Capillary electrophoresis method for plasmatic determination of imatinib mesylate in chronic myeloid leukemia patients

Imatinib (IMAT) is a tyrosine kinase inhibitor that has been used for the treatment of chronic myeloid leukemia (CML). Despite the efficacy of IMAT therapy, some cases of treatment resistance have been described in CML. Developing a plasma method is important since there are several studies that provided a higher correlation between IMAT plasma concentration and response to treatment. Therefore, in this investigation we validated a method by CE as an alternative, new, simple and fast electrophoretic method for IMAT determination in human plasma. The analysis was performed using a fused silica capillary (50 mm id x 46.5 cm total length, 38.0 cm effective length); 50 mmol/L sodium phosphate buffer, pH 2.5, as BGE; hydrodynamic injection time of 20 s (50 mbar); voltage of 30 kV; capillary temperature of 35 degrees C and detection at 200 nm. Plasma samples pre-treatment involved liquid-liquid extraction with methyl-tert-butyl ether as the extracting solvent. The method was linear from 0.125 to 5.00 mg/mL. The LOQ was 0.125 mg/mL. Mean absolute recovery of IMAT was 67%. The method showed to be precise and accurate with RSD and relative error values lower than 15%. Furthermore, the application of the method was performed in the analysis of plasma samples from CML patients undergoing treatment with IMAT.

Comparative analysis of the gas-phase reactions of cylindrospermopsin and the difference in the alkali metal cation mobility

Cylindrospermopsin (CYN) belongs to a group of toxins produced by several strains of freshwater cyanobacteria. It is a compact zwitterionic molecule composed of a uracil section and a tricyclic guanidinium portion with a primarily hepatotoxic effect. Using low multi-stage and high-resolution mass spectrometry, the gas-phase reactions of this toxin have been investigated. Our data show that collision-induced dissociation (CID) spectra of CYN are dominated by neutral losses, and three major initial fragmentation pathways are clearly distinguishable. Interestingly, comparative analysis of protonated and cationizated molecules showed a significant difference in the balance of the SO(3) and terminal ring elimination. These data indicate that the differential ion mobility of H(+), Li(+), Na(+) and K(+) leads to different fragmentation pathways, giving rise to mass spectra with different profiles. Copyright (C) 2008 John Wiley & Sons, Ltd.

Online identification of chlorogenic acids, sesquiterpene lactones, and flavonoids in the Brazilian arnica Lychnophora ericoides Mart. (Asteraceae) leaves by HPLC-DAD-MS and HPLC-DAD-MS/MS and a validated HPLC-DAD method for their simultaneous analysis

Lychnophora ericoides Mart. (Asteraceae, Vernonieae) is a plant, endemic to Brazil, with occurrence restricted to the ""cerrado"" biome. Traditional medicine employs alcoholic and aqueous-alcoholic preparations of leaves from this species for the treatment of wounds, inflammation, and pain. Furthermore, leaves of L. ericoides are also widely used as flavorings for the Brazilian traditional spirit ""cachaca"". A method has been developed for the extraction and HPLC-DAD analysis of the secondary metabolites of L. ericoides leaves. This analytical method was validated with 11 secondary metabolites chosen to represent the different classes and polarities of secondary metabolites occurring in L. ericoides leaves, and good responses were obtained for each validation parameter analyzed. The same HPLC analytical method was also employed for online secondary metabolite identification by HPLC-DAD-MS and HPLC-DAD-MS/MS, leading to the identification of di-C-glucosylflavones, coumaroylglucosylflavonols, flavone, flavanones, flavonols, chalcones, goyazensolide, and eremantholide-type sesquiterpene lactones and positional isomeric series of chlorogenic acids possessing caffeic and/or ferulic moieties. Among the 52 chromatographic peaks observed, 36 were fully identified and 8 were attributed to compounds belonging to series of caffeoylferuloylquinic and diferuloylquinic acids that could not be individualized from each other.

The bimolecular sensitization of nitric oxide release from weak interacting ruthenium units

This work reports on the bimolecular sensitization of nitric oxide release from cis-[Ru(bpy)(2)(iso)-NO](PF(6))(3) (1) (iso = isoquinoline and bpy = 2,2`- bipyridine) by irradiating the MLCT transition of the chloro analog cis-[Ru(bpy) 2(iso) Cl] PF6 (2). The compounds displayed peaks in the ESI-MS spectra at m/z 749.1 and m/z 578.1 ascribed, respectively, to ([1(NO(o))-2PF(6)center dot CH(3)OH](2+)) and ([2-PF(6)](+)). In the cyclic voltammograms, the nitrosyl complex presented two redox waves related to the NO ligand at 0.48 and -0.37 V (versus Ag/AgCl, NO(+/0/-1) processes), while the sensitizer showed two reversible waves at 0.79 and -1.46 V (versus Ag/AgCl, Ru(2+/3+) and bpy(0/-1), respectively). The most important feature of this system is that the nitrosyl compound does not have significant absorption in the visible region, while the sensitizer has an intense band centered at 496 nm. The irradiation of an equimolar mixture of the two compounds in an ethanol: water solution (v: v) with light of lambda > 500 nm leads to NO release, as probed by amperometric measurements. The variational method was applied, showing that the two compounds self-assembly in solution with a 1: 1 stoichiometry. Fluorescence spectra acquired at 77 K provided the E(0-0) for the system and, from the thermodynamic cycle it was estimated that the photoinduced electron transfer between the species has a Delta G value of -1.59 eV. (C) 2011 Elsevier B. V. All rights reserved.

Olfactory eavesdropping between two competing stingless bee species

Foragers can improve search efficiency, and ultimately fitness, by using social information: cues and signals produced by other animals that indicate food location or quality. Social information use has been well studied in predator-prey systems, but its functioning within a trophic level remains poorly understood. Eavesdropping, use of signals by unintended recipients, is of particular interest because eavesdroppers may exert selective pressure on signaling systems. We provide the most complete study to date of eavesdropping between two competing social insect species by determining the glandular source and composition of a recruitment pheromone, and by examining reciprocal heterospecific responses to this signal. We tested eavesdropping between Trigona hyalinata and Trigona spinipes, two stingless bee species that compete for floral resources, exhibit a clear dominance hierarchy and recruit nestmates to high-quality food sources via pheromone trails. Gas chromatography-mass spectrometry of T. hyalinata recruitment pheromone revealed six carboxylic esters, the most common of which is octyl octanoate, the major component of T. spinipes recruitment pheromone. We demonstrate heterospecific detection of recruitment pheromones, which can influence heterospecific and conspecific scout orientation. Unexpectedly, the dominant T. hyalinata avoided T. spinipes pheromone in preference tests, while the subordinate T. spinipes showed neither attraction to nor avoidance of T. hyalinata pheromone. We suggest that stingless bees may seek to avoid conflict through their eavesdropping behavior, incorporating expected costs associated with a choice into the decision-making process.

Assessment of Ametryn Contamination in River Water, River Sediment, and Mollusk Bivalves in Sao Paulo State, Brazil

Sao Paulo state, Brazil, is one of the main areas of sugar cane agriculture in the world. Herbicides, in particular, ametryn, are extensively used in this extensive area, which implies that this herbicide is present in the environment and can contaminate the surface water by running off. Thereby, residues of ametryn were analyzed in samples of river water an river sediment and in freshwater bivalves obtained from the rivers Sapucai, Pardo and Mogi-Guacu in Sao Paulo State, Brazil. Samples were taken in the winter of 2003 and 2004 in two locations in each river. The specimens of freshwater bivalves collected and analyzed were Corbicula fluminea, an exotic species, and Diplodon fontaineanus, a native species. Additionally, the evaluation of the ability of bioconcentration and depuration of ametryn by the freshwater bivalve Corbicula fluminea was also performed. Ametryn concentrations in the samples were measured by liquid chromatography coupled to mass spectrometry. Residues of ametryn in water (50 ng/L) and in freshwater bivalves (2-7 ng/g) were found in the Mogi-Guacu River in 2004, and residues in river sediments were found in all rivers in 2003 and 2004 (0.5-2 ng/g). The observation of the aquatic environment through the analysis of these matrixes, water, sediment, and bivalves, revealed the importance of the river sediment in the accumulation of the herbicide ametryn, which can contaminate the biota.

Isolation, functional, and partial biochemical characterization of galatrox, an acidic lectin from Bothrops atrox snake venom

Snake venom lectins have been studied in regard to their chemical structure and biological functions. However, little is known about lectins isolated from Bothrops atrox snake venom. We report here the isolation and partial functional and biochemical characterization of an acidic glycan-binding protein called galatrox from this venom. This lectin was purified by affinity chromatography using a lactosyl-sepharose column, and its homogeneity and molecular mass were evaluated by high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The purified galatrox was homogeneous and characterized as an acidic protein (pI 5.2) with a monomeric and dimeric molecular mass of 16.2 and 32.5 kDa, respectively. Alignment of N-terminal and internal amino acid sequences of galatrox indicated that this protein exhibits high homology to other C-type snake venom lectins. Galatrox showed optimal hemagglutinating activity at a concentration of 100 mu g/ml and this effect was drastically inhibited by lactose, ethylenediaminetetraacetic acid, and heating, which confirmed galatrox`s lectin activity. While galatrox failed to induce the same level of paw edema or mast cell degranulation as B. atrox crude venom, galatrox did alter cellular viability, which suggested that galatrox might contribute to venom toxicity by directly inducing cell death.

New constituents from Mikania laevigata Shultz Bip. ex Baker

A new phenylpropanoid and two new diterpenes were isolated from the leaves of the plant Mikania laevigata Shultz Bip. ex Baker. The structures of these compounds were established by 1D- and 2D-nuclear magnetic resonance spectroscopic techniques and mass spectrometry data. Taraxerol, lupeol, coumarin, syringaldehyde, trans-melilotoside, cis-melilotoside, adenosine, patuletin 3-O-beta-D-glucopyranoside, kaempferol 3-O-beta-D-glucopyranoside, quercetin 3-O-beta-D-glucopyranoside, methyl 3,5-di-O-caffeoyl quinate, and 3,3`,5-trihydroxy-4`,6,7-trimethoxyflavone were isolated too. In addition, the compounds dihydrocoumarin, spathulenol, caryophyllene oxide, kaurenoic acid, beyerenoic acid, and lupeol acetate were identified by GC-MS. (C) 2010 Elsevier Ltd. All rights reserved.

Simultaneous determination of rosiglitazone and its metabolites in rat liver microsomal fraction using hollow-fiber liquid-phase microextraction for sample preparation

A three-phase hollow-fiber liquid-phase microextraction method for the analysis of rosiglitazone and its metabolites N-desmethyl rosiglitazone and p-hydroxy rosiglitazone in microsomal preparations is described for the first time. The drug and metabolites HPLC determination was carried out using an X-Terra RP-18 column, at 22 degrees C. The mobile phase was composed of water, acetonitrile and acetic acid (85:15:0.5, v/v/v) and the detection was performed at 245 nm. The hollow-fiber liquid-phase microextraction procedure was optimized using multifactorial experiments and the following optimal condition was established: sample agitation at 1750 rpm, extraction for 30 min, hydrochloric acid 0.01 mol/L as acceptor phase, 1-octanol as organic phase, and donor phase pH adjustment to 8.0. The recovery rates, obtained by using 1 mL of microsomal preparation, were 47-70%. The method presented LOQs of 50 ng/mL and it was linear over the concentration range of 50-6000 ng/mL, with correlation coefficients (r) higher than 0.9960, for all analytes. The validated method was employed to study the in vitro biotransformation of rosiglitazone using rat liver microsomal fraction.

Dissociation of deprotonated microcystin variants by collision-induced dissociation following electrospray ionization

Microcystins (MC) are a family of hepatotoxic cyclic heptapeptides produced by a number of different cyanobacterial species. Considering the recent advances in the characterization of deprotonated peptides by mass spectrometry, the fragmentation behavior of four structurally related microcystin compounds was investigated using collision-induced dissociation (CID) experiments on an orbitrap mass spectrometer. It is demonstrated in this study that significant structural information can be obtained from the CID spectra of deprotonated microcystins. A predominant ring-opening reaction at the isoMeAsp residue, as well as two major complementary fragmentation pathways, was observed, reducing the complexity of the product ion spectra in comparison with spectra observed from protonated species. This proposed fragmentation behavior was applied to characterize [Leu(1)]MC-LR from a cyanobacterial cell extract. In conclusion, CID spectra of microcystins in the negative ion mode provide rich structurally informative mass spectra which greatly enhance confidence in structural assignments, in particular when combined with complementary positive ion CID spectra. Copyright (C) 2011 John Wiley & Sons, Ltd.

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.

Electrospray MS-based characterization of beta-carbolines - mutagenic constituents of thermally processed meat

The beta-carbolines 1-methyl-9H-pyrido [3,4-b]indole and 9H-pyrido[3,4b]indole have been implicated as having causative roles in a number of human diseases, such as Parkinson`s disease and cancer. As they can be formed during the heating of protein-rich food, a number of analytical methodologies have been proposed for their detection and quantification in foodstuff For this purpose, LC-MS and LC-MS/MS have emerged as the most specific analytical methods, and the quantification is based on the occurrence of unusual ions, such as [M+H-(H(center dot))](+) and [M + H-2H](+). In this study, we have investigated the formation of these ions by accurate-mass electrospray MS/MS and demonstrated that these ions are formed from gas-phase ion-molecule reactions between water vapor present in the collision cell and the protonated molecule of 1-methyl-9H-pyrido [3,4-b]indole and 9H-pyrido[3,4b]indole. Although this reaction has been previously described for heterocyclic amine ions, it has been overlooked in the most of recent LC-MS and LC-MS/MS studies, and no complete data of the fragmentation are reported. Our results demonstrate that additional attention should be given with respect to eliminating water vapor residues in the mass spectrometer when analysis of beta-carbolines is performed, as this residue may affect the reliability in the results of quantification.

Enantioselective analysis of mirtazapine, demethylmirtazapine and 8-hydroxy mirtazapine in human urine after solid-phase microextraction

A selective and reproducible off-line solid-phase microextraction procedure was developed for the simultaneous enantioselective determination of mirtazapine (MRT), demethylmirtazapine and 8-hydroxymirtazapine in human urine. CE was used for optimization of the extraction procedure whereas LC-MS was used for method validation and application. The influence of important factors in the solid-phase microextraction efficiency is discussed, such as the fiber coatings, extraction time, pH, ionic strength, temperature and desorption time. Before extraction, human urine samples were submitted to enzymatic hydrolysis at 37 degrees C for 16 h. Then, the enzyme was precipitated with trichloroacetic acid and the pH was adjusted to 8 with 1 mol/L pH 11 phosphate buffer solution. In the extraction, the analytes were transferred from the aqueous solution to the polydimethylsiloxane-divinylbenzene fiber coating and then desorbed in methanol. The mean recoveries were 5.4, 1.7 and 1.0% for MRT, demethylmirtazapine and 8-hydroxymirtazapine enantiomers, respectively. The method was linear over the concentration range of 62-1250 ng/mL. The within-day and between-day assay precision and accuracy were lower than 15%. The method was successfully employed in a preliminary cumulative urinary excretion study after administration of racemic MRT to a healthy volunteer.

Enantioselective Analysis of Fluoxetine and Norfluoxetine by LC in Culture Medium for Application in Biotransformation Studies Employing Fungi

A liquid chromatography method is described for the analysis of fluoxetine and norfluoxetine enantiomers in fungi cultures. The analytes were separated simultaneously by LC employing a serial system. The resolution was performed using a mobile phase of ethanol: 15 mM ammonium acetate buffer solution, pH 5.9: acetonitrile (77.5:17.5:5, v/v/v). UV detection was at 227 nm. Hexane: isoamyl alcohol (98:2, v/v) was used as extractor solvent. The calibration curves were linear over the concentration range of 12.5-3,750 ng mL(-1) (r a parts per thousand yen 0.996). The values for intra- and inter-day precision and accuracy were a parts per thousand currency sign10% for all analytes. The validated method was used to evaluate fluoxetine biotransformation to its mammalian metabolite, norfluoxetine, by selected endophytic fungi. Although the desired biotransformation was not observed in the conditions used here, the method could be used to evaluate the biotransformation of fluoxetine by other fungi or to be extended to other matrices with adequate procedures for sample preparation.

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.