Solid-phase extraction method with high-performance liquid chromatography and electrochemical detection for the quantitative analysis of oxycodone in human plasma

A sensitive and reproducible solid-phase extraction (SPE) method for the quantification of oxycodone in human plasma was developed. Varian Certify SPE cartridges containing both C-8 and benzoic acid functional groups were the most suitable for the extraction of oxycodone and codeine (internal standard), with consistently high (greater than or equal to 80%) and reproducible recoveries. The elution mobile phase consisted of 1.2 ml of butyl chloride-isopropanol (80:20, v/v) containing 2% ammonia. The quantification limit for oxycodone was 5.3 pmol on-column. Within-day and inter-day coefficients of variation were 1.2% and 6.8% respectively for 284 nM oxycodone and 9.5% and 6.2% respectively for 28.4 nM oxycodone using 0.5-ml plasma aliquots. (C) 1998 Elsevier Science BN. All rights reserved.

Solid-phase microextraction using poly(pyrrole) film and liquid chromatography with UV detection for analysis of antidepressants in plasma samples

Poly(pyrrole) (PPY) coating was prepared on a stainless-steel (SS) wire for solid-phase microextraction (SPME) by electrochemical deposition (cyclic voltammetric). The PPY was evaluated by analyzing new-generation antidepressants (mirtazapine, citalopram, paroxetine, duloxetine, fluoxetine, and sertraline) in plasma sample by SPME and liquid chromatography with UV detection (LC-UV). The effect of electrolyte Solution (lithium perchlorate or tetrabutylammonium perchlorate) and the number of cycles (50, 100 or 200) applied during the polymerization process on the SPME performance was evaluated. Important factors in the optimization of SPME efficiency such as extraction time, temperature, pH, influence of plasma proteins on sorption mechanisms, and desorption conditions are discussed. The SPME-PPY/LC method showed to be linear in concentrations ranging from the limit of quantification (LOQ) to 1200 ng mL(-1). The LOQ values range from 16 to 25 ng mL-1. The inter-day precision of the SPME-PPY/LC method presented coefficient of variation (CV) lower than 15%. Based on analytical validation results, the SPME-PPY/LC methodology showed to be adequate for antidepressant analysis, from therapeutic to toxic levels. In order to evaluate the proposed method for clinical use, the SPME-PPY/LC method was applied to the analysis of plasma samples from elderly depressed patients. (c) 2009 Elsevier B.V. All rights reserved,

In-tube solid-phase microextraction coupled to liquid chromatography (in-tube SPME/LC) analysis of nontricyclic antidepressants in human plasma

A sensitive, selective, and reproducible in-tube solid-phase microextraction and liquid chromatographic (in-tube SPME/LC-UV) method for simultaneous determination of mirrazapine, citalopram, paroxetine, duloxetine, fluoxetine, and sertraline in human plasma was developed, validated and further applied to the analysis of plasma samples from elderly patients undergoing therapy with antidepressants. Important factors in the optimization of in-tube SPME efficiency are discussed, including the sample draw/eject volume, draw/eject cycle number, draw/eject flow-rate, sample pH, and influence of plasma proteins. The quantification limits of the in-tube SPME/LC method varied between 20 and 50 ng/mL, with a coefficient of variation lower than 10%. The response of the in-tube SPME/LC method for most of the drugs was linear over a dynamic range from 50 to 500 ng/mL, with correlation coefficients higher than 0.9985. The in-tube SPME/LC can be successfully used to analyze plasma samples from ageing patients undergoing therapy with nontricyclic antidepressants. (c) 2007 Elsevier B.V. All rights reserved.

Microextraction in packed sorbent for analysis of antidepressants in human plasma by liquid chromatography and spectrophotometric detection

A sensitive and reproducible method by microextraction packed sorbent and liquid chromatography with UV detection (MEPS/LC-UV) is described for the determination of new generation antidepressants (sertraline, mirtazapine, fluoxetine, citalopram and paroxetine) in human plasma samples. The MEPS variables, such as sample volume, pH, number of extraction cycles (draw-eject), and desorption conditions (solvent and solvent volume of elution) influenced the MEPS/LC efficiency significantly. Important factors in the optimization of MEPS efficiency, as well as washing steps and carryover effect are discussed. The analyses were carried out using small sample volumes (400 mu L.), and in a short time period (3 min for the entire sample preparation step). The MEPS/LC-UV method was shown to be linear at concentrations ranging from the limit of quantification (LOQ) to 1000 ng mL(-1). The LOQ values ranged from 10 to 25 ng mL(-1). The inter-day precision of the method presented coefficient of the variation ranging from 1.3% to 8.7%. On the basis of analytical validation, it is shown that the MEPS/LC-UV methodology is adequate for antidepressant analysis, from therapeutic to toxic levels. In order to evaluate the proposed method for clinical use, the MEPS/LC-UV method was applied to analysis of plasma samples from elderly depressed patients. (C) 2010 Elsevier B.V. All rights reserved.

A proteome analysis of conditioned media from human neonatal fibroblasts used in the maintenance of human embryonic stem cells

The pathways involved in the maintenance of human embryonic stem (hES) cells remain largely unknown, although some signaling pathways have been identified in mouse embryonic stem (mES) cells. Fibroblast feeder layers are used to maintain the undifferentiated growth of hES cells and an examination of the conditioned media (CM) of human neonatal fibroblasts (HNFs) could provide insights into the maintenance of hES cells. The neonatal foreskin fibroblast line (HNF02) used in this study was shown to have a normal 2n = 46, XY chromosomal complement and to support the undifferentiated growth of the Embryonic Stem Cell International Pte. Ltd.-hES3 cell line. The CM of HNF02 was examined using two-dimensional liquid chromatography-tandem mass spectrometry (2-D LCMS) and two-dimensional electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (2-DE/MALDI). A total of 102 proteins were identified, 19 by 2-DE/MALDI, 53 by 2-D LCMS and 30 by both techniques. These proteins were classified into 15 functional groups. Proteins identified in the extracellular matrix and differentiation and growth factor functional categories were considered most likely to be involved in the maintenance of hES cell growth, differentiation and pluripotency as these groups contained proteins involved in a variety of events including cell adhesion, cell proliferation and inhibition of cell proliferation, Writ signaling and inhibition of bone morphogenetic proteins.

Wavelet correlation between subjects: A time-scale data driven analysis for brain mapping using fMRI

Functional magnetic resonance imaging (fMRI) based on BOLD signal has been used to indirectly measure the local neural activity induced by cognitive tasks or stimulation. Most fMRI data analysis is carried out using the general linear model (GLM), a statistical approach which predicts the changes in the observed BOLD response based on an expected hemodynamic response function (HRF). In cases when the task is cognitively complex or in cases of diseases, variations in shape and/or delay may reduce the reliability of results. A novel exploratory method using fMRI data, which attempts to discriminate between neurophysiological signals induced by the stimulation protocol from artifacts or other confounding factors, is introduced in this paper. This new method is based on the fusion between correlation analysis and the discrete wavelet transform, to identify similarities in the time course of the BOLD signal in a group of volunteers. We illustrate the usefulness of this approach by analyzing fMRI data from normal subjects presented with standardized human face pictures expressing different degrees of sadness. The results show that the proposed wavelet correlation analysis has greater statistical power than conventional GLM or time domain intersubject correlation analysis. (C) 2010 Elsevier B.V. All rights reserved.

Analysis of Detergent-Insoluble and Whole Cell Lysate Fractions of Resting Neutrophils Using High-Resolution Mass Spectrometry

Neutrophilic granulocytes play a major role in the initiation and resolution of the inflammatory response, and demonstrate significant transcriptional and translational activity. Although much was known about neutrophils prior to the introduction of proteomics, the use of MS-based methodologies has provided an unprecedented tool to confirm and extend previous findings. In the present study, we performed a Gel-LC-MS/MS analysis of neutrophil detergent insoluble and whole cell lysate fractions of resting neutrophils. We achieved a set of identifications through the use of high-resolution mass spectrometry and validation of its data. We identified a total of 1249 proteins with a wide range of intensities from both detergent-insoluble and whole cell lysate fractions, allowing a mapping of proteins such as those involved in intracellular transport (Rab and Sec family proteins) and cell signaling (S100 proteins). These results represent the most comprehensive proteomic characterization of resting human neutrophils to date, and provide important information relevant for further studies of the immune system in health and disease. The methods applied here can be employed to help us understand how neutrophils respond to various physiologic and pathophysiologic conditions and could be extended to protein quantitation after cell activation.

Methods for exploring the morpho-functional relations of the aortic depressor nerve in experimental diabetes

The present study investigated morpho-functional relations of the aortic depressor nerve (ADN) 5, 15 and 120 days after the onset of streptozotocin-induced diabetes in rats. Time control animals received vehicle. Under pentobarbital anesthesia, ADN activity was recorded simultaneously with arterial pressure. After the recordings, nerves were prepared for light microscopy study and morphometry. ADN function was accessed by means of pressure-nerve activity curve (fitted by sigmoidal regression) and cross-spectral analysis between mean arterial pressure (MAP) and ADN activity. The relation between morphological (myelinated fibers number and density, total myelin area, total fiber area and percentage of occupancy) and functional (gain, signal/noise relation, frequency) parameters were accessed by linear regression analysis and correlation coefficient calculations. Functional parameters obtained by means of the sigmoidal regression curve as well as by cross-spectral analysis were similar in diabetic and control rats. Morphometric parameters of the ADN were similar between groups 5 days after the onset of diabetes. Average myelin area and myelinated fiber area were significantly smaller on diabetic rats 15 and 120 days after the onset of diabetes, being the myelinated fiber and respective axons area and diameter also smaller on 120 days group. Nevertheless, G ratio (ratio between axon and fiber diameter) was nearly 0.6 and not different between groups or experimental times. No significant relationship between morphological and functional parameters was detected in all experimental groups. The present study suggests that ADN diabetic neuropathy was time-dependent, with damage to myelinated fibers to be the primary event, not evidenced by physiological methods. (C) 2010 Elsevier B.V. All rights reserved.

Proteomic analysis of low- to high-grade astrocytomas reveals an alteration of the expression level of raf kinase inhibitor protein and nucleophosmin

Proteomic approaches have been useful for the identification of aberrantly expressed proteins in complex diseases such as cancer. These proteins are not only potential disease biomarkers, but also targets for therapy. The aim of this study was to identify differentially expressed proteins in diffuse astrocytoma grade II, anaplastic astrocytoma grade III and glioblastoma multiforme grade IV in human tumor samples and in non-neoplastic brain tissue as control using 2-DE and MS. Tumor and control brain tissue dissection was guided by histological hematoxylin/eosin tissue sections to provide more than 90% of tumor cells and astrocytes. Six proteins were detected as up-regulated in higher grade astrocytomas and the most important finding was nucleophosmin (NPM) (p < 0.05), whereas four proteins were down-regulated, among them raf kinase inhibitor protein (RKIP) (p < 0.05). We report here for the first time the alteration of NPM and RKIP expression in brain cancer. Our focus on these proteins was due to the fact that they are involved in the PI3K/AKT/mTOR and RAS/RAF/MAPK pathways, known for their contribution to the development and progression of gliomas. The proteomic data for NPM and RKIP were confirmed by Western blot, quantitative real-time PCR and immunohistochemistry. Due to the participation of NPM and RKIP in uncontrolled proliferation and evasion of apoptosis, these proteins are likely targets for drug development.

Analysis of the Ontogenetic Variation in the Venom Proteome/Peptidome of Bothrops jararaca Reveals Different Strategies to Deal with Prey

Previous studies have demonstrated that the pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Variation in the venom proteome is a well-documented phenomenon; however, variation in the venom peptidome is poorly understood. We report a comparative proteomic and peptidomic analysis of venoms from newborn and adult specimens of B. jararaca and correlate it with the evaluation of important venom features. We demonstrate that newborn and adult venoms have similar hemorrhagic activities, while the adult venom has a slightly higher lethal activity in mice; however, the newborn venom is extremely more potent to kill chicks. The coagulant activity of newborn venom upon human plasma is 10 times higher than that of adult venom. These differences were clearly reflected in their different profiles of SDS-PAGE, gelatin zimography, immunostaining using specific antibodies, glycosylation pattern, and concanavalin A-binding proteins. Furthermore, we report for the first time the analysis of the peptide fraction of newborn and adult venoms by MALDI-TOF mass spectrometry and LC-MS/MS, which revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles and were detected in the venoms showing their canonical sequences and also novel sequences corresponding to BPPs processed from their precursor protein at sites so far not described. As a result of these studies, we demonstrated that the ontogenetic shift in diet, from ectothermic prey in early life to endothermic prey in adulthood, and in animal size are associated with changes in the venom proteome in B. jararaca species.

A simple, inexpensive and easily reproducible model of spinal cord injury in mice: Morphological and functional assessment

Spinal cord injury (SCI) causes motor and sensory deficits that impair functional performance, and significantly impacts life expectancy and quality. Animal models provide a good opportunity to test therapeutic strategies in vivo. C57BL/6 mice were subjected to laminectomy at T9 and compression with a vascular clip (30 g force, 1 min). Two groups were analyzed: injured group (SCI, n = 33) and laminectomy only (Sham, n = 15). Locomotor behavior (Basso mouse scale-BMS and global mobility) was assessed weekly. Morphological analyses were performed by LM and EM. The Sham group did not show any morphofunctional alteration. All SCI animals showed flaccid paralysis 24 h after injury. with subsequent improvement. The BMS score of the SCI group improved until the intermediate phase (2.037 +/- 1.198): the Sham animals maintained the highest BMS score (8.981 +/- 0.056). p < 0.001 during the entire time. The locomotor speed was slower in the SCI animals (5.581 +/- 0.871) than in the Sham animals (15.80 +/- 1.166), p < 0.001. Morphological analysis of the SCI group showed, in the acute phase, edema, hemorrhage, multiple cavities, fiber degeneration, cell death and demyelination. In the chronic phase we observed glial scarring, neuron death, and remyelination of spared axons by oligodendrocytes and Schwann cells. In conclusion, we established a simple, reliable, and inexpensive clip compression model in mice, with functional and morphological reproducibility and good validity. The availability of producing reliable injuries with appropriate outcome measures represents great potential for studies involving cellular mechanisms of primary injury and repair after traumatic SCI. (C) 2008 Elsevier B.V. All rights reserved.

Quantitative analysis of the time courses of enzyme-catalyzed reactions

The catalytic properties of enzymes are usually evaluated by measuring and analyzing reaction rates. However, analyzing the complete time course can be advantageous because it contains additional information about the properties of the enzyme. Moreover, for systems that are not at steady state, the analysis of time courses is the preferred method. One of the major barriers to the wide application of time courses is that it may be computationally more difficult to extract information from these experiments. Here the basic approach to analyzing time courses is described, together with some examples of the essential computer code to implement these analyses. A general method that can be applied to both steady state and non-steady-state systems is recommended. (C) 2001 academic Press.

Introducing ethnomethodological analysis to the field of music education

In recent years qualitative research methods have been adopted within in the field of music education and have received widespread acceptance. However, the theoretical framework provided by ethnomethodology (Garfinkel, 1974, in R. Turner, Ethnomethodology , Penguin, Middlesex, UK) and the tools of conversational analysis (Sacks, 1992, Lectures on Conversation , edited by Gail Jefferson, Blackwell, Oxford, UK) have, to this point, been overlooked by researchers in the field of music education. In this paper I argue that the application of ethnomethodological and conversation analytical approaches in the field of research in music education can provide fresh insights into the work of music teachers and how this work is accomplished in institutional settings. Here I demonstrate how a conversation analytical perspective drawing on an ethnomethodological framework might be used to investigate transcripts of audio-recorded interview talk. This type of analysis can illuminate aspects of members' roles in relation to, and perceptions about music education in school settings that might be overlooked in other types of analysis. A conversation analytical approach to the examination of talk-in-interaction explicates in fine-grained detail how members orient to matters at hand in the context of research settings, as well as revealing features of the cultural world of music teaching. Further application of the approach to research problems in other school settings, I argue, will inform the field of music education in ways yet to be realised.

Quantification of sulfur and sulfur-containing compounds in wastewaters by means of a combination of liquid chromatographic methods

Low-micromolar concentrations of sulfite, thiosulfate and sulfide, present in synthetic wastewater or anaerobic digester effluent, were quantified by means of derivatization with monobromobimane, followed by HPLC separation with fluorescence detection. The concentration of elemental sulfur was determined, after its extraction with chloroform from the derivatized sample, by HPLC with UV detection. Recoveries of sulfide (both matrices), and of thiosulfate and sulfite (synthetic wastewater) were between 98 and 103%. The in-run RSDs on separate derivatizations were 13 and 19% for sulfite (two tests), between 1.5 and 6.6% for thiosulfate (two tests) and between 4.1 and 7.7% for sulfide (three tests). Response factors for derivatives of sulfide and thiosulfate, but not sulfite, were steady over a 13-month period during which 730 samples were analysed. Dithionate and tetrathionate did not seem to be detectable with this method. The distinctness of the elemental sulfur and the derivatizing-agent peaks was improved considerably by detecting elution at 297 instead of 263 nm. (C) 2002 Elsevier Science B.V. All rights reserved.