Microwave-assisted digestion procedures for biological samples with diluted nitric acid: Identification of reaction products

Microwave-assisted sample preparation using diluted nitric acid solutions is an alternative procedure for digesting organic samples. The efficiency of this procedure depends on the chemical properties of the samples and in this work it was evaluated by the determination of crude protein amount. fat and original carbon. Soybeans grains, bovine blood. bovine muscle and bovine viscera were digested in a cavity-microwave oven using oxidant mixtures in different acid concentrations. The digestion efficiency was evaluated based on the determination of residual carbon content and element recoveries using inductively coupled plasma optical emission spectrometry (ICP OES). In order to determine the main residual organic compounds, the digests were characterized by nuclear magnetic resonance (1 H NMR). Subsequently, studies concerning separation of nitrobenzoic acid isomers were performed by ion pair reversed phase liquid chromatography using a C18 stationary phase, water:acetonitrile:methanol (75:20:5, v/v/v) +0.05% (v/v) TFA as mobile phase and ultraviolet detection at 254 nm. Sample preparation based on diluted acids proved to be feasible and a recommendable alternative for organic sample digestion, reducing both the reagent volumes and the variability of the residues as a result of the process of decomposition. It was shown that biological matt-ices containing amino acids, proteins and lipids in their composition produced nitrobenzoic acid isomers and other organic compounds after cleavage of chemical bonds. (C) 2009 Elsevier B.V. All rights reserved.

An improved and fast UHPLC-PDA methodology for determination of L-ascorbic and dehydroascorbic acids in fruits and vegetables: evaluation of degradation rate during storage

This study provides a versatile validated method to determine the total vitamin C content, as the sum of the contents of L-ascorbic acid (L-AA) and dehydroascorbic acid (DHAA), in several fruits and vegetables and its degradability with storage time. Seven horticultural crops from two different origins were analyzed using an ultrahigh-performance liquid chromatographic–photodiode array (UHPLC-PDA) system, equipped with a new trifunctional high strength silica (100% silica particle) analytical column (100 mm×2.1 mm, 1.7 μm particle size) using 0.1% (v/v) formic acid as mobile phase, in isocratic mode. This new stationary phase, specially designed for polar compounds, overcomes the problems normally encountered in HPLC and is suitable for the analysis of large batches of samples without L-AA degradation. In addition, it proves to be an excellent alternative to conventional C18 columns for the determination of L-AA in fruits and vegetables. The method was fully validated in terms of linearity, detection (LOD) and quantification (LOQ) limits, accuracy, and inter/intraday precision. Validation experiments revealed very good recovery rate of 96.6±4.4% for L-AA and 103.1±4.8 % for total vitamin C, good linearity with r2-values >0.999 within the established concentration range, excellent repeatability (0.5%), and reproducibility (1.6%) values. The LOD of the method was 22 ng/mL whereas the LOQ was 67 ng/mL. It was possible to demonstrate that L-AA and DHAA concentrations in the different horticulture products varied oppositely with time of storage not always affecting the total amount of vitamin C during shelf-life. Locally produced fruits have higher concentrations of vitamin C, compared with imported ones, but vegetables showed the opposite trend. Moreover, this UHPLC-PDA methodology proves to be an improved, simple, and fast approach for determining the total content of vitamin C in various food commodities, with high sensitivity, selectivity, and resolving power within 3 min of run analysis.

Effect of supplementation of diets for quails with vitamins A, D and E on performance of the birds and quality and enrichment of eggs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

High concentrations of the melatonin metabolite, N-1-acetyl-N (2)-formyl-5-methoxykynuramine, in cerebrospinal fluid of patients with meningitis: a possible immunomodulatory mechanism

We evaluated the presence of the melatonin metabolite N-1-acetyl-N-2-formyl-5-methoxykynuramine (AFMK), in cerebrospinal fluid (CSF) of patients with viral meningitis (n = 20) and control samples (n = 8) and correlate AFMK levels with inflammatory markers such as cellularity, protein, tumor necrosis factor (TNF)-alpha, interleukin (IL)-8 and IL-1 beta levels. A portion of the CSF was extracted with dichloromethane (1:5) and analyzed by high-performance liquid chromatography (HPLC) under standardized conditions for AFMK. AFMK was detected in 16 of 20 CSF samples of patients with viral meningitis; the concentration of AFMK was found to be above the quantification limit (50 nmol/L) in six of these samples. AFMK was not detected in any of the eight control samples. The samples were classified into groups according to AFMK levels: undetectable (< 10 nmol/L, group I), detectable but below the quantification limit (< 50 nmol/L, group II), and quantified (> 50 nmol/L, group III). Group II presented the highest levels of proteins and IL-8, whereas group III showed the lowest levels of the inflammatory parameters. This study supports our hypothesis that inflammation favors the formation of AFMK and that this compound has immunomodulatory activity in vivo.

Supercritical fluid extraction for pesticide multiresidue analysis in honey: determination by gas chromatography with electron-capture and mass spectrometry detection

An analytical procedure using supercritical fluid extraction (SFE) and capillary gas chromatography with electron-capture detection was developed to determine simultaneously residues of different pesticides (organochlorine, organophosphorus, organonitrogen and pyrethroid) in honey samples. Fortification experiments were conducted to test conventional extraction (liquid-liquid) and optimize the extraction procedure in SFE by varying the CO2-modifier, temperature, extraction time and pressure. Best efficiency was achieved at 400 bar using acetonitrile as modifier at 90 degreesC. For the clean-up step, Florisil cartridges were used for both methods LLE and SFE. Recoveries for majority of pesticides from fortified samples of honey at fortification level of 0.01-0.10 mg/kg ranged 75-94% from both methods. Limits of detection found were less than 0.01 mg/kg for ECD and confirmation of pesticide identity was performed by gas chromatography-mass spectrometry in selected-ion monitoring mode. The multiresidue methods in real honey samples were applied and the results of developed methods were compared. (C) 2004 Elsevier B.V. All rights reserved.

Off line extraction of phenol from human urine sample with isoamyl alcohol and determination by HPLC

This method has been developed for extraction and determination of phenol in a urine sample by high performance liquid chromatography.After acid hydrolysis, the free phenol was extracted with isoamyl alcohol solvent, followed by back extraction with 0.5 mol.L-1 sodium hydroxide solution and analyzed by an isocratic HPLC Varian System, equipped with reverse-phase column (MicroPak-C-18). The mobile phase was acetonitrile in 0.01 mol.L-1 hydrochloric acid solution, (20:80 v/v), and at a now-rate of 1.0 mL.min-1. The chromatogram was monitored at 220 nm in room temperature. The identification was based on retention time and the quantification was performed by automatic peak-area determination, corrected for the external standards method.The recovery was higher than 99.5 % for phenol and reproducibility of method was shown to be 2.3% standard deviation and 5.6% coefficient of variance (n=20). The limit detection was 0.05 mgL(-1) and a range of 0.05 to 20.0 mgL(-1) of phenol for linearity.

The identification of beta-thalassemia mutants in Brazilians with high Hb F levels

As hemoglobinopatias são um grupo de afecções genéticas que representam problema de saúde pública em muitos países em que sua incidência é alta, com significativa morbidade. Objetivamos identificar defeitos moleculares que pudessem explicar o perfil laboratorial obtido por eletroforese e HPLC com Hb F elevada, em um grupo de indivíduos adultos sem sinais ou sintomas de anemia. Encontramos cinco diferentes mutações que originam beta talassemia por PCR-ASO: três casos com CD 6 (-A), um CD 39, um IVS 1-5, um -87 todas de origem mediterrânea, e um IVS II-654 de origem asiática. As mutações CD 6 (-A), -87 e IVS II-654 foram descritas pela primeira vez na população brasileira.

Chlorination treatment of aqueous samples reduces, but does not eliminate, the mutagenic effect of the azo dyes Disperse Red 1, Disperse Red 13 and Disperse Orange 1

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Secondary Metabolites of Miconia rubiginosa

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mutagenic compounds generated from the chlorination of disperse azo-dyes and their presence in drinking water

The water produced by the Cristais River Drinking Water Treatment Plant (CR-DWTP) repeatedly produced mutagenic responses that could not be explained by the presence of disinfection byproducts (DBPs) generated by the reaction of humic acids and chlorine. In order to determine the possible role of chlorinated dye products in this mutagenic activity, solutions of a black dye commercial product (BDCP) composed of C. I. Disperse Blue 373, C. I. Disperse Orange 37, C. I. Disperse Violet 93, and chemically reduced BDCP (R-BDCP) were chlorinated in a manner similar to that used by the CR-DWTP. The resulting solutions were extracted with XAD-4 along with one drinking water sample collected from the CR-DWTP. All extracts showed mutagenic activity in the Salmonella/microsome assay. Dye components of the BDCP as well as its reduced chlorinated (Cl-R-BDCP) derivative were detected in the drinking water sample by analysis with a high performance liquid chromatography/diode array detector (HPLC/DAD). The mutagenicity results of these products suggest that they are, at least in part, accounting for the mutagenic activity detected in the drinking water samples from the Cristais River. The data obtained in this study have environmental and health implications because the chlorination of the BDCP and the R-BDCP leads to the formation of mutagenic compounds (Cl-BDCP and Cl-R-BDCP), which are potentially important disinfection byproducts that can contaminate the drinking water as well as the environment.

Incidence of patulin in fruits and fruit juices marketed in Campinas, Brazil

One hundred and eleven samples of processed fruit juices (apple, grape, pineapple, papaya, guava, banana and mango) and 38 samples of sound fruits (apple, papaya, mango, pear and peach) produced and marketed in Brazil, were analysed for patulin by HPLC. Only one out of 30 samples of apple juice was found positive at 17 μg/l. Patulin was not detected in the other foodstuffs. It was found in 14 samples of spoiled fruit samples of apple (150-267 μg/kg), pear (134-245 μg/kg) and peach (92-174 μg/kg). Confirmation of the identity of patulin was based on the UV spectrum obtained by the HPLC diode array detector, compared with that of standard patulin, TLC developed by several solvent systems and sprayed with 3-methyl-2-benzothiazolinone hydrazone, and by acetylation with acetic anhydride.

HPLC-EICD: An Useful Tool for the Pursuit of Novel Analytical Strategies for the Detection of Antioxidant Secondary Metabolites

Living cells are continuously exposed to a variety of challenges that exert oxidative stress and are directly related with senescence and the onset of various pathological conditions such as coronary heart disease, rheumatoid arthritis and cancer. Nevertheless, living organisms have developed a complex antioxidant network to counteract reactive species that are detrimental to life. With the aim of bio-prospecting plant species from the Brazilian Cerrado and Atlantic Forest, we have established a methodology to detect secondary antioxidant metabolites in crude extracts and fractions obtained from plant species. Combining HPLC with an electrochemical detector allowed us to detect micromolecules that showed antioxidant activities in Chimarrhis turbinata (DC) leaf extracts. Comparison with purified flavonoid standards led us to identify the compounds in their natural matrices giving valuable information on their antioxidant capacity.

A comparison of mycolic acid analysis for nontuberculous mycobacteria identification by thin-layer chromatography and molecular methods

The development of fast, inexpensive, and reliable tests to identify nontuberculous mycobacteria (NTM) is needed. Studies have indicated that the conventional identification procedures, including biochemical assays, are imprecise. This study evaluated a proposed alternative identification method in which 83 NTM isolates, previously identified by conventional biochemical testing and in-house M. avium IS1245-PCR amplification, were submitted to the following tests: thin-layer chromatography (TLC) of mycolic acids and PCR-restriction enzyme analysis of hsp65 (PRA). High-performance liquid chromatography (HPLC) analysis of mycolic acids and Southern blot analysis for M. avium IS1245 were performed on the strains that evidenced discrepancies on either of the above tests. Sixty-eight out of 83 (82%) isolates were concordantly identified by the presence of IS1245 and PRA and by TLC mycolic acid analysis. Discrepant results were found between the phenotypic and molecular tests in 12/83 (14.4%) isolates. Most of these strains were isolated from non-sterile body sites and were most probably colonizing in the host tissue. While TLC patterns suggested the presence of polymycobacterial infection in 3/83 (3.6%) cultures, this was the case in only one HPLC-tested culture and in none of those tested by PRA. The results of this study indicated that, as a phenotypic identification procedure, TLC mycolic acid determination could be considered a relatively simple and cost-effective method for routine screening of NTM isolates in mycobacteriology laboratory practice with a potential for use in developing countries. Further positive evidence was that this method demonstrated general agreement on MAC and M. simiae identification, including in the mixed cultures that predominated in the isolates of the disseminated infections in the AIDS patients under study. In view of the fact that the same treatment regimen is recommended for infections caused by these two species, TLC mycolic acid analysis may be a useful identification tool wherever molecular methods are unaffordable.

Extraction and quantification of phenolic acids and flavonols from Eugenia pyriformis using different solvents

The recovery of phenolic compounds of Eugenia pyriformis using different solvents was investigated in this study. The compounds were identified and quantified by reverse-phase high-performance liquid chromatography coupled with ultraviolet-visible diode-array detector (RP-HPLC-DAD/UV-vis). Absolute methanol was the most effective extraction agent of phenolic acids and flavonols (588.31 mg/Kg) from Eugenia pyriformis, although similar results (p ≤ 0.05) were observed using methanol/water (1:1 ratio). Our results clearly showed that higher contents of phenolic compounds were not obtained either with the most or the least polar solvents used. Several phenolic compounds were identified in the samples whereas gallic acid and quercetin were the major compounds recovered. © 2012 Association of Food Scientists & Technologists (India).

DEVELOPMENT OF A BIOMIMETIC SENSOR MODIFIED WITH HEMIN AND GRAPHENE OXIDE FOR MONITORING OF CARBOFURAN IN FOOD

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)