Quantitation of malondialdehyde in gingival crevicular fluid by a high-performance liquid chromatography-based method

Lipid peroxidation (LPO) has been associated with periodontal disease, and the evaluation of malondialdehyde (MDA) in the gingival crevicular fluid (GCF), an inflammatory exudate from the surrounding tissue of the periodontium, may be useful to clarify the role of LPO in the pathogenesis of periodontal disease. We describe the validation of a method to measure MDA in the GCF using high-performance liquid chromatography. MDA calibration curves were prepared with phosphate-buffered solution spiked with increasing known concentrations of MDA. Healthy and diseased GCF samples were collected from the same patient to avoid interindividual variability. MDA response was linear in the range measured, and excellent agreement was observed between added and detected concentrations of MDA. Samples' intra- and interday coefficients of variation were below 6.3% and 12.4%, respectively. The limit of quantitation (signal/noise = 5) was 0.03 mu M. When the validated method was applied to the GCF, excellent agreement was observed in the MDA quantitation from healthy and diseased sites, and diseased sites presented more MDA than healthy sites (P < 0.05). In this study, a validated method for MDA quantitation in GCF was established with satisfactory sensitivity, precision, and accuracy. (C) 2012 Elsevier Inc. All rights reserved.

DEVELOPMENT AND VALIDATION OF AN ANALYTICAL METHOD FOR QUANTITATION OF THE DRUG BEVACIZUMAB BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

DEVELOPMENT AND VALIDATION OF AN ANALYTICAL METHOD FOR QUANTITATION OF THE DRUG BEVACIZUMAB BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY. In this study, an analytical method was developed and validated for quantitation of the drug bevacizumab (Avastin (R)) by high performance liquid chromatography (HPLC). The HPLC column was a BioSuite 250 (R) HR SEC, 300 x 7.8 mm x 5 mu m (Waters, USA). The mobile phase consisted of phosphate buffered saline (PBS). The results revealed that the method was specific, precise. accurate, robust and linear (r(2) = 0.998) from 5 to 75 mu g mL(-1). Therefore, this method can be used in drug release studies or in quality control ampoules of the drug.

A Validated Reverse Phase HPLC Analytical Method for Quantitation of Glycoalkaloids in Solanum lycocarpum and Its Extracts

Solanum lycocarpum (Solanaceae) is native to the Brazilian Cerrado. Fruits of this species contain the glycoalkaloids solasonine (SN) and solamargine (SM), which display antiparasitic and anticancer properties. A method has been developed for the extraction and HPLC-UV analysis of the SN and SM in different parts of S. lycocarpum, mainly comprising ripe and unripe fruits, leaf, and stem. This analytical method was validated and gave good detection response with linearity over a dynamic range of 0.77-1000.00 mu g mL(-1) and recovery in the range of 80.92-91.71%, allowing a reliable quantitation of the target compounds. Unripe fruits displayed higher concentrations of glycoalkaloids (1.04% +/- 0.01 of SN and 0.69% +/- 0.00 of SM) than the ripe fruits (0.83% +/- 0.02 of SN and 0.60% +/- 0.01 of SM). Quantitation of glycoalkaloids in the alkaloidic extract gave 45.09% +/- 1.14 of SN and 44.37% +/- 0.60 of SM, respectively.

Plasmodium vivax: Reverse transcriptase real-time PCR for gametocyte detection and quantitation in clinical samples

The proportion of Plasmodium vivax-infected subjects that carry mature gametocytes, and thus are potentially infectious, remains poorly characterized in endemic settings. Here, we describe a quantitative reverse transcriptase (RI) real-time PCR (qRT-PCR) that targets transcripts of the mature gametocyte-specific pvs25 gene. We found mature gametocytes in 42 of 44 (95.4%) P. vivax infections diagnosed during an ongoing cohort study in northwestern Brazil. SYBR green qRT-PCR was more sensitive than a conventional RT-PCR that targets the same gene. Molecular detection of gametocytes failed, however, when dried bloodspots were used for RNA isolation and complementary DNA synthesis. Estimating the number of pvs25 gene transcripts allowed for examining the potential infectiousness of gametocyte carriers in a quantitative way. We found that most (61.9%) gametocyte carriers were either asymptomatic or had subpatent parasitemias and would have been missed by routine malaria control strategies. However, potentially undiagnosed gametocyte carriers usually had low-density infections and contributed a small fraction (up to 4%) to the overall gametocyte burden in the community. Further studies are required to determine the relative contribution to malaria transmission of long-lasting but low-density gametocytemias in asymptomatic carriers that are left undiagnosed and untreated. (C) 2012 Elsevier Inc. All rights reserved.

Real-time PCR quantitation of glucocorticoid receptor alpha isoform

Abstract Background The expression of glucocorticoid-receptor (GR) seems to be a key mechanism in the regulation of glucocorticoid (GC) sensitivity and is potentially involved in cases of GC resistance or hypersensitivity. The aim of this study is to describe a method for quantitation of GR alpha isoform (GRα) expression using real-time PCR (qrt-PCR) with analytical capabilities to monitor patients, offering standard-curve reproducibility as well as intra- and inter-assay precision. Results Standard-curves were constructed by employing standardized Jurkat cell culture procedures, both for GRα and BCR (breakpoint cluster region), as a normalizing gene. We evaluated standard-curves using five different sets of cell culture passages, RNA extraction, reverse transcription, and qrt-PCR quantification. Intra-assay precision was evaluated using 12 replicates of each gene, for 2 patients, in a single experiment. Inter-assay precision was evaluated on 8 experiments, using duplicate tests of each gene for two patients. Standard-curves were reproducible, with CV (coefficient of variation) of less than 11%, and Pearson correlation coefficients above 0,990 for most comparisons. Intra-assay and inter-assay were 2% and 7%, respectively. Conclusion This is the first method for quantitation of GRα expression with technical characteristics that permit patient monitoring, in a fast, simple and robust way.

CRITICAL APPLICATIONS OF SW 846 US EPA METHODS TO EVALUATION OF MARINE SAMPLES QUALITY

Technical evaluation of analytical data is of extreme relevance considering it can be used for comparisons with environmental quality standards and decision-making as related to the management of disposal of dredged sediments and the evaluation of salt and brackish water quality in accordance with CONAMA 357/05 Resolution. It is, therefore, essential that the project manager discusses the environmental agency`s technical requirements with the laboratory contracted for the follow-up of the analysis underway and even with a view to possible re-analysis when anomalous data are identified. The main technical requirements are: (1) method quantitation limits (QLs) should fall below environmental standards; (2) analyses should be carried out in laboratories whose analytical scope is accredited by the National Institute of Metrology (INMETRO) or qualified or accepted by a licensing agency; (3) chain of custody should be provided in order to ensure sample traceability; (4) control charts should be provided to prove method performance; (5) certified reference material analysis or, if that is not available, matrix spike analysis, should be undertaken and (6) chromatograms should be included in the analytical report. Within this context and with a view to helping environmental managers in analytical report evaluation, this work has as objectives the discussion of the limitations of the application of SW 846 US EPA methods to marine samples, the consequences of having data based on method detection limits (MDL) and not sample quantitation limits (SQL), and present possible modifications of the principal method applied by laboratories in order to comply with environmental quality standards.

Development and validation of a simple and sensitive high performance liquid chromatographic method for the simultaneous determination of anastrozole, bicalutamide, tamoxifen, and their synthetic impurities

A simple and sensitive analytical method for simultaneous determination of anastrozole, bicalutamide, and tamoxifen as well as their synthetic impurities, anastrozole pentamethyl, bicalutamide 3-fluoro-isomer, and tamoxifen e-isomer, was developed and validated by using high performance liquid chromatography (HPLC). The separation was achieved on a Symmetry (R) C-8 column (100 x 4.6 mm i.d., 3.5 mu m) at room temperature (+/- 24 degrees C), with a mobile phase consisting of acetonitrile/water containing 0.18% N,N dimethyloctylamine and pH adjusted to 3.0 with orthophosphoric acid (46.5/53.5, v/v) at a flow rate of 1.0 mL min(-1) within 20 min. The detection was made at a wavelength of 270 nm by using ultraviolet (UV) detector. No interference peaks from excipients and relative retention time indicated the specificity of the method. The calibration curve showed correlation coefficients (r) > 0.99 calculated by linear regression and analysis of variance (ANOVA). The limit of detection (LOD) and limit of quantitation (LOQ), respectively, were 2.2 and 6.7 mu g mL(-1) for anastrozole, 2.61 and 8.72 mu g mL(-1) for bicalutamide, 2.0 and 6.7 mu g mL(-1) for tamoxifen, 0.06 and 0.22 mu g mL(-1) for anastrozole pentamethyl, 0.02 and 0.07 mu g mL(-1) for bicalutamide 3-fluoro-isomer, and 0.002 and 0.007 mu g mL(-1) for tamoxifen e-isomer. Intraday and interday relative standard deviations (RSDs) were <2.0% (drugs) and <10% (degradation products) as well as the comparison between two different analysts, which were calculated by f test. (C) 2012 Elsevier B.V. All rights reserved.

Bacterial leakage along the implant-abutment interface: culture and DNA Checkerboard hybridization analyses

Objective Bacterial species have been found harboring the internal surface of dental implants as consequence of their failed connections. The aim of the present study was to compare the detection frequency of bacterial leakage from human saliva through the implantabutment interface, under non-loading conditions, using either DNA Checkerboard or culture method. Materials and methods Thirty dental implants with hexagonal platforms were connected to pre-machined abutments according to the manufacturers specifications. The assemblies were individually incubated in human saliva under anaerobic conditions for 7 similar to days at 37 degrees C. Afterward, contents from the inner parts of the implants were collected and evaluated with either DNA Checkerboard (s similar to=similar to 15) or culture (n similar to=similar to 15). Subsequently, identification and quantitation of bacterial species from saliva and implants were carried out for the group evaluated with the DNA Checkerboard method. Results Both DNA Checkerboard and culture showed positive signals of bacterial leakage in 6 of the 15 evaluated samples. Capnocytophaga gingivalis and Streptococcus mutans were the most frequently detected species harboring the internal surface of the implants followed by Veillonella parvula. Conclusion Occurrence of bacterial leakage along the implantabutment interface is comparably detected with both DNA Checkerboard hybridization and conventional culture methods.

A new HPLC-DAD-MS/MS method for the simultaneous determination of major compounds in the crude extract of Lychnophora salicifolia Mart. (Brazilian arnicao) leaves: Application to chemical variability evaluation

Lychnophora salicifolia Mart., which occurs in the Brazilian Cerrado in the states of Bahia and Minas Gerais as well as in the southeast of the state of Goias, is the most widely distributed and also the most polymorphic species of the genus. This plant is popularly known to have anti-inflammatory and analgesic activities. In this work, we have studied the variation in terms of polar metabolites of ninety-three Lychnophora salicifolia Mart, specimens collected from different regions of the Brazilian Cerrado. Identification of the constituents of this mixture was carried out by analysis of the UV spectra and MS data after chromatographic separation. Twenty substances were identified, including chlorogenic acid derivatives, a flavonoid C-glucoside, and other sesquiterpenes. The analytical method was validated, and the reliability and credibility of the results was ensured for the purposes of this study. The concentration range required for analysis of content variability within the analyzed group of specimens was covered with appropriate values of limits of detection and quantitation, as well as satisfactory precision and recovery. A quantitative variability was observed among specimens collected from the same location, but on average they were similar from a chemical viewpoint. In relation to the study involving specimens from different locations, there were both qualitative and quantitative differences among plants collected from different regions of Brazil. Statistical analysis revealed that there is a correlation between geographical localization and polar metabolites profile for specimens collected from different locations. This is evidence that the pattern of metabolites concentration depends on the geographical distribution of the specimens. (C) 2012 Elsevier B.V. All rights reserved.

On the formation of carbonate adducts of fatty alcohols, sterols, and sugars in biological conditions

The formation and properties of carbonate adducts of some organic hydroxy compounds in aqueous medium were investigated. Fatty alcohols and sugars were chosen as representative classes of biological interest, and the medium was carbonated aqueous solution with pH ranging from 3.0 to 8.3. Capillary electrophoresis with two capacitively coupled contactless conductivity detectors (C4Ds) was used for quantitation and to obtain the mobility of the monoalkyl carbonates (MACs), which were used to determine the equilibrium and kinetic constants of the reaction as well as the diffusion coefficients. For increasing chain length of the alcohols, the equilibrium constant tends to the unit, which suggests that fatty alcohols can form the corresponding MACs. The formation of MACs for cyclohexanol and cyclopentanol also suggest the existence of similar species for sterols. Carbonate adducts of fructose, glucose, and sucrose were also detected, which suggests that these counterparts of the well-known phosphates can also occur in the cytosol. Our calculations suggest that one in 1000 to one in 10 000 molecules of these hydroxy compounds would be available as the corresponding MAC in such a medium. Experiments carried out at pH values less than 3.0 showed that there is a catalytic effect of hydronium on the interconversion of bicarbonate and a MAC. Taking into account the great number of hydroxy compounds similar to the ones investigated and that bicarbonate is ubiquitous in living cells, one can anticipate the existence of a whole new class of carbonate adducts of these metabolites.

Solid-phase microextraction combined with comprehensive two-dimensional gas chromatography for fatty acid profiling of cell wall phospholipids

The combination of solid-phase microextraction (SPME) with comprehensive two-dimensional gas chromatography is evaluated here for fatty acid (FA) profiling of the glycerophospholipid fraction from human buccal mucosal cells. A base-catalyzed derivatization reaction selective for polar lipids such as glycerophospholipid was adopted. SPME is compared to a miniaturized liquidliquid extraction procedure for the isolation of FA methyl esters produced in the derivatization step. The limits of detection and limits of quantitation were calculated for each sample preparation method. Because of its lower values of limits of detection and quantitation, SPME was adopted. The extracted analytes were separated, detected, and quantified by comprehensive two-dimensional gas chromatography with flame ionization detection (FID). The combination of SPME and comprehensive two-dimensional gas chromatography with FID, using a selective derivatization reaction in the preliminary steps, proved to be a simple and fast procedure for FA profiling, and was successfully applied to the analysis of adult human buccal mucosal cells.

Enantioselective analysis of unbound tramadol, O-desmethyltramadol and N-desmethyltramadol in plasma by ultrafiltration and LC-MS/MS: Application to clinical pharmacokinetics

This study describes the enantioselective analysis of unbound and total concentrations of tramadol and its main metabolites O-desmethyltramadol (M1) and N-desmethyltramadol (M2) in human plasma. Sample preparation was preceded by an ultrafiltration step to separate the unbound drug. Both the ultrafiltrate and plasma samples were submitted to liquid/liquid extraction with methyl t-butyl ether. Separation was performed on a Chiralpak (R) AD column and tandem mass spectrometry consisting of an electrospray ionization source, positive ion mode and multiple reaction monitoring was used as the detection system. Linearity was observed in the following ranges: 0.2-600 and 0.5-250 ng/mL for analysis of total and unbound concentrations of the tramadol enantiomers, respectively, and 0.1-300 and 0.25-125 ng/mL for total and unbound concentrations of the M1 and M2 enantiomers, respectively. The lower limits of quantitation were 0.2 and 0.5 ng/mL for analysis of total and unbound concentration of each tramadol enantiomer, respectively, and 0.1 and 0.25 ng/mL for total and unbound concentrations of M1 and M2 enantiomers, respectively. Intra- and interassay reproducibility and inaccuracy did not exceed 15%. Clinical application of the method to patients with neuropathic pain showed plasma accumulation of (+)-tramadol and (+)-M2 after a single oral dose of racemic tramadol. Fractions unbound of tramadol, M1 or M2 were not enantioselective in the patients investigated. (C) 2011 Elsevier B.V. All rights reserved.

Molecular and phylogenetic analyses of human Parvovirus B19 isolated from Brazilian patients with sickle cell disease and beta-thalassemia major and healthy blood donors

Human Parvovirus B19 (B19V) is a recognized cause of life-threatening conditions among patients with hemoglobinopathies. This study investigates B19V infection in patients with sickle cell disease and beta-thalassemia using different experimental approaches. A total of 183 individuals (144 with sickle cell disease and 39 with beta-thalassemia major) and 100 healthy blood donors were examined for B19V using anti-B19V IgG enzyme immunoassay, quantitative PCR, DNA sequencing, and phylogenetic analysis. Viremia was documented in 18.6% of patients and 1% of donors, and was generally characterized by low viral load (VL); however, acute infections were also observed. Anti-B19V IgG was detected in 65.9% of patients with sickle cell disease and in 60% of donors, whereas the patients with thalassemia exhibited relatively low seroreactivity. The seroprevalence varied among the different age groups. In patients, it progressively increased with age, whereas in donors it reached a plateau. Based on partial NS1 fragments, all isolates detected were classified as subgenotype 1A with a tendency to elicit genetically complex infections. Interestingly, quasispecies occurred in the plasma of not only patients but also donors with even higher heterogeneity. The partial NS1 sequence examined did not exhibit positive selection. Quantitation of B19V with a conservative probe is a technically and practically useful approach. The extensive spread of B19V subgenotype 1A in patients and donors and its recent introduction into the countryside of the Sao Paulo State, Brazil were demonstrated; however, it is difficult to establish a relationship between viral sequences and the clinical outcomes of the infection. J. Med. Virol. 84:16521665, 2012. (c) 2012 Wiley Periodicals, Inc.

Depletion study and estimation of the withdrawal period for oxytetracycline in tilapia cultured in Brazil

Defining pharmacokinetic parameters and depletion intervals for antimicrobials used in fish represents important guidelines for future regulation by Brazilian agencies of the use of these substances in fish farming. This article presents a depletion study for oxytetracycline (OTC) in tilapias (Orechromis niloticus) farmed under tropical conditions during the winter season. High performance liquid chromatography, with fluorescence detection for the quantitation of OTC in tilapia fillets and medicated feed, was developed and validated. The depletion study with fish was carried out under monitored environmental conditions. OTC was administered in the feed for five consecutive days at daily dosages of 80 mg/kg body weight. Groups of ten fish were slaughtered at 1, 2, 3, 4, 5, 8, 10, 15, 20, and 25 days after medication. After the 8th day posttreatment, OTC concentrations in the tilapia fillets were below the limit of quantitation (13 ng/g) of the method. Linear regression of the mathematical model of data analysis presented a coefficient of 0.9962. The elimination half- life for OTC in tilapia fillet and the withdrawal period were 1.65 and 6 days, respectively, considering a percentile of 99% with 95% of confidence and a maximum residue limit of 100 ng/g. Even though the study was carried out in the winter under practical conditions where water temperature varied, the results obtained are similar to others from studies conducted under controlled temperature.

Correlates of human immunodeficiency virus cervicovaginal shedding among postmenopausal and fertile-aged women

Objective: The aims of this study were to compare the intensity of human immunodeficiency virus (HIV)-RNA genital shedding among postmenopausal (PM) and fertile-aged (F) women and to investigate the association between viral shedding and gynecological features, HIV plasma viral loads, and other markers of HIV disease progression. Methods: We interviewed 146 HIV-infected women (73 PM/73 F) in search of gynecological complaints and signs and symptoms of HIV disease and obtained additional information concerning HIV infection by medical chart review. Cervicovaginal lavages (CVLs) were collected for assessment of HIV shedding. Laboratory analyses included CD4(+) cell counts, HIV-RNA quantitation in plasma and CVL, and screening for concurrent genital infections. Results: HIV-RNA genital shedding was detected in 16.4% of PM and 21.9% of F women (P = 0.400), and the intensity of HIV shedding did not differ between both groups (means-PM: 1.4log/mL; F: 1.4log/mL; P = 0.587). Three women (2 PM/1 F) exhibited viral shedding in the absence of detectable viremia. HIV plasma viral loads correlated with HIV shedding in both groups. In multivariable analysis, HIV plasma viral loads were independently associated with HIV shedding in both groups. Moreover, the intensity of shedding was independently associated with vaginal pH, tumor necrosis factor a concentrations in CVL, and HIV plasma viral loads. Conclusions: Despite significant changes that occur in the vaginal mucosa of PM women, HIV cervicovaginal shedding was not significantly influenced by this state in our cohort. In contrast, increased vaginal pH and genital inflammation, evidenced by increased tumor necrosis factor alpha concentrations in CVL and HIV plasma viral loads, were independently associated with the intensity of HIV shedding in PM and F women.