National Institute of Science and Technology in Bioanalytics

Campinas, Brazil

National Institute of Science and Technology in Bioanalytics

Campinas, Brazil
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Orlando E.A.,Institute of Food Technology | Costa Roque A.G.,Institute of Food Technology | Losekann M.E.,EMBRAPA - Empresa Brasileira de Pesquisa Agropecuária | Colnaghi Simionato A.V.,University of Campinas | Colnaghi Simionato A.V.,National Institute of Science and Technology in Bioanalytics
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2016

Despite the benefits to fish farmers, the use of antimicrobials in aquaculture has concerned consumers and competent authorities. The indiscriminate use of such substances promotes the emergence of resistant microorganisms, decreases the effectiveness of treatments, and causes possible toxic effects in humans. In Brazil, florfenicol is the only antimicrobial registered for use in aquaculture and is often used in tilapia in cage creation. Thus, this study aimed to develop a method for determination of florfenicol residues and its metabolite florfenicol amine in tilapia fillet by UPLC–MS/MS. Analytes were extracted with ethyl acetate, followed by liquid-liquid partition clean-up with hexane and SPE. The sorbents C18, phenyl and HLB-Oasis were evaluated by SPE. Phenyl sorbent showed the best results, and the extraction conditions were optimized in the sample matrix with fractional factorial design 24−1. The analytes were separated on a C18 chromatographic column (50 × 2.1 mm × 1.7 μm) using water (A) and acetonitrile (B) as mobile phase at a flow rate of 0.3 mL min−1 with a linear gradient (in% B): 0–2.0 min: 20%; 2.0–2.5 min: increase to 90%; 2.5–3.5 min: 90%; 3.0–3.5 min: decrease to 20%; 4.0–5.0 min: 20%. The analytes were monitored in a MS/MS triple quadrupole system by MRM mode with transitions at m/z 356.1 > 336.1 (florfenicol) and m/z 248.1 > 130.1 (florfenicol amine). The optimized method was validated obtaining LOQ values of 3 and 25 ng g−1 for florfenicol and florfenicol amine, respectively, precision between 20 and 36%, absolute extraction efficiency between 38 and 80%, and adequate linearity. The method was applied to samples intended for human consumption, and within the 15 evaluated samples, only one showed florfenicol residue at 30 ng g−1, which is below the maximum residue limit established in Brazil. © 2016

De Campos R.P.S.,University of Kansas | De Campos R.P.S.,University of Campinas | De Campos R.P.S.,National Institute of Science and Technology in Bioanalytics | Siegel J.M.,University of Kansas | And 5 more authors.
Analytical and Bioanalytical Chemistry | Year: 2015

Superoxide, a naturally produced reactive oxygen species (ROS) in the human body, is involved in many pathological and physiological signaling processes. However, if superoxide formation is left unregulated, overproduction can lead to oxidative damage to important biomolecules, such as DNA, lipids, and proteins. Superoxide can also lead to the formation of peroxynitrite, an extremely hazardous substance, through its reaction with endogenously produced nitric oxide. Despite its importance, quantitative information regarding superoxide production is difficult to obtain due to its high reactivity and low concentrations in vivo. MitoHE, a fluorescent probe that specifically reacts with superoxide, was used in conjunction with microchip electrophoresis (ME) and laser-induced fluorescence (LIF) detection to investigate changes in superoxide production by RAW 264.7 macrophage cells following stimulation with phorbol 12-myristate 13-acetate (PMA). Stimulation was performed in the presence and absence of the superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC) and 2-metoxyestradiol (2-ME). The addition of these inhibitors resulted in an increase in the amount of superoxide specific product (2-OH-MitoE+) from 0.08 ± 0.01 fmol (0.17 ± 0.03 mM) in native cells to 1.26 ± 0.06 fmol (2.5 ± 0.1 mM) after PMA treatment. This corresponds to an approximately 15-fold increase in intracellular concentration per cell. Furthermore, the addition of 3-morpholino-sydnonimine (SIN-1) to the cells during incubation resulted in the production of 0.061 ± 0.006 fmol (0.12 ± 0.01 mM) of 2-OH-MitoE+ per cell on average. These results demonstrate that indirect superoxide detection coupled with the use of SOD inhibitors and a separation method is a viable method to discriminate the 2-OH-MitoE+ signal from possible interferences. © 2015 Springer-Verlag Berlin Heidelberg.

Orlando E.A.,University of Campinas | Simionato A.V.C.,University of Campinas | Simionato A.V.C.,National Institute of Science and Technology in Bioanalytics
Journal of Chromatography A | Year: 2013

Anti-microbials have been used to control the quality of the aquatic environment for both prophylactic and therapeutic purposes. Tetracyclines are among the main antimicrobials used in aquaculture, and present a particular difficulty for extraction, due to a complex structure and high interaction with components of the biological matrix. In this study, different techniques of extraction and clean-up of antimicrobials of the tetracycline class in tilapia filets have been optimized and compared, followed by validation of the methodology using the best procedure. Oxytetracycline, doxycycline, tetracycline and chlortetracycline were analyzed by HPLC-fluorescence under the following conditions: organic mobile phase composed of methanol:acetonitrile (1:1, v/v) and aqueous mobile phase containing sodium acetate (0.0375molL-1), calcium chloride (0.0175molL-1) and EDTA (0.0125molL-1) at pH 7.00. The chromatographic analysis was performed using a mobile phase gradient with a flow rate of 1mLmin-1 and detection wavelength of 385/528nm (λexc/λem). Four extraction methods have been evaluated, namely: liquid-liquid partition; solid phase extraction (SPE) using phenyl, C18 and polymeric Oasis-HLB stationary phases; dispersive SPE (dSPE) using polymeric adsorbent XAD 16 resin; and QuEChERS. The methods have been optimized with fractional factorial experimental design and compared by the extraction efficiency. The liquid-liquid extraction and the QuEChERS methods showed low extraction efficiencies (14-30%) for the analytes. The use of dSPE showed good efficiency (40-60%), but with low precision and high consumption of time. Among the evaluated extraction techniques the use of SPE showed the best results, with emphasis on the phenyl phase (58-76%), and has been validated for analysis of residues of tetracyclines in tilapia muscle regarding selectivity, linearity, precision and limits of detection and quantification. The validated method was adequate for the investigation of the analytes at residue levels. © 2013 Elsevier B.V.

Buzatto A.Z.,University of Campinas | De Sousa A.C.,University of Campinas | Guedes S.F.,University of Campinas | Cieslarova Z.,University of Campinas | And 2 more authors.
Electrophoresis | Year: 2014

Metabolomics is one of the most recent trends in the "omics" era that investigates the end products of an organism activity, that is, all metabolites in a biological system, which are small molecules (less than 1000 Da) from different chemical classes. Metabolomics represents a tool to assess the biochemical activity of a living system through the analysis of substrates and products processed during the metabolism. The analysis of the metabolic profile (nontargeted analysis, i.e. a comparison between samples profiles of individuals) and of specific metabolites (targeted analysis, which quantifies a selected group of metabolites) in biological samples provides an insight into the metabolic state and the biochemical processes of the organism and, therefore, may indicate the onset and the stage of different diseases. An early and accurate diagnosis is essential for successful treatment and probable cure of most illnesses; hence, the investigation of metabolites as disease biomarkers has increased considerably in recent years. This review aims to present the most relevant works that address the nontargeted and targeted analysis of metabolites in different diseases for the past 10 years, including kidney and neurological disorders, cardiovascular diseases, diabetes, and cancer, using CE and LC coupled with the accurate detection of mass spectroscopy. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Leitao A.A.,University of Campinas | de Lacerda Miranda P.C.M.,University of Campinas | Simionato A.V.C.,University of Campinas | Simionato A.V.C.,National Institute of Science and Technology in Bioanalytics
Electrophoresis | Year: 2011

Leaf-cutting ants cause large losses in several crops around the world. In the ant species Atta sexdens, each colony comprises up to 5 million individuals. In order to keep a close connection among such a large number of individuals, an efficient chemical communication system is necessary. Among other different substances, these animals use alkylpyrazines to mark their trails and to guide ant workers from the nest to their sources of food. In this study, CE-UV was used to apply a method for qualitative analysis of venom gland components of leaf-cutting ants. Mobility of these compounds proved to be a function of the ionization capability of these bases as well as their volumes. Migration order was thoroughly explained in terms of such parameters. The best analysis conditions were achieved with a BGE composed by 0.8% formic acid plus 20% methanol in water, hydrodynamic injection, and application of external pressure. Such analysis conditions may be easily applied in CE-MS analyses as well. CE-UV analyses proved to be as adequate as GC to analyze such compounds due to system detectability (LD ≅ 0.005mmol/L), separation efficiency (from 5.07×104 to 1.23×105 theoretical plates), and resolution (minimum of 2.35). In addition, analysis time was ca. 15min, which shows another advantage of CE analysis when compared with GC. Although the analytes are found in concentrations as low as 50ng/venom glands, four putative pyrazine ring moiety substances could be detected in real samples, due to sample stacking and use of a capillary with extended detection cell. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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