Effects of chlorogenic acid on neutrophil locomotion functions in response to inflammatory stimulus

Aim of the study: Species of Lychnophora are used in Brazilian folk medicine as analgesic and anti-inflammatory agents. Chlorogenic acid (CGA) and their analogues are important components of polar extracts of these species, as well in several European and Asian medicinal plants. Some of these phenolic compounds display anti-inflammatory effects. In this paper we report the isolation of CGA from Lychnophora salicifolia and its effects on functions involved in neutrophils locomotion. Materials and methods: LC-MS(n) data confirmed the presence of CGA in the plant. Actions of CGA were investigated on neutrophils obtained from peritoneal cavity of Wistar rats (4h after 1% oyster glycogen solution injection; 10 ml), and incubated with vehicle or with 50, 100 or 1000 mu M CGA in presence of lipopolysaccharide from Escherichia coil (LPS, 5 mu g/ml). Nitric oxide (NO; Griess reaction); prostaglandin E(2) (PGE(2)), interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha [TNF-alpha; enzyme-linked immunosorbent assay (EIA)]; protein (flow cytometry) and gene (RT-PCR) expression of L-selectin, beta(2)integrin and platelet-endothelial cell adhesion molecule-1 (PECAM-1) were quantified. In vitro neutrophil adhesion to primary culture of microvascular endothelial cell (PMEC) and neutrophil migration in response to formyl-methionil-leucil-phenilalanine (fMLP, 10(-8)M, Boyden chamber) was determined. Results: CGA treatment did not modify the secretion of inflammatory mediators, but inhibited L-selectin cleavage and reduced beta(2) integrin, independently from its mRNA synthesis, and reduced membrane PECAM-1 expression: inhibited neutrophil adhesion and neutrophil migration induced by fMLP. Conclusions: Based on these findings, we highlight the direct inhibitory actions of CGA on adhesive and locomotion properties of neutrophils, which may contribute to its anti-inflammatory effects and help to explain the use of Lychnophora salicifolia as an anti-inflammatory agent. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Negative ion `chip-based` nanospray tandem mass spectrometry for the analysis of flavonoids in glandular trichomes of Lychnophora ericoides Mart. (Asteraceae)

This paper reports a method for the analysis of secondary metabolites stored in glandular trichomes, employing negative ion `chip-based` nanospray tandem mass spectrometry. The analyses of glandular trichomes from Lychnophora ericoides, a plant endemic to the Brazilian `cerrado` and used in traditional medicine as an anti-inflammatory and analgesic agent, led to the identification of five flavonoids (chrysin, pinocembrin, pinostrobin, pinobanksin and 3-O-acetylpinobanksin) by direct infusion of the extracts of glandular trichomes into the nanospray ionisation source. All the flavonoids have no oxidation at ring B, which resulted in a modification of the fragmentation pathways compared with that of the oxidised 3,4-dihydroflavonoids already described in the literature. The absence of the anti-inflammatory and antioxidant di-C-glucosylflavone vicenin-2, or any other flavonoid glycosides, in the glandular trichomes was also demonstrated. The use of the,`chip-based` nanospray QqTOF apparatus is a new fast and useful tool for the identification of secondary metabolites stored in the glandular trichomes, which can be useful for chemotaxonomic studies based on metabolites from glandular trichomes. Copyright (C) 2008 John Wiley & Sons, Ltd.

Enantioselective analysis of zopiclone and its metabolites in plasma by liquid chromatography/tandem mass spectrometry

A high-performance liquid chromatographic method with triple-quadrupole mass spectrometry detection (LC-MS-MS) was developed and validated for the first time for the simultaneous quantification of zopiclone and its metabolites in rat plasma samples. The analytes were isolated from rat plasma by liquid-liquid extraction and separated using a chiral stationary phase based on an amylose derivative, Chiralpak ADR-H column, and ethanol-methanol-acetonitrile (50:45:5, v/v/v) plus 0.025% diethylamine as the mobile phase, at a flow-rate of 1.0 mL min(-1). Moclobemide was used as the internal standard. The developed method was linear over the concentration range of 7.5-500 ng mL(-1). The mean absolute recoveries were 74.6 and 75.7; 61.6 and 56.9; 72.5, and 70.7 for zopiclone enantiomers, for N-desmethyl zopiclone enantiomers and for zopiclone-N-oxide enantiomers, respectively, and 75.9 for the internal standard. Precision and accuracy were within acceptable levels of confidence (<15%). The method application in a pilot study of zopiclone kinetic disposition in rats showed that the levels of (+)-(S)-zopiclone were always higher than those of (-)-R-zopiclone. Higher concentrations were also observed for (+)-(S)-N-desmethyl zopiclone and (+)-(S)-N-oxide zopiclone, confirming the stereoselective disposition of zopiclone.

Hollow fiber-based liquid-phase microextraction (HF-LPME) of isradipine and its main metabolite followed by chiral HPLC analysis: application to an in vitro biotransformation study

An enantioselective liquid chromatographic method using two-phase hollow fiber liquid-phase microextraction (HF-LPME-HPLC) was developed for the determination of isradipine (ISR) enantiomers and its main metabolite (pyridine derivative of isradipine, PDI) in microsomal fractions isolated from rat liver. The analytes were extracted from 1 mL of microsomal medium using a two-phase HF-LPME procedure with hexyl acetate as the acceptor phase, 30 min of extraction, and sample agitation at 1,500 rpm. For the first time, ISR enantiomers and PDI were resolved. For this separation, a ChiralpakA (R) AD column with hexane/2-propanol/ethanol (94:04:02, v/v/v) as the mobile phase at a flow rate of 1.5 mL min(-1) was used. The column was kept at 23 A +/- 2 A degrees C. The drug and metabolite detection was performed at 325 nm and the internal standard oxybutynin was detected at 225 nm. The recoveries were 23% for PDI and 19% for each ISR enantiomer. The method presented quantification limits (LOQ) of 50 ng mL(-1) and was linear over the concentration range of 50-5,000 and 50-2,500 ng mL(-1) for PDI and each ISR enantiomer, respectively. The validated method was employed to an in vitro biotransformation study of ISR using rat liver microsomal fraction showing that (+)-(S)-ISR is preferentially biotransformed.

LC-MS-MS determination of ibuprofen, 2-hydroxyibuprofen enantiomers, and carboxyibuprofen stereoisomers for application in biotransformation studies employing endophytic fungi

The purpose of this study was the development and validation of an LC-MS-MS method for simultaneous analysis of ibuprofen (IBP), 2-hydroxyibuprofen (2-OH-IBP) enantiomers, and carboxyibuprofen (COOH-IBP) stereoisomers in fungi culture medium, to investigate the ability of some endophytic fungi to biotransform the chiral drug IBP into its metabolites. Resolution of IBP and the stereoisomers of its main metabolites was achieved by use of a Chiralpak AS-H column (150 x 4.6 mm, 5 mu m particle size), column temperature 8 degrees C, and the mobile phase hexane-isopropanol-trifluoroacetic acid (95: 5: 0.1, v/v) at a flow rate of 1.2 mL min(-1). Post-column infusion with 10 mmol L(-1) ammonium acetate in methanol at a flow rate of 0.3 mL min(-1) was performed to enhance MS detection (positive electrospray ionization). Liquid-liquid extraction was used for sample preparation with hexane-ethyl acetate (1:1, v/v) as extraction solvent. Linearity was obtained in the range 0.1-20 mu g mL(-1) for IBP, 0.05-7.5 mu g mL(-1) for each 2-OH-IBP enantiomer, and 0.025-5.0 mu g mL(-1) for each COOH-IBP stereoisomer (r >= 0.99). The coefficients of variation and relative errors obtained in precision and accuracy studies (within-day and between-day) were below 15%. The stability studies showed that the samples were stable (p > 0.05) during freeze and thaw cycles, short-term exposure to room temperature, storage at -20 degrees C, and biotransformation conditions. Among the six fungi studied, only the strains Nigrospora sphaerica (SS67) and Chaetomium globosum (VR10) biotransformed IBP enantioselectively, with greater formation of the metabolite (+)-(S)-2-OH-IBP. Formation of the COOH-IBP stereoisomers, which involves hydroxylation at C3 and further oxidation to form the carboxyl group, was not observed.

Determination of beta-artemether and its main metabolite dihydroartemisinin in plasma employing liquid-phase microextraction prior to liquid chromatographic-tandem mass spectrometric analysis

A method for the determination of artemether (ART) and its main metabolite dihydroartemisinin (DHA) in plasma employing liquid-phase microextraction (LPME) for sample preparation prior to liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed. The analytes were extracted from 1 nil, of plasma utilizing a two-phase LPME procedure with artemisinin as internal standard. Using the optimized LPME conditions, mean absolute recovery rates of 25 and 32% for DHA and ART, respectively, were achieved using toluene-n-octanol (1:1, viv) as organic phase with an extraction time of 30 min. After extraction, the analytes were resolved within 5 min using a mobile phase consisting of methanol-ammonium acetate (10 mmol L(-1) pH 5.0, 80:20. v/v) on a laboratory-made column based on poly(methyltetradecylsiloxane) attached to a zirconized-silica support. MS-MS detection was employed using an electrospray interface in the positive ion mode. The method developed was linear over the range of 5-1000 ng mL(-1) for both analytes. Precision and accuracy were within acceptable levels of confidence (<15%). The assay was applied to the determination of these analytes in plasma from rats treated with ART. The two-phase LPME procedure is affordable and the solvent consumption was very low compared to the traditional methods of sample preparation. (C) 2010 Elsevier B.V. All rights reserved.

LC-MS-MS Identification and Determination of the Flavone-C-Glucoside Vicenin-2 in Rat Plasma Samples Following Intraperitoneal Administration of Lychnophora Extract

The flavone C-glucoside, vicenin-2, in semi-purified extracts of the leaves of Lychnophora ericoides was quantified in rat plasma samples using a method based on reversed-phase high performance liquid chromatography coupled to tandem mass spectrometry. Vicenin-2 was analyzed on a LiChrospher (R) RP18 column using an isocratic mobile phase consisting of a mixture of methanol: water (30:70, v/v) plus 2.0% glacial acetic acid at a flow rate of 0.8 mL min(-1). Genistein was used as internal standard. The mass spectrometer was operated in positive ionization mode and analytes were quantified by multiple reaction monitoring at m/z 595 > 457 for vicenin-2 and m/z 271 > 153 for internal standard. Prior to the analysis, each rat plasma sample was acidified with 200 mu L of 50 mmol L(-1) acetic acid solution and extracted by solid-phase extraction using a C18 cartridge. The absolute recoveries were reproducible and the coefficients of variation values were lower than 5.2%. The method was linear over the 12.5 - 1500 ng mL(-1) concentration range and the quantification limit was 12.5 ng mL(-1). Within-day and between-day assay precision and accuracy were studied at three concentration levels (40, 400 and 800 ng mL(-1)) and were lower than 15%. The developed and validated method seems to be suitable for analysis of vicenin-2 in plasma samples obtained from rats that receive a single i.p. dose of 200 mg kg(-1) vicenin-2 extract.

Enantioselective analysis of praziquantel and trans-4-hydroxypraziquantel in human plasma by chiral LC-MS/MS: Application to pharmacokinetics

A simple enantioselective method for the determination of praziquantel (PZQ) and trans-4-hydroxypraziquantel (4-OHPZQ) in human plasma was developed and validated by high-performance liquid chromatography/mass spectrometry. The plasma samples were prepared by liquid-liquid extraction using a mixture of methyl-tert-butylether/dichloromethane (2:1, v/v) as extraction solvent. The direct resolution of PZQ and 4-OHPZQ enantiomers was performed on a Chiralpak AD column using hexane-isopropanol (75:25, v/v) as the mobile phase. Diazepam was used as internal standard. The method described here is simple and reproducible. The quantitation limit of 1.25 ng/ml for each PZQ enantiomer and of 12.5 ng/ml for each 4-OHPZQ enantiomer permits the use of the method in studies investigating the kinetic disposition of a single dose of 1.5g racemic PZQ. Enantioselectivity in the kinetic disposition of PZQ and 4-OHPZQ was observed in the clinical study. with the demonstration of a higher proportion of the (+)-(S)-PZQ and (-)-(R)-4-OHPZQ enantiomers in plasma. (C) 2009 Elsevier B.V. All rights reserved.

A fast sample preparation procedure for mercury speciation in hair samples by high-performance liquid chromatography coupled to ICP-MS

A simple method for mercury speciation in hair samples with a fast sample preparation procedure using high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry is proposed. Prior to analysis, 50 mg of hair samples were accurately weighed into 15 mL conical tubes. Then, an extractant solution containing mercaptoethanol, L-cysteine and HCl was added to the samples following sonication for 10 min. Quantitative mercury extraction was achieved with the proposed procedure. Separation of inorganic mercury (Ino-Hg), methylmercury (Met-Hg) and ethylmercury (Et-Hg) was accomplished in less than 8 min on a C18 reverse phase column with a mobile phase containing 0.05% v/v mercaptoethanol, 0.4% m/v L-cysteine, 0.06 mol L(-1) ammonium acetate and 5% v/v methanol. The method detection limits were found to be 15 ng g(-1), 10 ng g(-1) and 38 ng g(-1), for inorganic mercury, methylmercury and ethylmercury, respectively. Sample throughput is 4 samples h(-1) (duplicate). A considerable improvement in the time of analysis was achieved when compared to other published methods. Method accuracy is traceable to Certified Reference Materials (CRMs) 85 and 86 human hair from the International Atomic Energy Agency (IAEA). Finally, the proposed method was successfully applied to the speciation of mercury in hair samples collected from fish-eating communities of the Brazilian Amazon.

Determination of total and inorganic mercury in whole blood by cold vapor inductively coupled plasma mass spectrometry (CV ICP-MS) with alkaline sample preparation

A simple method with a fast sample preparation procedure for total and inorganic mercury determinations in blood samples is proposed based on flow injection cold vapor inductively coupled plasma mass spectrometry (FI-CVICP-MS). Aliquots of whole blood (500 mL) are diluted 1 + 1 v/v with 10.0% v/v tetramethylammonium hydroxide (TMAH) solution, incubated for 3 h at room temperature and then further diluted 1 + 4 v/v with 2.0% v/v HCl. The inorganic Hg was released by online addition of L-cysteine and then reduced to elemental Hg by SnCl(2). On the other hand, total mercury was determined by on-line addition of KMnO(4) and then reduced to elemental Hg by NaBH(4). Samples were calibrated against matrix-matching. The method detection limit was found to be 0.80 mu g L(-1) and 0.08 mu g L(-1) for inorganic and total mercury, respectively. Sample throughput is 20 samples h(-1). The method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). For additional validation purposes, human whole blood samples were analyzed by the proposed method and by an established CV AAS method, with no statistical difference between the two techniques at 95% confidence level on applying the t-test.

Biotype stability of Candida albicans isolates after culture storage determined by randomly amplified polymorphic DNA and phenotypical methods

P>Typing methods to evaluate isolates in relation to their phenotypical and molecular characteristics are essential in epidemiological studies. In this study, Candida albicans biotypes were determined before and after storage in order to verify their stability. Twenty C. albicans isolates were typed by Randomly Amplified Polymorphic DNA (RAPD), production of phospholipase and proteinase exoenzymes (enzymotyping) and morphotyping before and after 180 days of storage in Sabouraud dextrose agar (SDA) and sterilised distilled water. Before the storage, 19 RAPD patterns, two enzymotypes and eight morphotypes were identified. The fragment patterns obtained by RAPD, on the one hand, were not significantly altered after storage. On the other hand, the majority of the isolates changed their enzymotype and morphotype after storage. RAPD typing provided the better discriminatory index (DI) among isolates (DI = 0.995) and maintained the profile identified, thereby confirming its utility in epidemiological surveys. Based on the low reproducibility observed after storage in SDA and distilled water by morphotyping (DI = 0.853) and enzymotyping (DI = 0.521), the use of these techniques is not recommended on stored isolates.

Trypanocidal, leislimanicidal and antifungal potential from marine red alga Bostrychia tenella J. Agardli (Rhodomelaceae, Ceraniiales)

Specimens of the red alga Bostrychia tenella J Agardh (Rhodomelaceae, Ceramiales) were collected from the Sao Paulo coast and submitted to loom temperature solvent extraction The resulting extract was fractionated by partitioning with organic solvent The n-hexane (BT-H) and dichloromethane (BT-D) fractions showed antiprotozoal potential in biological tests with Trypanosoma cruzi and Leishmania amazonensis and presented high activity in an antifungal assay with the phytopathogenic fungi Cladosporium cladosporioides and Cladosporium sphaerospermum Chromatography methods were used to generate subfractions from BT-H (H01 to H11) and from BT-D (D01 to 019) The subtractions were analyzed by gas chromatography-mass spectrometry (GC/MS). and the substances were identified by retention index (Kovats) and by comparison to databases of commercial mass spectra The volatile compounds found in marine algae were identified as fatty acids, low molecular mass hydrocarbons, esters and steroids, some of these have been previously described in the literature based on other biological activities Moreover, uncommon substances. such as neophytadiene were also identified In a trypanocidal assay, fractions BT-H and BT-D showed IC(50) values of 168 and 19 1 mu g/mL. respectively, and were mote active than the gentian violet standard (31 mu g/ml.); subfractions H02. H03, D01 and D02 were active against L amasonensis, exhibiting IC(50) values of 1 S. 2 7, 4 4. and 4 3 mu g/mL., respectively (standard amphotericin B IC(50) = 13 mu g/mL.) All fractions showed antifungal potential this work reports the biological activity and identification of compounds by GC/MS for the marine red alga B tenella for the first time (C) 2010 Elsevier B V All lights reserved

Determination of mitragynine in rat plasma by LC-MS/MS: Application to pharmacokinetics

This study used for the first time LC-MS/MS for the analysis of mitragynine (MIT), a mu-opioid agonist with antinociceptive and antitussive properties, in rat plasma. Mitragynine and the internal standard (amitriptyline) were extracted from plasma with hexane-isoamyl alcohol and resolved on a Lichrospher (R) RP-SelectB column (9.80 and 12.90 min, respectively). The quantification limit was 0.2 ng/mL within a linear range of 0.2-1000 ng/mL The method was applied to quantify mitragynine in plasma samples of rats (n = 8 per sampling time) treated with a single oral dose of 20 mg/kg. The following pharmacokinetic parameters were obtained (mean): maximum plasma concentration: 424 ng/mL; time to reach maximum plasma concentration: 1.26 h; elimination half-life: 3.85 h, apparent total clearance: 6.35 L/h/kg, and apparent volume of distribution: 37.90 L/kg. (C) 2009 Elsevier B.V. All rights reserved.

Assessing the competitiveness of loss allocation methods in a deregulated electricity market

This paper critically assesses several loss allocation methods based on the type of competition each method promotes. This understanding assists in determining which method will promote more efficient network operations when implemented in deregulated electricity industries. The methods addressed in this paper include the pro rata [1], proportional sharing [2], loss formula [3], incremental [4], and a new method proposed by the authors of this paper, which is loop-based [5]. These methods are tested on a modified Nordic 32-bus network, where different case studies of different operating points are investigated. The varying results obtained for each allocation method at different operating points make it possible to distinguish methods that promote unhealthy competition from those that encourage better system operation.

A framework for electricity price spike analysis with advanced data mining methods

There are many techniques for electricity market price forecasting. However, most of them are designed for expected price analysis rather than price spike forecasting. An effective method of predicting the occurrence of spikes has not yet been observed in the literature so far. In this paper, a data mining based approach is presented to give a reliable forecast of the occurrence of price spikes. Combined with the spike value prediction techniques developed by the same authors, the proposed approach aims at providing a comprehensive tool for price spike forecasting. In this paper, feature selection techniques are firstly described to identify the attributes relevant to the occurrence of spikes. A simple introduction to the classification techniques is given for completeness. Two algorithms: support vector machine and probability classifier are chosen to be the spike occurrence predictors and are discussed in details. Realistic market data are used to test the proposed model with promising results.