cholinesterase and butyryl-cholinesterase inhibitors with IC(50) 62.46 +/- 1.58, 153.31 +/- 1.9, 74.5 +/- 1.05 and 150.04 +/- 0.45, 12.99 +/- 0.31, 78.3 +/- 1.86 microM, respectively.     

A simple micellar electrokinetic capillary chromatographic method for the quantitative analysis of organic expectorants

A simple and rapid micellar electrokinetic capillary chromatographic method is described for the separation and quantification of five expectorant drugs, including ambroxol (AMB), bromhexine (BRM), carbocysteine, guaiacol and guaifenesin. The drugs were separated in a mixed solution of phosphate buffer (35 mM; pH 3.90) and acetonitrile (75:25, v/v) with sodium dodecylsulfate (120 mM) as the micellar source, and the separated drugs were directly monitored with UV detector (200 nm). Several key parameters affecting the separation and analysis of the drugs were studied and optimized. Based on the corrected peak-area ratios of the drugs to an internal standard (7-hydroxy-4-methyl-coumarin) versus the concentration of the drugs, the established method is applicable to quantify AMB and BRM each over 20-150 microM, carbocysteine over 100-1500 microM, guaiacol and guaifenesin each over 10-150 microM. The detection limits (S/N=3; 0.5 psi, 5 s injection) of the method for the analytes are in the range of 3.0-5.0 microM (except that of carbocysteine at 30 microM). The precision (relative standard deviation) and accuracy (relative error) of the method for the intra-day (n=3) and inter-day (n=5) analyses of the analytes at three levels are all below 4%. The method is speedy with a run time of about 6 min for the analysis of the five analytes. Application of this method to the analysis of AMB and BRM in pharmaceutical preparations or AMB in the urine of a dosed subject proved simple and effective.     

Comparative studies on conventional and microwave synthesis of some benzimidazole, benzothiazole and indole derivatives and testing on inhibition of hyaluronidase

We have synthesized twelve 2-substituted benzimidazole, benzothiazole and indole derivatives using on both microwave irradiation and conventional heating methods. The microwave method was observed to be more beneficial as it provides an increase of yield from 3% to 113% and a 95 to 98 % reduction in time. All compounds were tested by a stains-all assay at pH 7 and by a Morgan-Elson assay at pH 3.5 for hyaluronidase inhibitory activity at a concentration of 100 microM. The most potent compound was 2-(4-hydroxyphenyl)-3-phenylindole (12) with an IC(50) value of 107 microM at both pH 7 and 3.5.     

The determination of hydrogen peroxide in aqueous solutions by square wave voltammetry

Hydrogen peroxide in aqueous solutions can be determined directly by square wave voltammetry. The method is applicable to samples with a large range of pH in matrices ranging from distilled de-ionized water to sea-water. Its dynamic range extends from 0.5 to at least 1000 microM and the precision is about +/-6% at 2.5 microM and +/-2% at 215 microM. In comparison to DC and differential pulse polarography, by using square wave voltammetry the scan time is reduced from minutes to a fraction of a second, the sensitivity is increased by several-fold and the dynamic range has been greatly expanded at both the lower and the upper end by at least an order of magnitude. The low detection limit allows this method to be applied to the determination of H(2)O(2) in some samples of rainwater.     

Effects of anonaine on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells

The effects of anonaine, an aporphine isoquinoline alkaloid, on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Anonaine at concentration ranges of 0.01-0.2 microM showed a significant inhibition of dopamine content at 24 h, with an IC(50) value of 0.05 microM. Anonaine at 0.05 microM inhibited tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) activities to 38.4-40.2% and 78.4-90.2% of control levels at 12-24 h and 3-6 h, respectively. TH activity was more influenced than AADC activity. Anonaine also decreased intracellular cyclic AMP levels, but not intracellular Ca(2+) concentrations. In addition, anonaine (0.05 microM) reduced L-DOPA (50 microM and 100 microM)-induced increases in dopamine content at 24 h. However, anonaine (0.05 microM) did not enhance L-DOPA (50 microM and 100 microM)-induced cell death after 24 h. These results suggest that anonaine inhibits dopamine biosynthesis by mainly reducing TH activity without aggravating L-DOPA-induced cytotoxicity in PC12 cells.     

Acetylcholinesterase and butyrylcholinesterase inhibitory compounds from Chelidonium majus (Papaveraceae)

The roots and aerial parts of Chelidonium majus L. were extracted with EtOH and fractionated using CHCl3 and EtOH. Repeated column chromatography, preparative TLC and crystallization led to the isolation of five isoquinoline alkaloids, stylopine (3), chelidonine (4), homochelidonine (5), protopine (6), and allocryptopine (7), along with two isolation artifacts 6-ethoxydihydrosanguinarine (1) and 6-ethoxydihydrochelerythrine (2). All isolated compounds were tested for human blood acetylcholinesterase (HuAChE) and human plasma butyrylcholinesterase (HuBuChE) inhibitory activity. The isolation artifacts exhibited the highest activity against HuAChE and HuBuChE with IC50 values of 0.83 +/- 0.04 microM and 4.20 +/- 0.19 microM for 6-ethoxydihydrochelerythrine and 3.25 +/- 0.24 microM and 4.51 +/- 0.31 microM for 6-ethoxydihydrosanguinarine. The most active of the naturally-occurring alkaloids was chelidonine, which inhibited both HuAChE and HuBuChE in a dose-dependent manner with IC50 values of 26.8 +/- 1.2 microM and 31.9 +/- 1.4 microM, respectively.     

Determination of cytosine-beta-D-arabinoside in plasma using capillary electrophoresis.

An assay for the antileukaemic agent cytosine-beta-D-arabinoside (ara-C) has been developed using capillary zone electrophoresis. Solid-phase extraction and on-capillary peak concentration are used to improve the detection limit. The electrophoretic separation time is less than 5 min. The limit of detection for ara-C in plasma is 0.5 microM (signal-to-noise ratio = 3). The assay has been validated for the determination of ara-C in human plasma over the concentration range 1-10 microM. The calibration curve was linear with a correlation coefficient r2 = 0.996. At an ara-C concentration of 8 microM the intra-day coefficient of variation was 9.1% and the inter-day coefficient of variation was 12.3%. At an ara-C concentration of 2 microM the coefficients of variation were 15.2 and 12.0%, respectively.     

Two new stereoisomers of Tetrahydrofuranoid lignans from the flower buds of Magnolia fargesii

Two new stereoisomers of tetrahydrofuranoid lignans, 7S,8R,7'S,8'R- (1) and 7R,8S,7'S,8'R-3,4,3',4'-tetramethoxy-9,7'-dihydroxy-8.8',7.O.9'-lignan (2) along with nine known lignans including tetrahydrofuranoids (3, 4) and tetrahydrofurofuranoids (5-11) were isolated from a CHCl(3)-soluble fraction of the flower buds of Magnolia fargesii. Two tetrahydrofuranoids, magnostellin A (3) and lariciresinol dimethyl ether (4) were isolated from this species for the first time. The structures of these compounds (1-11) were identified by spectroscopic methods as well as by comparison with published values. Absolute configurations of new stereoisomers (1, 2) were determined by the Mosher's esterification method and Circular Dichroism (CD) studies. All the isolates (1-11) were evaluated for their antioxidant activities using modified superoxide radical-scavenging assay. Compounds 5-8 showed the potent superoxide radical-scavenging activities with the ED(50) values of 19.2, 19.2, 16.5, and 27.7 microM, respectively, as compared with standard antioxidants (BHA: 22.8 microM; Trolox: 940 microM).     

Acetylcholinesterase and butyrylcholinesterase inhibitory compounds from Corydalis cava (Fumariaceae)

Tubers of Corydalis cava were extracted with ethanol and fractionated using n-hexane, chloroform and ethanol. Repeated column chromatography, preparative TLC and crystallization led to the isolation of fifteen isoquinoline alkaloids. The chemical structures of the isolated compounds were determined on the basis of spectroscopic techniques and by comparison with literature data. All isolated compounds were tested for human blood acetylcholinesterase (HuAChE) and human plasma butyrylcholinesterase (HuBuChE) inhibitory activity. (+)-Canadaline inhibited acetylcholinesterase as well as butyrylcholinesterase in a dose-dependent manner with IC50 values of 20.1 +/- 1.1 microM and 85.2 +/- 3.2 microM, respectively. (+)-Canadine, with an IC50 value of 12.4 +/- 0.9 microM, was the most potent inhibitor of acetylcholinesterase, whilst (+/-)-corycavidine and (+)-bulbocapnine were effective inhibitors of butyrylcholinesterase with IC50 values of 46.2 +/- 2.4 microM and 67.0 +/- 2.1 microM, respectively. The other isolated alkaloids were considered inactive (IC50 > 100 microM).     

In-channel dual-electrode amperometric detection in electrophoretic chips with a palladium film decoupler

An electrophoretic microchip integrated with a Pd-film decoupler and a series-dual electrode was proven practical (200-800 V/cm) for routine amperometric detection. In fluidic systems, amperometric enhancement of parallel-opposed dual-electrode detection is due to redox cycling of analytes between the electrodes. We, however, found that the oxidation current of catecholamines was enhanced significantly (1.9-3.8 folds) by switching from the single electrode mode to dual-series mode. This novel finding was unexpected because the unidirectional flow characteristic of the microfluidic system should eliminate the possibility for analytes physically migrating back and forth between the upstream and downstream electrodes. We attribute the enhancement to turbulence generated by impinging of the flow onto the edge of the downstream electrode. The linear range, sensitivity, limit of detection (S/N = 3) and number of theoretical plates for DA and CA are, respectively, 0.5-50 microM, 47 pA/microM, 0.25 microM, 7000 m(-1) and 1.0-100 microM, 28 pA/microM, 0.49 microM, 15,000 m(-1).     

Determination of enterostatin in human cerebrospinal fluid by capillary electrophoresis with laser induced fluorescence detection

A capillary electrophoresis (CE) method with laser induced fluorescence (LIF) detection is described for quantification of enterostatin (Val-Pro-Asp-Pro-Arg), a pentapeptide involved in appetite regulation and insulin secretion. Enterostatin and two other pentapeptides belonging to the enterostatin family (i.e. Ala-Pro-Gly-Pro-Arg and Val-Pro-Gly-Pro-Arg) were well separated from each other. The peptides were fluorescently tagged with naphthalene-2,3- dicarboxaldehyde (NDA) and separated by micellar electrokinetic chromatography (MEKC) in the presence of methanol as an organic modifier. Coupled with LIF detection, the method had a detection limit of 4.8 x 10(-6) M for enterostatin. The relative standard deviation was to be 4.0% from five determinations of enterostatin at 37.2 microM in a human cerebrospinal fluid (CSF) sample. Twenty-three human CSF samples were analyzed. The level of enterostatin ranged from 24 microM to 51 microM with a mean (+/- SEM) value of 41.7 +/- 2.0 microM.     

EFFECT OF CALCIUM ON DARK ADAPTATION IN Phycornyces PHOTOTROPISM

Adaptation processes enable phototropism of Phycomyces to operate over a 10-decade range of blue-light intensity (1 nW m-2-10 W m-2). To investigate the influence of calcium on dark adaptation, the phototropic latency method was employed with the modification that sporangiophores were temporarily immersed in solutions containing CaCl2 or LaCl3. Following such treatment, the time course of bending was found to have two components with distinct latencies and bending rates. After immersion in darkness for 30 min in LaCl3 solution or 1 h in a solution of CaCl2, MgCl2, or the calcium chelator EGTA, each sporangiophore was adapted to a blue light beam (1 W m-2) for 45 min by rotation around its vertical axis. Cessation of rotation defined the onset of the phototropic stimulus, at which time the intensity was reduced by as much as 10(3)-fold. For a 10(2)-fold reduction (to 10(-2) W m-2), immersion in CaCl2 (10-100 microM) reduces the latency 13 min for the early bending component and 18 min for the late component, whereas treatment with the calcium-channel blocker lanthanum (0.1-11 microM LaCl3) increases the latency 12 min for the early component and 13 min for the late component. EGTA (10 microM) also had an inhibitory effect, increasing the latency of the first and the second components by 7 and 10 min, respectively. In experiments performed similarly, but without the light adaptation treatment after immersion, no differences between calcium-treated and control sporangiophores were found. The bending rates of both components show only a weak dependence on calcium.(ABSTRACT TRUNCATED AT 250 WORDS)