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.

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 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 propranolol and 4-hydroxypropranolol by CE with application to biotransformation studies employing endophytic fungi

A CE method is described for the enantioselective analysis of propranolol (Prop) and 4-hydroxypropranolol (4-OH-Prop) in liquid Czapek medium with application in the study of the enantioselective biotransformation of Prop by endophytic fungi. The electrophoretic conditions previously optimized were as follows: an uncoated fused-silica capillary, 4%w/v carboxymethyl-beta-CD in 25 mmol/L triethylamine/phosphoric acid (H(3)PO(4)) buffer at pH 9 as running electrolyte and 17 kV of voltage. UV detection was carried out at 208 nm. Liquid-liquid extraction using diethyl ether: ethyl acetate (1:1 v/v) as extractor solvent was employed for sample preparation. The calibration curves were linear over the concentration range of 0.25-10.0 mu g/mL for each 4-OH-Prop enantiomer and 0.10-10.0 mu g/mL for each Prop enantiomer (r >= 0.995). Within-day and between-day relative standard deviations and relative errors for precision and accuracy were lower than 15% for all the enantiomers. Finally, the validated method was used to evaluate Prop biotransformation in its mammalian metabolite 4-OH-Prop by some selected endophytic fungi. The screening of five strains of endophytic fungi was performed and all of them could biotransform Prop to some extent. Specifically, Glomerella cingulata (VA1) biotransformed 47.8% of (-)-(S)-Prop to (-)-(S)-4-OH-Prop with no formation of (+)-(R)4-OH-Prop in 72 h of incubation.

Development and validation of HPLC method for imiquimod determination in skin penetration studies

A simple, rapid and sensitive analytical procedure for the measurement of imiquimod in skin samples after in vitro penetration studies has been developed and validated. In vitro penetration studies were carried out in Franz diffusion cells with porcine skin. Tape stripping technique was used to separate the stratum corneum (SC) from the viable epidermis and dermis. Imiquimod was extracted from skin samples using a 7:3 (v/v) methanol:acetate buffer (100 mm, pH 4.0) solution and ultrasonication. Imiquimod was analyzed by H-PLC using C(8) column and UV detection at 242 ran. The mobile phase used was acetonitrile:acetate buffer (pH 4.0, 100 mM):diethylamine (30:69.85:0.15, v/v) with flow rate 1 mL/min. Imiquimod eluted at 4.1 min and the running time was limited to 6.0 min. The procedure was linear across the following concentration ranges: 100-2500 ng/mL for both SC and tape-stripped skin and 20-800 ng/mL for receptor solution. Intra-day and inter-day accuracy and precision values were lower than 20% at the limit of quantitation. The recovery values ranged from 80 to 100%. The method is adequate to assay imiquimod from skin samples, enabling the determination of the cutaneous penetration profile of uniquimod by in vitro studies. Copyright (C) 2008 John Wiley & Sons, Ltd.

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.

Excised Porcine Cornea Integrity Evaluation in an in vitro Model of Iontophoretic Ocular Research

Background/Aims: It is a challenge to adapt traditional in vitro diffusion experiments to ocular tissue. Thus, the aim of this work was to present experimental evidence on the integrity of the porcine cornea, barrier function and maintenance of electrical properties for 6 h of experiment when the tissue is mounted on an inexpensive and easy-to-use in vitro model for ocular iontophoresis. Methods: A modified Franz diffusion cell containing two ports for the insertion of the electrodes and a receiving compartment that does not need gassing with carbogen was used in the studies. Corneal electron transmission microscopy images were obtained, and diffusion experiments with fluorescent markers were performed to examine the integrity of the barrier function. The preservation of the negatively charged corneal epithelium was verified by the determination of the electro-osmotic flow of a hydrophilic and non-ionized molecule. Results: The diffusion cell was able to maintain the temperature, homogenization, porcine epithelial corneal structure integrity, barrier function and electrical characteristics throughout the 6 h of permeation experiment, without requiring CO(2) gassing when the receiving chamber was filled with 25 m M of HEPES buffer solution. Conclusion: The system described here is inexpensive, easy to handle and reliable as an in vitro model for iontophoretic ocular delivery studies. Copyright (C) 2010 S. Karger AG, Basel

Quantum-chemical, NMR and X-ray diffraction studies on (+/-)-1-[3,4-(methylenedioxy)phenyl]-2-methylaminopropane

Time-averaged conformations of (+/-)-1-[3,4-(methylenedioxy)phenyl]-2-methylaminopropane hydrochloride (MDMA, ""ecstasy"") in D(2)O, and of its free base and trifluoroacetate in CDCl(3), were deduced from their (1)H NMR spectra and used to calculate their conformer distribution. Their rotational potential energy surface (PES) was calculated at the RHF/6-31G(d,p), 133LYP/6-31G(d,p), B3LYP/cc-pVDZ and AM1 levels. Solvent effects were evaluated using the polarizable continuum model. The NMR and theoretical studies showed that, in the free base, the N-methyl group and the ring are preferentially trans. This preference is stronger in the salts and corresponds to the X-ray structure of the hydrochloride. However, the energy barriers separating these forms are very low. The X-ray diffraction crystal structures of the anhydrous salt and its monohydrate differed mainly in the trans or cis relationship of the N-methyl group to the a-methyl, although these two forms interconvert freely in solution. (C) 2007 Elsevier Inc. All rights reserved.

Genotoxic studies in hypertensive and normotensive rats treated with amiodarone

Amiodarone, a benzofuran derivative. is a very effective antiarrhythmic medication, but has potential to cause side effects. Although its cytotoxicity potential is very well-known, there are few reports about its genotoxicity effects. Since amiodarone has not been investigated in genotoxicity studies, and the spontaneously hypertensive rat (SHR) is a well-characterized model for hypertension, the aim of the present study was to perform cytogenetic analysis on chromosome aberrations in bone marrow cells of SHRs and normotensive Wistar-Kyoto rats (WKYs) that received oral amiodarone treatment for 4 weeks. Amiodarone activity was also monitored using electrocardiograms. The presence of bradycardia in amiodarone-treated rats confirmed that this drug was really active. Metaphase analysis on bone marrow cells showed that there were significant differences in total chromosomal damage and percentage abnormal metaphase between WKY and SHR negative controls. In the SHR negative control, the frequencies of basal chromosomal aberrations and abnormal metaphases were significantly higher (p < 0.05). There were high numbers of chromosomal aberrations in all amiodarone-treated groups, compared with negative controls. In amiodarone-treated groups, the most frequent chromosomal aberration was chromatid breaks. More chromosomal aberrations were found in WKYs that received amiodarone, with a statistically significant difference in comparison with negative controls (p < 0.05). However, in SHR rats there was no significant difference between the amiodarone and negative groups regarding chromosomal damage induction. These results showed that treatment with amiodarone was genotoxic in WKYs, but not in SHRs. Further studies are needed to confirm whether amiodarone is genotoxic or efficient and harmless, among humans undergoing therapy. (c) 2008 Published by Elsevier B.V.

Quality of Reporting of Observational Longitudinal Research

Observational longitudinal research is particularly useful for assessing etiology and prognosis and for providing evidence for clinical decision making. However, there are no structured reporting requirements for studies of this design to assist authors, editors, and readers. The authors developed and tested a checklist of criteria related to threats to the internal and external validity of observational longitudinal studies. The checklist criteria concerned recruitment, data collection, biases, and data analysis and descriptive issues relevant to study rationale, study population, and generalizability. Two raters independently assessed 49 randomly selected articles describing stroke research published from 1999 to 2003 in six journals: American Journal of Epidemiology, Journal of Epidemiology and Community Health, Stroke, Annals of Neurology, Archives of Physical Medicine and Rehabilitation, and American Journal of Physical Medicine and Rehabilitation. On average, 17 of the 33 checklist criteria were reported. Criteria describing the study design were better reported than those related to internal validity. No relation was found between study type (etiologic or prognostic) or word count and quality of reporting. A flow diagram for summarizing participant flow through a study was developed. Editors and authors should consider using a checklist and flow diagram when reporting on observational longitudinal research.

Aboriginal and Torres Strait Islander Studies Unit Annual Report

Welcome to the 2002 Aboriginal and Torres Strait Islander Studies Unit Annual Report. This report is a brief summary of Unit activities during the 2002 calendar year. The Unit provides personal and academic support for Aboriginal and Torres Strait Islander students and specifically aims to increase the recruitment, retention, academic performance and graduation rates of Indigenous students. The Unit also administers schemes to help Indigenous students gain access to, and receive support in, tertiary studies such as the Alternative Entry scheme and the federally funded Aboriginal Tutorial Assistance Scheme (ATAS). The Unit is also the focus for teaching and research in Aboriginal and Torres Strait Islander Studies at the University of Queensland.

Aboriginal and Torres Strait Islander Studies Unit Annual Report 2003

Welcome to the 2003 Aboriginal and Torres Strait Islander Studies Unit Annual Report. This report is a brief summary of Unit activities during the 2003 calendar year. The Unit provides personal and academic support for Aboriginal and Torres Strait Islander students and specifically aims to increase the recruitment, retention, academic performance and graduation rates of Indigenous students. The Unit also administers schemes to help Indigenous students gain access to, and receive support in, tertiary studies such as the Alternative Entry scheme and the federally funded Aboriginal Tutorial Assistance Scheme (ATAS). The Unit is also the focus for teaching and research in Aboriginal and Torres Strait Islander Studies at the University of Queensland.

Aboriginal and Torres Strait Islander Studies Unit Annual Report 2005

Welcome to the 2005 Aboriginal and Torres Strait Islander Studies Unit Annual Report. This report is a brief summary of Unit activities during the 2005 calendar year. The Unit provides personal and academic support for Aboriginal and Torres Strait Islander students and specifically aims to increase the recruitment, retention, academic performance and graduation rates of Indigenous students. The Unit also administers schemes to help Indigenous students gain access to, and receive support in, tertiary studies such as the Alternative Entry scheme and the federally-funded Indigenous Tutorial Assistance Scheme (ITAS). The Unit is also the focus for teaching and research in Aboriginal and Torres Strait Islander Studies at the University of Queensland.

One size doesn't fit all: Research methodologies in a language variation study of Sudanese teens

The research reported here draws on a study of five teenagers from a Dinka-speaking community of Sudanese settling in Australia. A range of factors including language proficiency, social network structure and language attitudes are examined as possible causes for the variability of language use. The results and discussion illustrate how the use of a triangular research approach captured the complexity of the participants' language situation and was critical to developing a full understanding of the interplay of factors influencing the teens' language maintenance and shift in a way that no single method could. Further, it shows that employment of different methodologies allowed for flexibility in data collection to ensure the fullest response from participants. Overall, this research suggests that for studies of non-standard communities, variability in research methods may prove more of a strength that the use of standardised instruments and approaches.

It's all very well, in theory: Theoretical perspectives and their applications in contemporary pedagogical research

Current debates about educational theory are concerned with the relationship between knowledge and power and thereby issues such as who possesses a truth and how have they arrived at it, what questions are important to ask, and how should they best be answered. As such, these debates revolve around questions of preferred, appropriate, and useful theoretical perspectives. This paper overviews the key theoretical perspectives that are currently used in physical education pedagogy research and considers how these inform the questions we ask and shapes the conduct of research. It also addresses what is contested with respect to these perspectives. The paper concludes with some cautions about allegiances to and use of theories in line with concerns for the applicability of educational research to pressing social issues.