Bioequivalence assay between orally disintegrating and conventional tablet formulations in healthy volunteers

The purpose of this study was to evaluate bioequivalence of two commercial 8 mg tablet formulations of ondansetrona available ill the Brazilian market. In this study, a simple, rapid, sensitive and selective liquid chromarography-tandem mass spectrometry method is described for the determination of ondansetron in human plasma samples. The method was validated over a concentration range of 2.5-60 ng/ml and used in a bioequivalence trial between orally disintegrating and conventional tablet ondansetron formulations, to assess its usefulness in this kind of Study. Vonau flash (R) (Biolab Sanus Farmaceutica, Brazil, as test formulations) and Zofran (R) (GlaxoSmithKline, Brazil, as reference formulation) were evaluated following a single 8 mg close to 23 healthy volunteers of both genders. The dose was administered after an overnight fast according to a two-way crossover design. Bioequivalence between the products was determinated by Calculating 90% confidence interval (90% CI) for the ratio of C(max), AUC(0-t) and AUC(0-(sic)) values for the test and reference products, using logarithmically transformed data. The 90% confidence interval for the ratio of C(max) (87.5-103.8%), AUC(0-t) (89.3-107.2%) and AUC(0--(sic)) (89.7-106.0%) values for the test and reference products is Within the 80-125% interval, proposed by FDA, EMEA and ANVISA. It was concluded that two ondansetron formulations are bioequivalent ill their rate and extent of absorption. (C) 2008 Elsevier B.V. All rights reserved.

Biochemical and biophysical characterization of lysozyme modified by PEGylation

PEGylation is a strategy that has been used to improve the biochemical properties of proteins and their physical and thermal stabilities. In this study, hen egg-white lysozyme (EC 3.2.1.17; LZ) was modified with methoxypolyethylene glycol-p-nitrophenyl carbonate (mPEG-pNP, MW 5000). This PEGylation of LZ produced conjugates that retained full enzyme activity with glycol chitosan, independent of degree of enzyme modification; its biological activity with the substrate Micrococcus lysodeikticus was altered according to its degree of modification. The conjugate obtained with a low degree of mPEG-pNP/NH(2) modification was studied by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), demonstrating a spectral peak at m/z 19,988 Da with 77% of its original enzymatic activity. Spectroscopic studies of Fourier transform infrared (FIR) and circular dichroism (CD) did not show any relevant differences in protein structure between the native and conjugate LZ. Studies of the effects of pH and temperature on PEGylated LZ indicated that the conjugate was active over a broad pH range, stable at 50 degrees C, and demonstrated resistance to proteolytic degradation. (C) 2010 Elsevier B.V. All rights reserved.

Biomonitoring method for the simultaneous determination of cadmium and lead in whole blood by electrothermal atomic absorption spectrometry for assessment of environmental exposure

The aim of this work is to propose a biomonitoring method for the simultaneous determination of Cd and Pb in whole blood by simultaneous electrothermal atomic absorption spectrometry for assessment of environmental levels. A volume of 200 mu L of whole blood was diluted in 500 mu L of 0.2% (w v(-1)) Triton(R) X-100 + 2.0% (v v(-1)) HNO3. Trichloroacetic acid was added for protein precipitation and the supernatant analyzed. A mixture of 250 mu g W + 200 mu g Rh as permanent and 2.0% (w v(-1)) NH4H2PO4 as co-injected modifiers were used. Characteristic masses and limits of detections (n = 20, 3s) for Cd and Pb were 1.26 and 33 pg and 0.026 mu g L-1 and 0.65 mu g L-1, respectively. Repeatability ranged from 1.8 to 6.8% for Cd and 1.2 to 1.7% for Pb. The trueness of method was checked by the analysis of three Reference Materials: Lyphocheck(R) Whole Blood Metals Control level 1 and Seronorm(TM) Trace Elements in Whole Blood levels 1 and 2. The found concentrations presented no statistical differences at the 95% confidence level. Blood samples from 40 volunteers without occupational exposure were analyzed and the concentrations ranged from 0.13 to 0.71 mu g L-1 (0.32 +/- 0.19 mu g L-1) for Cd and 9.3 to 56.7 mu g L-1 (25.1 +/- 10.8 mu g L-1) for Pb. (C) 2007 Elsevier B.V. All rights reserved.

Enantioselective determination of chloroquine and its n-dealkylated metabolites in plasma using liquid-phase microextraction and LC-MS

A selective method using three-phase liquid-phase microextraction (LPME) in conjunction with LC-MS-MS was devised for the enantioselective determination of chloroquine and its n-dealkylated metabolites in plasma samples. After alkalinization of the samples, the analytes were extracted into n-octanol immobilized in the pores of a polypropylene hollow fiber membrane and back extracted into the acidic acceptor phase (0.1 M TFA) filled into the lumen of the hollow fiber. Following LPME, the analytes were resolved on a Chirobiotic V column using methanol/ACN/glacial aceti acid/diethylamine (90:10:0.5:0.5 by volume) as the mobile phase. The MS detection was carried out using multiple reaction monitoring with ESI in the positive ion mode. The optimized LPME method yielded extraction recoveries ranging from 28 to 66%. The method was linear over 5 - 500 ng/mL and precision (RSD) and accuracy (relative error) values were below 15% for all analytes. The developed method was applied to the determination of the analytes in rat plasma samples after oral administration of the racemic drug.

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.

Visualizing inhibition of fatty acid synthase through mass spectrometric analysis of mitochondria from melanoma cells

Fatty acid synthase (FASN) is the metabolic enzyme responsible for the endogenous synthesis of the saturated long-chain fatty acid palmitate. In contrast to most normal cells, FASN is overexpressed in a variety of human cancers including cutaneous melanoma, in which its levels of expression are associated with a poor prognosis and depth of invasion. Recently, we have demonstrated the mitochondrial involvement in FASN inhibition-induced apoptosis in melanoma cells. Herein we compare, via electrospray ionization mass spectrometry (ESI-MS), free fatty acids (FFA) composition of mitochondria isolated from control (EtOH-treated cells) and Orlistat-treated B16-F10 mouse melanoma cells. Principal component analysis (PCA) was applied to the ESI-MS data and found to separate the two groups of samples. Mitochondria from control cells showed predominance of six ions, that is, those of m/z 157 (Pelargonic, 9:0), 255 (Palmitic, 16:0), 281 (Oleic, 18:1), 311 (Arachidic, 20:0), 327 (Docosahexaenoic, 22:6) and 339 (Behenic, 22:0). In contrast, FASN inhibition with Orlistat changes significantly mitochondrial FFA composition by reducing synthesis of palmitic acid, and its elongation and unsaturation products, such as arachidic and behenic acids, and oleic acid, respectively. ESI-MS of mitochondria isolated from Orlistat-treated cells presented therefore three major ions of m/z 157 (Pelargonic, 9:0), 193 (unknown) and 199 (Lauric, 12:0). These findings demonstrate therefore that FASN inhibition by Orlistat induces significant changes in the FFA composition of mitochondria. Copyright (C) 2011 John Wiley & Sons, Ltd.

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.

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.

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.

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.

Chemical Signals in the Stingless Bee, Frieseomelitta varia, Indicate Caste, Gender, Age, and Reproductive Status

Chemical compounds on the cuticle are a rich source of information used during interactions among social insects. Despite the multitude of studies on these substances and their function in ants, wasps, and honeybees, little is known about this subject in stingless bees (Hymenoptera: Apidae, Meliponini). We studied the chemical composition of the cuticle of the stingless bee, Frieseomelitta varia, by gas chromatography-mass spectrometry (GC-MS), to investigate potential chemical variation among castes, gender, age, and reproductive status. We found differences in the cuticular hydrocarbon composition among workers, males, and queens, recording both qualitative and quantitative differences among individuals of different ages and gender. The cuticle of physogastric queens presented a chemical profile that was distinct from all other groups in the analysis, with high relative abundances of alkenes and alkadienes with 27, 29, and 31 carbon atoms. We discuss the possibility that these compounds signal a queen`s presence to the colony, thereby initiating all vital worker-queen interactions.

3`3-Ditrifluoromethyldiphenyl diselenide: A new organoselenium compound with interesting antigenotoxic and antimutagenic activities

The trace element selenium (Se), once known only for its potential toxicity, is now a well-established essential micronutrient for mammals. The organoselenium compound diphenyl diselenide (DPDS) has shown interesting antioxidant and neuroprotective activities. On the other hand, this compound has also presented pro-oxidant and mutagenic effects. The compound 3`3-ditrifluoromethyldiphenyl diselenide (DFDD), a structural analog of diphenyl diselenide, has proven antipsychotic activity in mice. Nevertheless, as opposed to DPDS, little is known on the biological and toxicological properties of DFDD. In the present study, we report the genotoxic effects of the organoselenium compound DFDD on Salmonella typhimurium, Saccharomyces cerevisiae and Chinese hamster lung fibroblasts (V79 cells). DFDD protective effects against hydrogen peroxide (H(2)O(2))-induced DNA damage in vitro are demonstrated. DFDD did not cause mutagenic effects on S. typhimurium or S. cerevisiae strains; however, it induced DNA damage in V79 cells at doses higher than 25 mu M, as detected by comet assay. DFDD protected S. typhimurium and S. cerevisiae against H(2)O(2)-induced mutagenicity, and, at doses lower than 12.5 mu M, prevented H(2)O(2)-induced genotoxicity in V79 cells. The in vitro assays demonstrated that DFDD mimics catalase activity better than DPDS, but neither presents Superoxide dismutase action. The products of the reactions of DFDD or DPDS with H(2)O(2) were different. as determined by electrospray mass spectrometry analysis (ESI-MS). These results suggest that DFDD is not mutagenic for bacteria or yeast; however, it may induce weak genotoxic effects on mammalian cells. In addition, DFDD has a protective effect against H(2)O(2)-induced damage probably by mimicking catalase activity, and the distinct products of the reaction DFDD with H(2)O(2) probably have a fundamental role in the protective effects of DFDD. (C) 2009 Elsevier B.V. All rights reserved.

Fragmentation studies and electrospray ionization mass spectrometry of lapachol: protonated, deprotonated and cationized species

Electrospray ionization mass spectrometric analysis of lapachol (2-hydroxy-3-(3-methy1-2-butenyl)-1,4-naphthoquinone) was accomplished in order to elucidate the gas-phase dissociation reactions of this important biologically active natural product. The occurrence of protonated and cationized species in the positive mode and of deprotonated species in the negative mode was explored by means of collision-induced dissociation (CID) experiments. For the protonated molecule, the H(2)O and C(4)H(8) losses occur by two competitive channels. For the deprotonated molecule, the even-electron rule is not conserved, and the radicalar species are eliminated by formation of distonic anions. The fragmentation mechanism for each ion was suggested on the basis of computational thermochemistry. Atomic charges, relative energies, and frontier orbitals were employed aiming at a better understanding of the gas-phase reactivity of lapachol. Potential energy surfaces for fragmentation reactions were obtained by the B3LYP/6-31+G(d,p) model. Copyright (C) 2010 John Wiley & Sons, Ltd.

A common matrix metalloproteinase (MMP)-2 polymorphism affects plasma MMP-2 levels in subjects environmentally exposed to mercury

Mercury (Hg) exposure is associated with disease conditions, including cardiovascular problems. Although the mechanisms implicated in these complications have not been precisely defined yet, matrix metalloproteinases (MMPs) may be involved. The gene encoding MMP-2 presents genetic polymorphisms which affect the expression and activity level of this enzyme. A common polymorphism of MMP-2 gene is the C(-1306)T (rs 243865), which is known to disrupt a Sp1-type promoter site (CCACC box), thus leading to lower promoter activity associated with the T allele. This study aimed at examining how this polymorphism affects the circulating MMP-2 levels and its endogenous inhibitor, the tissue inhibitor of metalloproteinase-2 (TIMP-2) in 210 subjects environmentally exposed to Hg. Total blood and plasma Hg concentrations were determined by inductively coupled plasma-mass spectrometry (ICP-MS). MMP-2 and TIMP-2 concentrations were measured in plasma samples by gelatin zymography and ELISA, respectively. Genotypes for the C(-1306)T polymorphism were determined by Taqman (R) Allele Discrimination assay. We found a positive association (p = 0.0057) between plasma Hg concentrations and MMP-2/TIMP-2 (an index of net MMP-2 activity). The C(-1306)T polymorphism modified MMP-2 concentrations (p = 0.0465) and MMP-2/TIMP-2 ratio (p = 0.0060) in subjects exposed to Hg, with higher MMP-2 levels been found in subjects carrying the C allele. These findings suggest a significant interaction between the C(-1306)T polymorphism and Hg exposure, possibly increasing the risk of developing diseases in subjects with the C allele. (C) 2011 Elsevier B.V. All rights reserved.

A functional matrix metalloproteinase (MMP)-9 polymorphism modifies plasma MMP-9 levels in subjects environmentally exposed to mercury

Mercury (Hg) exposure causes health problems including cardiovascular diseases. Although precise mechanisms have not been precisely defined yet, matrix metalloproteinases (MMPs) may be involved. The gene encoding MMP-9 presents genetic polymorphisms which affect the expression and activity level of this enzyme. Two polymorphisms in the promoter region [C(-1562)T and (CA)(n)] are functionally relevant, and are implicated in several diseases. This study aimed at examining how these polymorphisms affect the circulating MMP-9 levels and its endogenous inhibitor, the tissue inhibitor of metalloproteinase-1 (TIMP-1) in 266 subjects environmentally exposed to Hg. Blood and plasma Hg concentrations were determined by inductively coupled plasma-mass spectrometry (ICP-MS). MMP-9 and TIMP-1 concentrations were measured in plasma samples by gelatin zymography and ELISA, respectively. Genotypes for the C(-1562)T and the microsatellite (CA)(n) polymorphisms were determined. We found a positive association (P<0.05) between plasma Hg concentrations and MMP-9/TIMP-1 ratio (an index of net MMP-9 activity). When the subjects were divided into tertiles with basis on their plasma Hg concentrations, we found that the (CA)(n) polymorphism modified MMP-9 concentrations and MMP-9/TIMP-1 ratio in subjects with the lowest Hg concentrations (first tertile), with the highest MMP-9 levels being found in subjects with genotypes including alleles with 21 or more CA repeats (H alleles) (P<0.05). Conversely, this polymorphism had no effects on subjects with intermediate or high plasma Hg levels (second and third tertiles, respectively). The C(-1562)T polymorphism had no effects on MMP-9 levels. These findings suggest a significant interaction between the (CA)(n) polymorphism and low levels of Hg exposure, possibly increasing the risk of developing diseases in subjects with H alleles. (c) 2010 Elsevier B.V. All rights reserved.