Application of high-resolution electrospray mass spectrometry for the elucidation of the disproportionation reaction of iodobenzene diacetate

Disproportionation reactions take place in solution of (diacetoxyiodo)benzene (DIB) in acetonitrile in the presence of water, giving iodine(V) and iodine(l) species. This redox reaction is accelerated by the presence of water and by increasing the temperature. Several species of the solution of DIB were identified by high-resolution ESI-MS/MS, which allowed the elucidation of the mechanisms of disproportionation for DIB in gas phase and in solution. Key species in the process are the dimers [PhI(CH)OlPh](+) at m/z 440.8864, [PhI(OAc)OlPh](+) at m/z 482.8947, and [PhI(O)(OAc)OlPh](+) at m/z 498.8887. (C) 2008 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.

Liquid-phase microextraction combined with high-performance liquid chromatography for the enantioselective analysis of mefloquine in plasma samples

A simple and rapid method, which involves liquid-phase microextraction (LPME) followed by HPLC analysis using Chiralpak AD column and UV detection, was developed for the enantioselective determination of mefloquine in plasma samples. Several factors that influence the efficiency of three-phase LPME were investigated and optimized. Under the optimal extraction conditions, the mean recoveries were 33.2 and 35.0% for (-)-(SR-)-mefloquine and (+)-(RS)-mefloquine, respectively. The method was linear over 50-1500 ng/ml range. Within-day and between-day assay precision and accuracy were below 15% for both enantiomers at concentrations of 150, 600 and 1200 ng/ml. Furthermore, no racemization or degradation were seen with the method described. (C) 2007 Elsevier B.V. All rights reserved.

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.

Assignment of the absolute configuration at the sulfur atom of thioridazine metabolites by the analysis of their chiroptical properties: The case of thioridazine 2-sulfoxide

Thioridazine (THD) is a commonly prescribed phenotiazine neuroleptic drug, which is extensively biotransformed in the organism producing as main metabolites sulfoxides and a sulfone by sulfur oxidation Significant differences have been observed in the activity of the THD enantiomers as well as for its main metabolites, and enantioselectivity phenomena have been proved in the metabolic pathway. Here the assignment of the absolute configuration at the sulfur atom of enantiomeric THD-2-sulfoxide (THD-2-SO) has been carried out by circular dichroism (CD) spectroscopy The stereoisomers were separated by HPLC on Chiralpak AS column, recording the CD spectra for the two collected enantiomeric fractions The theoretical electronic CD spectrum has been obtained by the TDDFT/B3LYP/6-31G*. as Boltzmann averaging of the contributions calculated for the most stable conformations of the drug The comparison of the simulated and experimental spectra allowed the absolute configuration at the sulfur atom of the four THD-2-SO stereoisomers to be assigned The developed method should be useful for a reliable correlation between stereochemistry and activity and/or toxicity

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.

Re-examination of the anion derivatives of isoflavones by radical fragmentation in negative electrospray ionization tandem mass spectrometry: experimental and computational studies

This paper reports theoretical and experimental studies of gas-phase fragmentation reactions of four naturally occurring isoflavones. The samples were analyzed in negative ion mode by direct infusion in ESI-QqQ, ESI-QqTOF and ESI-Orbitrap systems. The MS/MS and MS(n) spectra are in agreement with the fragmentation proposals and high-resolution analyses have confirmed the formulae for each ion observed. As expected, compounds with methoxyl aromatic substitution have showed a radical elimination of center dot CH(3) as the main fragmentation pathway. A second radical loss (center dot H) occurs as previously observed for compounds which exhibit a previous homolytic center dot CH(3) cleavage (radical anion) and involves radical resonance to stabilize the anion formed. However, in this study we suggest another mechanism for the formation of the main ions, on the basis of the enthalpies for each species. Compounds without methoxy substituent dissociate at the highest energies and exhibit the deprotonated molecule as the most intense ion. Finally, energy-resolved experiments were carried out to give more details about the gas-phase dissociation reaction of the isoflavones and the results are in agreement with the theoretical approaches. Copyright (C) 2011 John Wiley & Sons, Ltd.

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.

In vitro metabolism study of a new nitrosyl ruthenium complex [Ru(NH center dot NHq)(terpy)NO](3+) nitric oxide donor using rat microsomes

A new nitrosyl ruthenium complex [Ru(NH center dot NHq)(terpy)NO](3+) nitric oxide donor was recently developed and due to its excellent vasodilator activity, it has been considered as a potential drug candidate. Drug metabolism is one of the main parameters that should be evaluated in the early drug development, so the biotransformation of this complex by rat hepatic microsomes was investigated. In order to perform the biotransformation study, a simple, sensitive and selective HPLC method was developed and carefully validated. The parameters evaluated in the validation procedure were: linearity, recovery, precision, accuracy, selectivity and stability. Except for the stability study, all the parameters evaluated presented values below the recommended by FDA guidelines. The stability study showed a time-dependent degradation profile. After method validation, the biotransformation study was accomplished and the kinetic parameters were determined. The biotransformation study obeyed the Michaelis-Menten kinetics. The V(max) and K(m) were, respectively, 0.1625 +/- 0.010 mu mol/mg protein/min and 79.97 +/- 11.52 mu M. These results indicate that the nitrosyl complex is metabolized by CYP450. (C) 2009 Elsevier Inc. All rights reserved.

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.

Brazilian Propolis: Seasonal Variation of the Prenylated p-Coumaric Acids and Antimicrobial Activity

Brazilian green propolis, which is used in food and beverages to improve health and to prevent diseases, demostrates antioxidant, antimutagenic, and antimicrobial activities. Most biological activities are thought to be related to the high levels of drupanin, artepillin C, and baccharin, which are compounds also present in Baccharis dracunculifolia D.C. (Asteraceae). Since propolis chemical composition depends on the region and the period of collection, as well as its plant origin, the effect of seasonal variation on the both content of prenylated p-coumaric acids and in vitro antimicrobial activity of Brazilian propolis from four different sites, was performed. The results showed that MIC values ranged from 100 to 300 mu g/mL against both Staphylococcus aureus and Kocuria rhizophila, while none of the propolis samples was active against Pseudomonas aeruginosa, Escherichia coli, and Candida albicans. HPLC analysis showed that the content of drupanin, artepillin C, and baccharin varied throughout the year, as well as among the different study sites. Also, it is suggested that Baccharis dracunculifolia is the main botanical source of Brazilian propolis in sites I and 2, while in sites 3 and 4, other plant species are also used by bees to produce propolis. All the evaluated propolis samples exhibited similar antibacterial activity, but different contents of prenylated p-coumaric acids throughout the year.

Quercetin in lyotropic liquid crystalline formulations: Physical, chemical and functional stability

The purpose of this study was to develop a lyotropic liquid crystalline formulation using the emulsifier vitamin E TPGS and evaluate its behavior after incorporation of a flavonoid, quercetin. The physical (macro and microscopic), chemical (determination of quercetin content by the HPLC method) and functional (determination of quercetin antioxidant activity by DPPH center dot assay) stability of the lamellar liquid crystalline formulation containing flavonoid was evaluated when stored at 4+/-2 degrees C; 30+/-2 degrees C/70+/-5% RH (relative humidity) and 40+/-2 degrees C/70+/-5% RH during 12 months. The lamellar liquid crystalline structure of the formulation was maintained during the experiment, however chemical and functional stability results showed a great influence of the storage period in all conditions tested. A significant decrease in quercetin content (approximately 40%) was detected during the first month of storage and a similar significant loss in antioxidant activity was detected after 6 months. The remaining flavonoid content was unchanged during the final 6 months of the experimental period. The results suggest possible interactions between quercetin and the liquid crystalline formulation, which could inhibit or reduce the quercetin activity incorporated in the system. In conclusion, the present study demonstrated that incorporation of quercetin (1%) did not affect the liquid crystalline structure composed of vitamin E TPGS/IPM/PG-H2O (1:1) at 63.75/21.25/15 (w/w/w). Nevertheless, of the total quercetin incorporated in the system only 60% was free to act as an antioxidant.

Trypanocidal structure-activity relationship for cis- and trans-methylpluviatolide

The trypanocidal activity of racemic mixtures of cis- and trans-methylpluviatolides was evaluated in vitro against trypomastigote forms of two strains of Trypanosoma cruzi, and in the enzymatic assay of T. cruzi gGAPDH. The cytotoxicity of the compounds was assessed by the MTT method using LLC-MK2 cells. The effect of the compounds on peroxide and NO production were also investigated. The mixture of the trans stereoisomers displayed trypanocidal activity (IC(50) similar to 89.3 mu M). Therefore, it was separated by chiral HPLC, furnishing the and (+) (-)-enantiomers. Only the (-)-enantiomer was active against the parasite (IC(50) similar to 18.7 mu M). Despite being inactive, the (+)-enantiomer acted as an antagonistic competitor. Trans-methylpluviatolide displayed low toxicity for LLC-MK(2) cells, with an IC(50) of 6.53 mM. Furthermore, methylpluviatolide neither inhibited gGAPDH activity nor hindered peroxide and NO production at the evaluated concentrations. (C) 2008 Elsevier Ltd. All rights reserved.