Determinação de isotiocianato de benzila em Carica papaya utilizando cromatografia gasosa com detectores seletivos

In the present work, a method was developed and validated for the quantification of benzyl isothiocyanate (BITC) in the fruits of Carica papaya. The quantification of this compound was carried out by gas chromatography (GC) with selective detectors - nitrogen phosphorus detector (NPD) and flame photometric detector (FPD). The performance of these detectors showed a higher sensitivity of the NPD with a broader linear range of detection. The LOD/LOQ were 0.038/0.100 µg/mL for NPD and 5.78/19.29 µg/mL for FPD. The recovery of the method for BITC was 90,64%. An average value of BITC concentration in all the analyzed samples was 16,23 µg BITC/g.

Uptake and translocation of nitrogen, phosphorus and calcium in soybean infected with Meloidogyne incognita and M. javanica

Two soybean (Glycine max) cultivars were used in this study, Ocepar 4, rated as moderately resistant to Meloidogyne incognita race 3 but susceptible to M. javanica, and 'BR 16', susceptible to both nematodes. The effect of nematodes infection on the uptake and transport of N, P and Ca to the shoot was studied in plants growing in a split root system. The upper half was inoculated with 0, 3,000, 9,000 or 27,000 eggs/plant while the lower half received 15N, 32P or 45Ca. Infected plants showed an increase of root but a decrease of shoot mass with increasing inoculum levels. In general, total endogenous nutrients increased in the roots and tended to decrease in the shoots with increasing inoculum levels. When concentrations were calculated, there was an increase in the three nutrients in the roots, and an increase of Ca but no significant variation of N and P was observed in the shoots. The total amount of 15N in the roots increased at the highest inoculum levels but 32P and 45Ca decreased. In the shoots there was a reduction of 32P and 45Ca. The specific concentrations of the labelled nutrients (abundance or radioactivity/tissue mass) also showed a decrease of 32P and 45Ca in the shoots and roots of infected plants and an increase of 15N in the shoots. Considering that overall nutrient concentrations reflect cumulative nutrient uptake and the data from labelled elements gave information at a specific moment of the infection, thus nematodes do interfere with nutrient uptake and translocation.

Investigations into the extraction and the determination of polycyclic aromatic hydrocarbons (PAHs) from water by solid-phase extraction and capillary gas chromatography/ mass spectrometry

Factors affecting the detennination of PAHs by capillary GC/MS were studied. The effect of the initial column temperature and the injection solvent on the peak areas and heights of sixteen PAHs, considered as priority pollutants, USillg crosslinked methyl silicone (DB!) and 5% diphenyl, 94% dimethyl, 1% vinyl polysiloxane (DBS) columns was examined. The possibility of using high boiling point alcohols especially butanol, pentanol, cyclopentanol, and hexanol as injection solvents was investigated. Studies were carried out to optimize the initial column temperature for each of the alcohols. It was found that the optimum initial column temperature is dependent on the solvent employed. The peak areas and heights of the PAHs are enhanced when the initial column temperature is 10-20 c above the boiling point of the solvent using DB5 column, and the same or 10 C above the boiling point of the solvent using DB1 column. Comparing the peak signals of the PAHs using the alcohols, p-xylene, n-octane, and nonane as injection solvents, hexanol gave the greatest peak areas and heights of the PAHs particularly the late-eluted peaks. The detection limits were at low pg levels, ranging from 6.0 pg for fluorene t9 83.6 pg for benzo(a)pyrene. The effect of the initial column temperature on the peak shape and the separation efficiency of the PARs was also studied using DB1 and DB5 columns. Fronting or splitting of the peaks was obseIVed at very low initial column temperature. When high initial column temperature was used, tailing of the peaks appeared. Great difference between DB! and.DB5 columns in the range of the initial column temperature in which symmetrical.peaks of PAHs can be obtained is observed. Wider ranges were shown using DB5 column. Resolution of the closely-eluted PAHs was also affected by the initial column temperature depending on the stationary phase employed. In the case of DB5, only the earlyeluted PAHs were affected; whereas, with DB1, all PAHs were affected. An analytical procedure utilizing solid phase extraction with bonded phase silica (C8) cartridges combined with GC/MS was developed to analyze PAHs in water as an alternative method to those based on the extraction with organic solvent. This simple procedure involved passing a 50 ml of spiked water sample through C8 bonded phase silica cartridges at 10 ml/min, dried by passing a gentle flow of nitrogen at 20 ml/min for 30 sec, and eluting the trapped PAHs with 500 Jll of p-xylene at 0.3 ml/min. The recoveries of PAHs were greater than 80%, with less than 10% relative standard deviations of nine determinations. No major contaminants were present that could interfere with the recognition of PAHs. It was also found that these bonded phase silica cartridges can be re-used for the extraction of PAHs from water.

Methoxypyrazine removal in grape juice: Development of a removal system using odorant and pheromone binding proteins coupled with bentonite fining

Multicoloured Asian Lady Beetles (MALB) and 7-spot Lady Beetles that infect vineyards can secrete alkyl-methoxypyrazines when they are processed with the grapes, resulting in wines containing a taint. The main methoxypyrazine associated with this taint is 3-isopropyl-2-methoxypyrazine (IPMP). The wines are described as having aroma and flavours of peanut butter, peanut shells, asparagus and earthy which collectively, have become known as “ladybug taint”. To date, there are no known fining agents used commercially added to juice or wine that are effective in removing this taint. The goal of this project was to use previously identified proteins with an ability to bind to methoxypyrazines at low pH, and subsequently develop a binding assay to test the ability of these proteins to bind to and remove methoxypyrazines from grape juice. The piglet odorant binding protein (plOBP) and mouse major urinary protein (mMUP) were identified, cloned and expressed in the Pichia pastoris expression system. Protein expression was induced using methanol and the proteins were subsequently purified from the induction media using anion exchange chromatography. The purified proteins were freeze-dried and rehydrated prior to use in the methoxypyrazine removal assay. The expression and purification system resulted in yields of approximately 78% of purified plOBP and 62% of purified mMUP from expression to rehydration. Purified protein values were 87 mg of purified plOPB per litre of induction media and 19 mg of purified mMUP per litre of induction medium. In order to test the ability of the protein to bind to the MPs, an MP removal assay was developed. In the assay, the purified protein is incubated with either IPMP or 3-isobutyl-2-methoxypyrazine (IBMP) for two hours in either buffer or grape juice. Bentonite is then used to capture the protein-MP complex and the bentonite-protein-MP complex is then removed from solution by filtration. Residual MP is measured in solution following the MP removal assay and compared to that in the starting solution by Gas Chromatography Mass Spectrometry (GC/MS). GC/MS results indicated that the mMUP was capable of removing IBMP and IPMP from 300 ng/L in buffer pH 4.0, buffer pH 3.5 and Riesling Juice pH 3.5 down to the limit of quantification of the instrument, which is 6ng/L and 2ng/L for IBMP and IPMP, respectively. The results for the plOBP showed that although it could remove some IBMP, it was only approximately 50-70 ng/L more than bentonite treatment followed by filtration, resulting in approximately 100 ng/L of the MPs being left in solution. pIOBP was not able to remove IPMP in buffer pH 3.5 using this system above that removed by bentonite alone. As well, the pIOBP was not able to remove any additional MPs from Chardonnay juice pH 3.5 above that already removed by the bentonite and filtration alone. The mouse MUP was shown to be a better candidate protein for removal of MPs from juice using this system.

Determination of fluoxetine in plasma by gas chromatography-mass spectrometry using stir bar sorptive extraction

This article presents a method employing stir bar sorptive extraction (SBSE) with in situ derivatization, in combination with either thermal or liquid desorption on-line coupled to gas chromatography-mass spectrometry for the analysis of fluoxetine in plasma samples. Ethyl chloroformate was employed as derivatizing agent producing symmetrical peaks. Parameters such as solvent polarity, time for analyte desorption, and extraction time, were evaluated. During the validation process, the developed method presented specificity, linearity (R-2 > 0.99), precision (R.S.D. < 15%), and limits of quantification (LOQ) of 30 and 1.37 pg mL(-1), when liquid and thermal desorption were employed, respectively. This simple and highly sensitive method showed to be adequate for the measurement-of fluoxetine in typical and trace concentration levels. (c) 2008 Elsevier B.V. All rights reserved.

Biocompatible in-tube solid phase microextraction coupled with liquid chromatography-fluorescence detection for determination of interferon alpha in plasma samples

The present work demonstrates the successful application of automated biocompatible in-tube solid-phase microextraction coupled with liquid chromatography (in-tube SPME/LC) for determination of interferon alpha(2a) (IFN alpha(2a)) in plasma samples for therapeutic drug monitoring. A restricted access material (RAM, protein-coated silica) was employed for preparation of a lab-made biocompatible in-tube SPME capillary that enables the direct injection of biological fluids as well as the simultaneous exclusion of macromolecules by chemical diffusion barrier and drug pre-concentration. The in-tube SPME variables, such as sample volume, draw/eject volume, number of draw-eject cycles, and desorption mode were optimized, to improve the sensitivity of the proposed method. The IFN alpha(2a) analyses in plasma sample were carried out within 25 min (sample preparation and LC analyses). The response of the proposed method was linear over a dynamic range, from 0.06 to 3.0 MIU mL(-1), with correlation coefficient equal to 0.998. The interday precision of the method presented coefficient of variation lower than 8%. The proposed automated method has adequate analytical sensitivity and selectivity for determination of IFN alpha(2a) in plasma samples for therapeutic drug monitoring. (C) 2010 Elsevier B.V. All rights reserved.

Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry combined with solid phase microextraction as a powerful tool for quantification of ethyl carbamate in fortified wines. The case study of Madeira wine

An analytical methodology based on headspace solid phase microextraction (HS-SPME) combined with comprehensive two-dimensional gas chromatography—time-of-flight mass spectrometry (GC × GC–ToFMS) was developed for the identification and quantification of the toxic contaminant ethyl carbamate (EC) directly in fortified wines. The method performance was assessed for dry/medium dry and sweet/medium sweet model wines, and for quantification purposes, calibration plots were performed for both matrices using the ion extraction chromatography (IEC) mode (m/z 62). Good linearity was obtained with a regression coefficient (r2) higher than 0.981. A good precision was attained (R.S.D. <20%) and low detection limits (LOD) were achieved for dry (4.31 μg/L) and sweet (2.75 μg/L) model wines. The quantification limits (LOQ) and recovery for dry wines were 14.38 μg/L and 88.6%, whereas for sweet wines were 9.16 μg/L and 99.4%, respectively. The higher performance was attainted with sweet model wine, as increasing of glucose content improves the volatile compound in headspace, and a better linearity, recovery and precision were achieved. The analytical methodology was applied to analyse 20 fortified Madeira wines including different types of wine (dry, medium dry, sweet, and medium sweet) obtained from several harvests in Madeira Island (Portugal). The EC levels ranged from 54.1 μg/L (medium dry) to 162.5 μg/L (medium sweet).

Investigation of urinary volatile organic metabolites as potential cancer biomarkers by solid-phase microextraction in combination with gas chromatography-mass spectrometry

BACKGROUND: Non-invasive diagnostic strategies aimed at identifying biomarkers of cancer are of great interest for early cancer detection. Urine is potentially a rich source of volatile organic metabolites (VOMs) that can be used as potential cancer biomarkers. Our aim was to develop a generally reliable, rapid, sensitive, and robust analytical method for screening large numbers of urine samples, resulting in a broad spectrum of native VOMs, as a tool to evaluate the potential of these metabolites in the early diagnosis of cancer. METHODS: To investigate urinary volatile metabolites as potential cancer biomarkers, urine samples from 33 cancer patients (oncological group: 14 leukaemia, 12 colorectal and 7 lymphoma) and 21 healthy (control group, cancer-free) individuals were qualitatively and quantitatively analysed. Dynamic solid-phase microextraction in headspace mode (dHS-SPME) using a carboxenpolydimethylsiloxane (CAR/PDMS) sorbent in combination with GC-qMS-based metabolomics was applied to isolate and identify the volatile metabolites. This method provides a potential non-invasive method for early cancer diagnosis as a first approach. To fulfil this objective, three important dHS-SPME experimental parameters that influence extraction efficiency (fibre coating, extraction time and temperature of sampling) were optimised using a univariate optimisation design. The highest extraction efficiency was obtained when sampling was performed at 501C for 60min using samples with high ionic strengths (17% sodium chloride, wv 1) and under agitation. RESULTS: A total of 82 volatile metabolites belonging to distinct chemical classes were identified in the control and oncological groups. Benzene derivatives, terpenoids and phenols were the most common classes for the oncological group, whereas ketones and sulphur compounds were the main classes that were isolated from the urine headspace of healthy subjects. The results demonstrate that compound concentrations were dramatically different between cancer patients and healthy volunteers. The positive rates of 16 patients among the 82 identified were found to be statistically different (Po0.05). A significant increase in the peak area of 2-methyl3-phenyl-2-propenal, p-cymene, anisole, 4-methyl-phenol and 1,2-dihydro-1,1,6-trimethyl-naphthalene in cancer patients was observed. On average, statistically significant lower abundances of dimethyl disulphide were found in cancer patients. CONCLUSIONS: Gas chromatographic peak areas were submitted to multivariate analysis (principal component analysis and supervised linear discriminant analysis) to visualise clusters within cases and to detect the volatile metabolites that are able to differentiate cancer patients from healthy individuals. Very good discrimination within cancer groups and between cancer and control groups was achieved.

In-depth search focused on furans, lactones, volatile phenols, and acetals as potential age markers of Madeira wines by comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry combined with solid phase microextraction

The establishment of potential age markers of Madeira wine is of paramount significance as it may contribute to detect frauds and to ensure the authenticity of wine. Considering the chemical groups of furans, lactones, volatile phenols, and acetals, 103 volatile compounds were tentatively identified; among these, 71 have been reported for the first time in Madeira wines. The chemical groups that could be used as potential age markers were predominantly acetals, namely, diethoxymethane, 1,1-diethoxyethane, 1,1-diethoxy-2-methyl-propane, 1-(1-ethoxyethoxy)-pentane, trans-dioxane and 2-propyl-1,3-dioxolane, and from the other chemical groups, 5-methylfurfural and cis-oak-lactone, independently of the variety and the type of wine. GC × GC-ToFMS system offers a more useful approach to identify these compounds compared to previous studies using GC−qMS, due to the orthogonal systems, that reduce coelution, increase peak capacity and mass selectivity, contributing to the establishment of new potential Madeira wine age markers. Remarkable results were also obtained in terms of compound identification based on the organized structure of the peaks of structurally related compounds in the GC × GC peak apex plots. This information represents a valuable approach for future studies, as the ordered-structure principle can considerably help the establishment of the composition of samples. This new approach provides data that can be extended to determine age markers of other types of wines.

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.

Effect of Ionic Liquid on the Determination of Aromatic Amines as Contaminants in Hair Dyes by Liquid Chromatography Coupled to Electrochemical Detection

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

Determination of pesticide residues in coconut water by liquid-liquid extraction and gas chromatography with electron-capture plus thermionic specific detection and solid-phase extraction and high-performance liquid chromatography with ultraviolet detection

Two simple methods were developed to determine, 11 pesticides in coconut water, a natural isotonic drink rich in salts, sugars and vitamins consumed by the people and athletes. The first procedure involves solid-phase extraction using Sep-Pak Vac C-18 disposable cartridges with methanol for elution. Isocratic analysis was carried out by means of high-performance liquid chromatography with ultraviolet detection at 254 nm to analyse captan, chlorothalonil, carbendazim, lufenuron and diafenthiuron. The other procedure is based on liquid-liquid extraction with hexane-dichloromethane (1:1, v/v), followed by gas chromatographic analysis with effluent splitting to electron-capture detection for determination of endosulfan, captan, tetradifon and trichlorfon and thermionic specific detection for determination of malathion, parathion-methyl and monocrotophos. The methods were validated with fortified samples at different concentration levels (0.01-12.0 mg/kg). Average recoveries ranged from 75 to 104% with relative standard deviations between 1.4 and 11.5%. Each recovery analysis was repeated at least five times. Limits of detection ranged from 0.002 to 2.0 mg/kg. The analytical procedures were applied to 15 samples and no detectable amounts of the pesticides were found in any samples under the conditions described. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Solid-phase extraction of sugar cane soot extract for analysis by gas chromatography with flame ionisation and mass spectrometric detection

The incomplete combustion of biomass is one of the most important sources of emissions of organic compounds into the atmosphere, like polycyclic aromatic hydrocarbons (PAHs) which show genotoxic activity. Since environmental samples generally contain interferents and trace amounts of PAHs of interest, concentration and clean-up procedures are usually required prior to the final chromatographic analysis. This paper discusses the performance of Sep-Pak cartridges (silica gel and RP18) on clean-up of sugar cane soot extract. The best results were obtained with a silica Sep-Pak cartridge. The recoveries ranged from 79% (benzo[b]fluoranthene) to 113% (benzo[e]pyrene). (C) 2000 Elsevier B.V. B.V. All rights reserved.

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.