Validation of a gas chromatographic method to quantify sesquiterpenes in copaiba oils

Copaifera species (Leguminoseae) are popularly known as ""copaiba"" or ""copaiva"". The oleoresins obtained from the trunk of these species have been extensively used in folk medicine and are commercialized in Brazil as crude oil and in several pharmaceutical and cosmetic products. This work reports a complete validated method for the quantification of beta-caryophyllene, alpha-copaene, and alpha-humulene in distinct copaiba oleoresins available commercially. Thus, essential oil samples (100 mu L) were dissolved in 20 mL of hexanes containing internal standard (1,2,4,5-tetramethylbenzene, 3.0 mM) in a 25 mL glass flask. A 1 mu L aliquot was injected into the GC-FID system. A fused-silica capillary column HP-5, coated with 5% phenylmethylsiloxane was used for this study. The developed method gave a good detection response with linearity in the range of 0.10-18.74 mM. Limits of detection and quantitation variety ranged between 0.003 and 0.091 mM. beta-Caryophyllene, alpha-copaene, and alpha-humulene were recovered in a range from 74.71% to 88.31%, displaying RSD lower than 10% and relative errors between -11.69% and -25.30%. Therefore, this method could be considered as an analytical tool for the quality control of different Copaifera oil samples and its products in both cosmetic and pharmaceutical companies. (C) 2010 Elsevier B.V. All rights reserved.

A HPLC method to evaluate the influence of photostabilizers on cosmetic formulations containing UV-filters and vitamins A and E

This paper reports a simple and reliable HPLC method to evaluate the influence of two currently available photostabilizers on cosmetic formulations containing combined UV-filters and vitamins A and E. Vitamins and UV-filters, widely encountered in products of daily use have to be routinely evaluated since photoinstability can lead to reductions in their efficacy and safety. UV-irradiated formulation samples were submitted to a procedure that included a reliable, precise and specific HPLC method employing a C18 column and detection at 325 and 235 nm. Methanol, isopropanol and water were the mobile phases in gradient elution. The method precision was between 0.28 and 5.07. The photostabilizers studied [diethylhexyl 2,6-naphthalate (DEHN) and benzotriazolyl dodecyl p-cresol (BTDC)], influenced the stability of octyl methoxycinnamate (OMC) associated with vitamins A and E. BTDC was considered the best photostabilizer to vitamins and OMC when the UV-filters were combined with both vitamins A and E. (C) 2010 Elsevier B.V. All rights reserved.

Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a combined method based on enrichment and 16S rRNA real-time PCR

Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eat foods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of those products may favor the growth of psychrotrophic bacteria, including the pathogen Listeria monocytogenes. In this work, minimally processed leafy vegetables samples (n = 162) from retail market from Ribeirao Preto, Sao Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immunoassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminated samples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN) with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCR with primers specific for L. monocytogenes using the commercial preparation ABSOLUTET (TM) QPCR SYBR (R) Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and no statistical significant difference was found in comparison with the conventional culture method (p < 0.05). Moreover, RTi-PCR was fist and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method. (C) 2009 Elsevier Ltd. 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,

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.

Determination of fluoxetine and norfluoxetine enantiomers in human plasma by polypyrrole-coated capillary in-tube solid-phase microextraction coupled with liquid chromatography-fluorescence detection

A sensitive, selective, and reproducible in-tube polypyrrole-coated capillary (PPY) solid-phase microextraction and liquid chromatographic method for fluoxetine and norfluoxetine enantiomers analysis in plasma samples has been 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. Separation of the analytes was achieved with a Chiralcel OD-R column and a mobile phase consisting of potassium hexafluorophosphate 7.5 mM and sodium phosphate 0.25 M solution, pH 3.0, and acetonitrile (75:25, v/v) in the isocratic mode, at a flow rate of 1.0 mL/min. Detection was carried out by fluorescence absorbance at Ex/Em 230/290 nm. The multifunctional porous surface structure of the PPY-coated film provided high precision and accuracy for enantiomers. Compared with other commercial capillaries, PPY-coated capillary showed better extraction efficiency for all the analytes. The quantification limits of the proposed method were 10 ng/mL for R- and S-fluoxetine, and 15 ng/mL for R- and S-norfluoxetine, with a coefficient of variation lower than 13%. The response of the method for enantiomers is linear over a dynamic range, from the limit of quantification to 700ng/mL, with correlation coefficients higher than 0.9940. The in-tube SPME/LC method can therefore be successfully used to analyze plasma samples from ageing patients undergoing therapy with fluoxetine. (C) 2009 Elsevier B.V. All rights reserved.

First detection of lead in black paper from intraoral film An environmental concern

Lead (Pb) contamination in the black paper that recovers intraoral films (BKP) has been investigated. BKP samples were collected from the Radiology Clinics of the Dental School of Ribeirao Preto, University of Sao Paulo, Brazil. For sake of comparison, four different methods were used. The results revealed the presence of high lead levels, well above the maximum limit allowed by the legislation. Pb contamination levels achieved after the following treatments: paper digestion in nitric acid, microwave treatment, DIN38414-54 method and TCLP method were 997 mu g g(-1), 189 mu g g(-1), 20.8 mu g g(-1), and 54.0 mu g g(-1), respectively. Flame atomic absorption spectrometry (FAAS) and inductively coupled plasma mass spectrometry (ICP-MS) were employed for lead determination according to the protocols of the applied methods. Lead contamination in used BKP was confirmed by scanning electron microscopy coupled with energy dispersive X-ray microanalysis (SEM-EDS). All the SEM imaging was carried out in the secondary electron mode (SE) and backscattered-electron mode (QBSD) following punctual X-ray fluorescence spectra. Soil contamination derived from this product revealed the urgent need of addressing this problem. These elevated Pb levels, show that a preliminary treatment of BKP is mandatory before it is disposed into the common trash. The high lead content of this material makes its direct dumping into the environment unwise. (C) 2009 Elsevier B.V. All rights reserved.

Usefulness of MLPA in the detection of SHOX deletions

SHOX haploinsufficiency causes a wide spectrum of short stature phenotypes, such as Leri-Weill dyschondrosteosis (LWD) and disproportionate short stature (DSS). SHOX deletions are responsible for approximately two thirds of isolated haploinsufficiency; therefore, it is important to determine the most appropriate methodology for detection of gene deletion. In this study, three methodologies for the detection of SHOX deletions were compared: the fluorescence in situ hybridization (FISH), microsatellite analysis and multiplex ligation-dependent probe amplification (MLPA). Forty-four patients (8 LWD and 36 DSS) were analyzed. The cosmid LLNOYCO3`M`34F5 was used as a probe for the FISH analysis and microsatellite analysis were performed using three intragenic microsatellite markers. MLPA was performed using commercial kits. Twelve patients (8 LWD and 4 DSS) had deletions in SHOX area detected by MLPA and 2 patients generated discordant results with the other methodologies. In the first case, the deletion was not detected by FISH. In the second case, both FISH and microsatellite analyses were unable to identify the intragenic deletion. In conclusion, MLPA was more sensitive, less expensive and less laborious; therefore, it should be used as the initial molecular method for the detection of SHOX gene deletion. (C) 2010 Elsevier Masson SAS. All rights reserved.

A quantitative chemiluminescent method for studying replicative and stress-induced premature senescence in cell cultures

beta-Galactosidase (beta-Gal) activity is a widely accepted biomarker to detect senescence both in situ and in vitro. A cytochemical assay based on production of a blue-dyed precipitate that results from the cleavage of the chromogenic substrate X-Gal is commonly used. Blue and nonblue cells are counted under the microscope and a semiquantitative percentage of senescent cells can be obtained. Here, we present a quantitative, fast, and easy to use chemiluminescent assay to detect senescence. The Galacton chemiluminescent method used to detect the prokaryotic beta-Gal reporter enzyme in transfection studies was adapted to assay mammalian beta-Gal. The assay showed linear production of luminescence in a time- and cell-number-dependent manner. The chemiluminescent assay showed significant correlation with the cytochemical assay in detecting replicative senescence (Pearson r = 0.8486, p < 0.005). Moreover, the chemiluminescent method (Galacton) also detected stress-induced senescence in cells treated with H2O2 similar to the cytochemical assay (X-Gal) (Galacton: control 25.207.3 +/- 6548.6. H2O, 52,487.4 +/- 16,284.9, p < 0.05; X-Gal: control 41.31 +/- 7.0%, H2O2 92.97 +/- 2.8%, p < 0.01). Thus, our method is well suited to the detection of replicative and stress-induced senescence in cell culture. (C) 2007 Elsevier Inc. All rights reserved.

Inhibin A Production after Gonadotropin Stimulus: A New Method to Detect Ovarian Tissue in Ovotesticular Disorder of Sex Development

Background/Aims: While laboratory methods for the detection of testicular tissue are well standardized, currently there is no available test to demonstrate the presence of ovarian tissue. We evaluated the effectiveness of gonadal stimulation with luteinizing hormone (LH)/follicle-stimulating hormone (FSH) for the detection of ovarian tissue in patients with disorders of sex development (DSD). Methods: Ten patients with congenital adrenal hyperplasia (CAH) as ovarian-positive controls, 10 with cryptorchidism (ovarian-negative controls), 13 patients with DSD of no defined etiology and 7 patients with ovotesticular DSD (true hermaphroditism, TH) were included in the study. They underwent a daily injection of both LH and FSH on 3 consecutive days. LH, FSH, estradiol, testosterone and inhibin A were measured before treatment, 24 h after the 1st dose and 24 h after the 3rd dose. Results: Estradiol increased in all CAH and TH patients, with a median value of 155.1 and 92.6 pg/ml, respectively, after the 3rd injection. Inhibin A also increased in all CAH and TH patients, with a median value of 70.4 and 32.2 pg/ml, respectively, after the 3rd injection. There was no change in these hormones in the other groups. Conclusion: The LH/FSH stimulation test might be a useful method to detect the presence of ovarian tissue. Copyright (C) 2009 S. Karger AG, Basel

Transcriptional Response of Peripheral Lymphocytes to Early Fibrosarcoma: A Model System for Cancer Detection Based on Hybridization Signatures

Since circulating leukocytes, mainly B and T cells, continuously maintain vigilant and comprehensive immune surveillance, these cells could be used as reporters for signs of infection or other pathologies, including cancer. Activated lymphocyte clones trigger a sensitive transcriptional response, which could be identified by gene expression profiling. To assess this hypothesis, we conducted microarray analysis of the gene expression profile of lymphocytes isolated from immunocompetent BALB/c mice subcutaneously injected with different numbers of tumorigenic B61 fibrosarcoma cells. Flow cytometry demonstrated that the number of circulating T (CD3(+)CD4(+) or CD3(+)CD8(+)) or B (CD19(+)) cells did not change. However, the lymphocytes isolated from tumor cell-injected animals expressed a unique transcriptional profile that was identifiable before the development of a palpable tumor mass. This finding demonstrates that the transcriptional response appears before alterations in the main lymphocyte subsets and that the gene expression profile of peripheral lymphocytes can serve as a sensitive and accurate method for the early detection of cancer. Exp Biol Med 234:802-812, 2009

Radon-domain detection of the nipple and the pectoral muscle in mammograms

In this paper, methods are presented for automatic detection of the nipple and the pectoral muscle edge in mammograms via image processing in the Radon domain. Radon-domain information was used for the detection of straight-line candidates with high gradient. The longest straight-line candidate was used to identify the pectoral muscle edge. The nipple was detected as the convergence point of breast tissue components, indicated by the largest response in the Radon domain. Percentages of false-positive (FP) and false-negative (FN) areas were determined by comparing the areas of the pectoral muscle regions delimited manually by a radiologist and by the proposed method applied to 540 mediolateral-oblique (MLO) mammographic images. The average FP and FN were 8.99% and 9.13%, respectively. In the detection of the nipple, an average error of 7.4 mm was obtained with reference to the nipple as identified by a radiologist on 1,080 mammographic images (540 MLO and 540 craniocaudal views).

Detection of Spinal Muscular Atrophy Carriers in a Sample of the Brazilian Population

Background: Spinal muscular atrophy is a common autosomal recessive neuromuscular disorder caused by mutations in the SMN1 gene. Identification of spinal muscular atrophy carriers has important implications for individuals with a family history of the disorder and for genetic counseling. The aim of this study was to determine the frequency of carriers in a sample of the nonconsanguineous Brazilian population by denaturing high-performance liquid chromatography (DHPLC). Methods: To validate the method, we initially determined the relative quantification of DHPLC in 28 affected patients (DHPLC values: 0.00) and 65 parents (DHPLC values: 0.49-0.69). Following quantification, we studied 150 unrelated nonconsanguineous healthy individuals from the general population. Results: Four of the 150 healthy individuals tested (with no family history of a neuromuscular disorder) presented a DHPLC value in the range of heterozygous carriers (0.6-0.68). Conclusions: Based on these results, we estimated there is a carrier frequency of 2.7% in the nonconsanguineous Brazilian population, which is very similar to other areas of the world where consanguineous marriage is not common. This should be considered in the process of genetic counseling and risk calculations. Copyright (C) 2011 S. Karger AG, Basel

Detection of Clonal Immunoglobulin and T-Cell Receptor Gene Rearrangements in Childhood Acute Lymphoblastic Leukemia Using a Low-Cost PCR Strategy

Background Imunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements function as specific markers for minimal residual disease (MRD) which is one of the best predictors of outcome in childhood acute lymphoblastic leukemia (ALL) We recently reported on the prognostic value of MRD during the induction of remission through a simplified PCR method Here we report on gene rearrangement frequencies and offer guidelines for the application of the technique Procedure Two hundred thirty three children had DNA extracted from bone marrow Ig and TCR gene rearrangements were amplified using consensus primers and conventional PCR PCR products were submitted to homo/heteroduplex analysis A computer program was designed to define combinations of targets for clonal detection using a minimum set of primers and reactions Results At least one clonal marker could be detected in 98% of the patients and two markers in approximately 80% The most commonly rear ringed genes in precursor B cell ALL were IgH (75%) TCRD (59%) IgK (55%), and TCRG (54%) The most commonly rearranged genes for TALL were TCRG (100%) and TCRD (24%) The sensitivity of primers was limited to the detection of 1 leukemic cell among 100 normal cells Conclusions We propose that eight PCR reactions per ALL subtype would allow for the detection of two markers in most cases In addition these reactions ire suitable for MRD monitoring especially when aiming the selection of patients with high MRD levels (>= 10(-2)) at the end of induction therapy Such an approach would be very useful in centers with limited financial resources Pediatr Blood Cancer 2010 55 1278-1286 (C) 2010 Wiley Liss Inc

Detection of Auditory Cortex Activity by fMRI Using a Dependent Component Analysis

Functional MRI (fMRI) data often have low signal-to-noise-ratio (SNR) and are contaminated by strong interference from other physiological sources. A promising tool for extracting signals, even under low SNR conditions, is blind source separation (BSS), or independent component analysis (ICA). BSS is based on the assumption that the detected signals are a mixture of a number of independent source signals that are linearly combined via an unknown mixing matrix. BSS seeks to determine the mixing matrix to recover the source signals based on principles of statistical independence. In most cases, extraction of all sources is unnecessary; instead, a priori information can be applied to extract only the signal of interest. Herein we propose an algorithm based on a variation of ICA, called Dependent Component Analysis (DCA), where the signal of interest is extracted using a time delay obtained from an autocorrelation analysis. We applied such method to inspect functional Magnetic Resonance Imaging (fMRI) data, aiming to find the hemodynamic response that follows neuronal activation from an auditory stimulation, in human subjects. The method localized a significant signal modulation in cortical regions corresponding to the primary auditory cortex. The results obtained by DCA were also compared to those of the General Linear Model (GLM), which is the most widely used method to analyze fMRI datasets.