Desenvolvimento de método para determinação de espécies de mercúrio empregando MSPD e GC-MS

Neste trabalho é proposto pela primeira vez, o desenvolvimento e validação de um método analítico baseado no emprego da dispersão da matriz em fase sólida (MSPD) modificada, para extração das espécies CH3Hg+ e Hg2+ em amostras de peixe e determinação por cromatografia em fase gasosa acoplada à espectrometria de massas (GC-MS). O método de extração utilizando a MSPD combina o rompimento da estrutura física da amostra, através da maceração e do uso de SiO2 como suporte sólido, com o método da extração ácida, utilizando uma solução de HCl 4,2 mol L-1 e NaCl 0,5 mol L-1. Para otimização da MSPD, foram avaliados parâmetros como massa de amostra, massa de suporte sólido, concentração de HCl, concentração de NaCl, tipo de suporte sólido e o tempo de agitação, com auxílio da metodologia de superfície de resposta. Além disso, a etapa de derivatização e a separação cromatográfica também foram otimizadas na determinação de CH3Hg+ e Hg2+ por GC-MS. O método mostrouse adequado para extração e determinação de espécies de mercúrio através da aplicação em materiais de referência certificados de fígado de peixe (DOLT-3) e músculo de peixe (DORM-2), apresentando boas concordâncias com os valores certificados e desvio padrão relativo inferior a 9,5%. Os limites de detecção foram de 0,06 e 0,12 µg g-1 para CH3Hg+ e Hg2+, respectivamente. Além disso, foi observado um significativo efeito de matriz e, por isso, a calibração foi feita com curvas preparadas com o extrato da MSPD. O método mostrou boa concordância na comparação entre a soma da concentração das espécies e a concentração de mercúrio total determinada por espectrometria de massas com plasma indutivamente acoplado com geração de vapor frio (CVG-ICP-MS), após digestão assistida por micro-ondas (MAD) em peixes do tipo atum (Thunnus thynnus), cação anjo (Squatina squatina) e cação viola (Rhinobatos blochii.).

Study on the inhibitory effects of native medicinal plants on growth and aflatoxin production by toxigenic Aspergilli isolated from rainbow trout feed

In the present study, natural occurrence of fungi and aflatoxin B1 (AFB1) in pellet feed and feed ingredients used for rainbow trout was investigated with emphasis to Aspergillus section Flavi members and medicinal plants inhibitory to Aspergillus growth and/or AF production. The feed samples were cultured on the standard isolation media including dichloran rosebengal chloramphenicol agar (DRCA) and Aspergillus flavus/parasiticus agar (AFPA) for 2 weeks at 28 °C. Identification of fungal isolates was implemented based on the macro- and microscopic morphological criteria. AFs were detected using high performance liquid chromatography (HPLC). Based on the results obtained, a total of 109 fungal isolates were identified of which Aspergillus was the prominent genus (57.0%), followed by Penicillium (12.84%), Absidia (11.01%) and Pseudallscheria (10.10%). The most frequent Aspergillus species was A. flavus (60.66%) isolated from all the feed ingredients as well as pellet feed. Among 37 A. flavus isolates, 19 (51.35%) were able to produce AFB1 on yeast extract-sucrose (YES) broth in the range of 10.2 to 612.8 [tg/g fungal dry weight. HPLC analyses of trout feed showed that pellet feed and all feed ingredients tested except gluten were contaminated with different levels of AFB1 in the range of 1.83 to 67.35 lig/kg. In order to finding natural inhibitors of fungal growth and/or AF production, essential oils (EOs) and extracts of 49 medicinal plants were studied against an aflatoxin-producing A. parasiticus using a microbioassay technique. The EOs was analyzed by gas chromatography/mass spectrometry (GC/MS). Based on the results obtained, Achillea millefolium sub sp. elborsensis, Ferula gummosa, Mentha spicata, Azadirachta indica, Conium maculatum and Artemisia dracunculus remarkably inhibited A. parasiticus growth without affecting AF production by the fungus. Besides of Thymus vulgaris and Citrus aurantifolia, the EO of Foeniculum vulgare significantly inhibited both fungal growth (-70.0%) and AFs B1 and G1 (-99.0%) production. The EO of Carum carvi and ethyl acetate extract of Platycladus orientalis suppressed AFs B1 and G1 by more than 90.0%, without any obvious effect on fungal growth. The IC50 values of bioactive plants for AFs B1 and G1 were determined in the ranges of 90.6 to 576.2 and 2.8 to 61.9 µg/ml, respectively. Overall, results of the present study indicate the importance of AF contamination of trout feed as a risk factor for fish farming and thus, an urgent necessity for constant monitoring of trout feed for any unacceptable levels of AF contamination. Likewise, antifungal activities of bioactive plants introduced here would be an important contribution to explain the use of these plants as effective antimicrobial candidates to protect feeds from toxigenic fungus growth and subsequent AF contamination.

Effets préventifs et thérapeutiques des polyphénols dans un modèle in vitro et in vivo de maladie inflammatoire de l’intestin : caractérisation des polyphénols de la pelure de pomme et de la canneberge par spectrométrie de masse

La muqueuse intestinale est exposée à des agents oxydants provenant de l’ingestion d’aliments modifiés, de cellules immuno-inflammatoires et de la flore intestinale. Une diète élevée en fruits et légumes peut diminuer le stress oxydant (SOx) ainsi que l’inflammation via plusieurs mécanismes. Ces effets bénéfiques peuvent être attribuables à leur contenu élevé en polyphénols. La première étude de mon doctorat consistait à tester l’hypothèse que les polyphénols extraits de pelures de pomme (DAPP) pouvaient diminuer le stress oxydant et l'inflammation impliqués dans les maladies inflammatoires de l'intestin (MII). Nous avons caractérisé les polyphénols des DAPP par spectrométrie de masse (LC-MS) et examiné leur potentiel antioxydant et anti-inflammatoire au niveau des cellules intestinales. L’identification des structures chimiques des polyphénols a été effectuée par LC-MS. Le SOx a été induit par l’ajout du complexe fer/ascorbate (Fe/Asc, 200 µM/2 mM) et l’inflammation par la lipopolysaccharide (LPS, 200µg/mL) à des cellules intestinales Caco-2/15 pré-incubées avec les DAPP (250 µg/mL). L’effet du SOx est déterminé par le dosage du malondialdéhyde (MDA), de la composition des acides gras polyinsaturés et de l’activité des enzymes antioxydantes endogènes (SOD et GPx). L’impact des DAPP sur l’inflammation a été testé par l’analyse de l’expression des marqueurs inflammatoires: cyclooxygénase-2 (COX-2), le facteur de nécrose tumorale alpha (TNF-a et l’interleukine-6 (IL-6) et les facteurs de transcription NF-KB, Nrf-2 et PGC1α par immunobuvardage. Nos données ont montré que les flavonols et les flavan-3-ols constituent les composés polyphénoliques majoritaires des DAPP. L’ajout de Fer2+/Asc a provoqué une augmentation de la peroxidation lipidique comparativement aux cellules contrôles, un appauvrissement des acides gras polyinsaturés n-3 et n-6, et une modulation des enzymes antioxydantes, se traduisant par une augmentation de l’activité de la SOD et une diminution de la GPx. En contrepartie, les DAPP ont exhibé leur potentiel à corriger la plupart des perturbations, y compris l’expression protéique anormalement élevée du COX-2 et la production de la prostaglandine E2 (PGE2), ainsi que l’inflammation telle que réflétée par les facteurs NF-κB, TNF-α et IL-6. Par ailleurs, les mécanismes sous-jacents à ces changements bénéfiques des DAPP ont fait intervenir les facteurs de transcription antioxydants (Nrf-2, PGC1α). Vraisemblablement, cette première étude a permis de démontrer la capacité des DAPP à amoindrir le SOx et à réduire l’inflammation, deux processus étroitement impliqués dans les MII. Dans la deuxième étape de mon doctorat, nous avons voulu comparer les résultats de DAPP à ceux des polyphénols dérivant de la canneberge qui est considérée par la communauté scientifique comme le fruit ayant le plus fort potentiel antioxydant. À cette fin, nous avons caractérisé l’effet des composés polyphénoliques de la canneberge (CPC) sur le SOx, la défense antioxydante et l’inflammation au niveau intestinal tout en définissant leur métabolisme intraluminal. Les différents CPC ont été séparés selon leur poids moléculaire par chromatographie et leurs structures chimiques ont été identifiées par LC-MS. Suite à une pré-incubation des cellules Caco-2/15 avec les extraits CPC (250 µg/mL), le Fe/Asc et la LPS ont été administrés comme inducteurs du SOx et de l’inflammation, respectivement. La caractérisation globale des CPC a révélé que les acides phénoliques composaient majoritairement l’extrait de canneberge de petit poids moléculaire (LC) alors que les flavonoïdes et les procyanidines dimériques/trimériques représentaient l’extrait de poids moléculaire moyen (MC) tout en laissant les procyanidines oligo et polymériques à l’extrait de haut poids moléculaire (HC). Les CPC ont permis de restaurer la plupart des perturbations engendrées dans les Caco-2/15 par le Fe/Asc et le LPS. Les CPC exhibaient le potentiel d’abaisser les niveaux de MDA, de corriger la composition des acides gras polyinsaturés n-3 et n-6, d’augmenter l’activité des enzymes antioxydantes (SOD, GPx et CAT) et d’élever l’expression de Nrf2 et PGC1α. En outre, les CPC pouvaient aussi réduire les niveaux élevés des protéines inflammatoires COX-2, TNF-α et IL-6 ainsi que la production des PGE2 par un mécanisme impliquant le NF-κB. Au niveau mitochondrial, les procyanidines oligomériques ont réussi à corriger les dysfonctions reliées à la production d’énergie (ATP), l’apoptose (Bcl-2, Cyt C et AIF) et le statut des facteurs de transcription mitochondriaux (mtTFA, mtTFB1, mtTFB2). Dans le but de bien comprendre les mécanismes d’action des CPC, nous avons défini par LC-MS les composés polyphénoliques qui ont été transportés ou absorbés par l’entérocyte. Nos analyses soulignent le transport (i) des acides cinnamiques et benzoïques (LC); (ii) la quercétine glycosylée et conjuguée et les procyanidines dimériques de type A (MC); et (iii) l’épicatéchine et les procyanidines oligomériques (HC). Les processus de métabolisation (méthylation, glucuronidation et sulfatation) au niveau de l’entérocyte ont probablement permis le transport de ces CPC surtout sous leur forme conjuguée. Les procyanidines oligomériques ayant un degré de polymérisation supérieur à 2 (HC) ont semblé adhérer aux cellules Caco-2/15. L’épicatéchine suivi par les procyanidines dimériques de type A ont été trouvés majoritaires au niveau des mitochondries. Même si nous ignorons encore l’action biologique de chaque composé polyphénolique, nous pouvons suggérer que leurs effets combinatoires exercent des fonctions antioxydantes, anti-inflammatoires et mitochondriales dans le modèle intestinal Caco-2/15. Dans une troisième étape, nous avons procédé à l’évaluation des aspects préventifs et thérapeutique des DAPP tout en sondant les mécanismes sous-jacents dans une étude préclinique. À cette fin, nous avons exploité le modèle de souris avec colite expérimentale provoquée par le Dextran Sulfate de Sodium (DSS). L’induction de l’inflammation intestinale chez la souris C57BL6 a été effectuée par l’administration orale de DSS à 2.5% pendant 10 jours. Des doses physiologiques et supra-physiologiques de DAPP (200 et 400 mg/kg/j, respectivement) ont été administrées par gavage pendant 10 jours pré- et post-DSS. L’inflammation par le DSS a provoqué une perte de poids, un raccourcissement du côlon, le décollement dystrophique de l’épithélium, l’exulcération et les infiltrations de cellules mono et polynucléaires au niveau du côlon. De plus, le DSS a induit une augmentation de la peroxidation lipidique, une régulation à la baisse des enzymes antioxydantes, une expression protéique à la hausse de la myéloperoxidase (MPO), du COX-2 et de la production des PGE2. Par ailleurs, les DAPP ont permis de corriger ou du moins d’alléger la plupart de ces anomalies en situation préventive ou thérapeutique, en plus d’abaisser l’expression protéique de NF-κB et des cytokines inflammatoires (TNF-a et l’IL-6) tout en stimulant les facteurs de transcription antioxydants (Nrf-2, PGC1α). Conséquemment, les polyphénols des DAPP ont exhibé leur puissant pouvoir antioxydant et anti-inflammatoire au niveau intestinal dans un modèle in vivo. Leurs actions sont associées à la régulation des voies de signalisation cellulaire et des changements dans la composition du microbiote. Ces trois projets de recherche permettent d’envisager l’évaluation des effets préventifs et thérapeutiques des DAPP cliniquement chez les patients avec des désordres inflammatoires de l’intestin.

First metabolomic approach of the epiphytic bacteria-marine diatom Haslea ostrearia relationships

The marine diatom Haslea ostrearia [1] produces a water-soluble blue-pigment named marennine [2] of economic interest. But the lack of knowledge of the ecological conditions, under which this microalga develops in its natural ecosystem, more especially bacteria H. ostrearia interactions, prevents any optimization of its culture in well-controlled conditions. The structure of the bacterial community was analyzed by PCR-TTGE before and after the isolation of H. ostrearia cells recovered from 4 localities, to distinguish the relative part of the biotope and the biocenose and eventually to describe the temporal dynamic of the structure of the bacterial community at two time-scales. The differences in genetic fingerprints, more especially high between two H. ostrearia isolates (HO-R and HO-BM) showed also the highest differences in the bacterial structure [3] as the result of specific metabolomics profiles. The non-targeted metabolomic investigation showed that these profiles were more distinct in case of bacteria-alga associations than for the H. ostrearia monoculture Here we present a Q-TOF LC/MS metabolomic fingerprinting approach [3]: - to investigate differential metabolites of axenic versus non axenic H. ostrearia cultures. - to focus on the specific metabolites of a bacterial surrounding associated with the activation or inhibition of the microalga growing. The Agilent suite of data processing software makes feature finding, statistical analysis, and identification easier. This enables rapid transformation of complex raw data into biologically relevant metabolite information.

Preliminary metabolomic approach on the bacterial structure community of Haslea ostrearia

The marine diatom Haslea ostrearia [1] produces a water-soluble blue-pigment named marennine [2] of economic interest. But the lack of knowledge of the ecological conditions, under which this microalga develops in its natural ecosystem, more especially bacteria H. ostrearia interactions, prevents any optimization of its culture in well-controlled conditions. The structure of the bacterial community was analyzed by PCR-TTGE before and after the isolation of H. ostrearia cells recovered from 4 localities, to distinguish the relative part of the biotope and the biocenose and eventually to describe the temporal dynamic of the structure of the bacterial community at two time-scales. The differences in genetic fingerprints, more especially high between two H. ostrearia isolates (HO-R and HO-BM) showed also the highest differences in the bacterial structure [3] as the result of specific metabolomics profiles. The non-targeted metabolomic investigation showed that these profiles were more distinct in case of bacteria-alga associations than for the H. ostrearia monoculture Here we present a Q-TOF LC/MS metabolomic fingerprinting approach [3]: - to investigate differential metabolites of axenic versus non axenic H. ostrearia cultures. - to focus on the specific metabolites of a bacterial surrounding associated with the activation or inhibition of the microalga growing. The Agilent suite of data processing software makes feature finding, statistical analysis, and identification easier. This enables rapid transformation of complex raw data into biologically relevant metabolite information.

Development of procedures for the triazole fungicides determination in fruits and liquid samples using microextraction techniques and chromatographic separation

Agricultural crops can be damaged by funguses, insects, worms and other organisms that cause diseases and decrease the yield of production. The effect of these damaging agents can be reduced using pesticides. Among them, triazole compounds are effective substances against fungus; for example, Oidium. Nevertheless, it has been detected that the residues of these fungicides in foods as well as in derivate products can affect the health of the consumers. Therefore, the European Union has established several regulations fixing the maximum residue of pesticide levels in a wide range of foods trying to assure the consumer safety. Hence, it is very important to develop adequate methods to determine these pesticide compounds. In most cases, gas or liquid chromatographic (GC, LC) separations are used in the analysis of the samples. But firstly, it is necessary to use proper sample treatments in order to preconcentrate and isolate the target analytes. To reach this aim, microextraction techniques are very effective tools; because allow to do both preconcentration and extraction of the analytes in one simple step that considerably reduces the source of errors. With these objectives, two remarkable techniques have been widely used during the last years: solid phase microextraction (SPME) and liquid phase microextraction (LPME) with its different options. Both techniques that avoid the use or reduce the amount of toxic solvents are convenient coupled to chromatographic equipments providing good quantitative results in a wide number of matrices and compounds. In this work simple and reliable methods have been developed using SPME and ultrasound assisted emulsification microextraction (USAEME) coupled to GC or LC for triazole fungicides determination. The proposed methods allow confidently determine triazole concentrations of μg L‐1 order in different fruit samples. Chemometric tools have been used to accomplish successful determinations. Firstly, in the selection and optimization of the variables involved in the microextraction processes; and secondly, to overcome the problems related to the overlapping peaks. Different fractional factorial designs have been used for the screening of the experimental variables; and central composite designs have been carried out to get the best experimental conditions. Trying to solve the overlapping peak problems multivariate calibration methods have been used. Parallel Factor Analysis 2 (PARAFAC2), Multivariate Curve Resolution (MCR) and Parallel Factor Analysis with Linear Dependencies (PARALIND) have been proposed, the adequate algorithms have been used according to data characteristics, and the results have been compared. Because its occurrence in Basque Country and its relevance in the production of cider and txakoli regional wines the grape and apple samples were selected. These crops are often treated with triazole compounds trying to solve the problems caused by the funguses. The peel and pulp from grape and apple, their juices and some commercial products such as musts, juice and cider have been analysed showing the adequacy of the developed methods for the triazole determination in this kind of fruit samples.

Fast Detection and Chemical Characterization of Gunshot Residues by CMV-GC-MS and LIBS

Gunshot residue (GSR) is the term used to describe the particles originating from different parts of the firearm and ammunition during the discharge. A fast and practical field tool to detect the presence of GSR can assist law enforcement in the accurate identification of subjects. A novel field sampling device is presented for the first time for the fast detection and quantitation of volatile organic compounds (VOCs). The capillary microextraction of volatiles (CMV) is a headspace sampling technique that provides fast results (< 2 min. sampling time) and is reported as a versatile and high-efficiency sampling tool. The CMV device can be coupled to a Gas Chromatography-Mass Spectrometry (GC-MS) instrument by installation of a thermal separation probe in the injection port of the GC. An analytical method using the CMV device was developed for the detection of 17 compounds commonly found in polluted environments. The acceptability of the CMV as a field sampling method for the detection of VOCs is demonstrated by following the criteria established by the Environmental Protection Agency (EPA) compendium method TO-17. The CMV device was used, for the first time, for the detection of VOCs on swabs from the hands of shooters, and non-shooters and spent cartridges from different types of ammunition (i.e., pistol, rifle, and shotgun). The proposed method consists in the headspace extraction of VOCs in smokeless powders present in the propellant of ammunition. The sensitivity of this method was demonstrated with method detection limits (MDLs) 4-26 ng for diphenylamine (DPA), nitroglycerine (NG), 2,4-dinitrotoluene (2,4-DNT), and ethyl centralite (EC). In addition, a fast method was developed for the detection of the inorganic components (i.e., Ba, Pb, and Sb) characteristic of GSR presence by Laser Induced Breakdown Spectroscopy (LIBS). Advantages of LIBS include fast analysis (~ 12 seconds per sample) and good sensitivity, with expected MDLs in the range of 0.1-20 ng for target elements. Statistical analysis of the results using both techniques was performed to determine any correlation between the variables analyzed. This work demonstrates that the information collected from the analysis of organic components has the potential to improve the detection of GSR.

Novel Analytical Methodologies for the Monitoring of Traditional and Non-traditional Pollutants in different Environmental Compartments of South Florida

Routine monitoring of environmental pollution demands simplicity and speed without sacrificing sensitivity or accuracy. The development and application of sensitive, fast and easy to implement analytical methodologies for detecting emerging and traditional water and airborne contaminants in South Florida is presented. A novel method was developed for quantification of the herbicide glyphosate based on lyophilization followed by derivatization and simultaneous detection by fluorescence and mass spectrometry. Samples were analyzed from water canals that will hydrate estuarine wetlands of Biscayne National Park, detecting inputs of glyphosate from both aquatic usage and agricultural runoff from farms. A second study describes a set of fast, automated LC-MS/MS protocols for the analysis of dioctyl sulfosuccinate (DOSS) and 2-butoxyethanol, two components of Corexit®. Around 1.8 million gallons of those dispersant formulations were used in the response efforts for the Gulf of Mexico oil spill in 2010. The methods presented here allow the trace-level detection of these compounds in seawater, crude oil and commercial dispersants formulations. In addition, two methodologies were developed for the analysis of well-known pollutants, namely Polycyclic Aromatic Hydrocarbons (PAHs) and airborne particulate matter (APM). PAHs are ubiquitous environmental contaminants and some are potent carcinogens. Traditional GC-MS analysis is labor-intensive and consumes large amounts of toxic solvents. My study provides an alternative automated SPE-LC-APPI-MS/MS analysis with minimal sample preparation and a lower solvent consumption. The system can inject, extract, clean, separate and detect 28 PAHs and 15 families of alkylated PAHs in 28 minutes. The methodology was tested with environmental samples from Miami. Airborne Particulate Matter is a mixture of particles of chemical and biological origin. Assessment of its elemental composition is critical for the protection of sensitive ecosystems and public health. The APM collected from Port Everglades between 2005 and 2010 was analyzed by ICP-MS after acid digestion of filters. The most abundant elements were Fe and Al, followed by Cu, V and Zn. Enrichment factors show that hazardous elements (Cd, Pb, As, Co, Ni and Cr) are introduced by anthropogenic activities. Data suggest that the major sources of APM were an electricity plant, road dust, industrial emissions and marine vessels.

First report of Ciguatoxins in two starfish species: Ophidiaster ophidianus and Marthasterias glacialis

Ciguatera fish poisoning (CFP) is a syndrome caused by the ingestion of fish contaminated with Ciguatoxins (CTXs). These phycotoxins are produced mainly by dinoflagellates that belong to the genus Gambierdiscus that are transformed in more toxic forms in predatory fish guts, and are more present in the Indo-Pacific and Caribbean areas. It is estimated that CFP causes per year more than 10,000 intoxications worldwide. With the rise of water temperature and anthropogenic intervention, it is important to study the prevalence of CFP in more temperate waters. Through inter- and subtidal sampling, 22 species of organisms were collected, in Madeira and Azores archipelagos and in the northwestern Moroccan coast, during September of 2012 and June and July of 2013. A total of 94 samples of 22 different species of bivalves, gastropods, echinoderms and crustaceans where analyzed by Ultra Performance Liquid Chromatography-Mass Spectometry-Ion Trap-Time of Flight (UPLC-MS-IT-TOF) and Ultra Performance Chromatography- Mass Spectrometry (UPLC-MS). Our main aim was to detect new vectors and ascertain if there were some geographical differences. We detected for the first time putative CTXs in echinoderms, in two starfish species—M. glacialis and O. ophidianus. We detected differences regarding uptake values by organisms and geographical location. Toxin amounts were significant, showing the importance and the need for continuity of these studies to gain more knowledge about the prevalence of these toxins, in order to better access human health risk. In addition, we suggest monitoring of these toxins should be extended to other vectors, starfish being a good alternative for protecting and accessing human health risk.

First report of Ciguatoxins in two starfish species: Ophidiaster ophidianus and Marthasterias glacialis

Ciguatera fish poisoning (CFP) is a syndrome caused by the ingestion of fish contaminated with Ciguatoxins (CTXs). These phycotoxins are produced mainly by dinoflagellates that belong to the genus Gambierdiscus that are transformed in more toxic forms in predatory fish guts, and are more present in the Indo-Pacific and Caribbean areas. It is estimated that CFP causes per year more than 10,000 intoxications worldwide. With the rise of water temperature and anthropogenic intervention, it is important to study the prevalence of CFP in more temperate waters. Through inter- and subtidal sampling, 22 species of organisms were collected, in Madeira and Azores archipelagos and in the northwestern Moroccan coast, during September of 2012 and June and July of 2013. A total of 94 samples of 22 different species of bivalves, gastropods, echinoderms and crustaceans where analyzed by Ultra Performance Liquid Chromatography-Mass Spectometry-Ion Trap-Time of Flight (UPLC-MS-IT-TOF) and Ultra Performance Chromatography- Mass Spectrometry (UPLC-MS). Our main aim was to detect new vectors and ascertain if there were some geographical differences. We detected for the first time putative CTXs in echinoderms, in two starfish species—M. glacialis and O. ophidianus. We detected differences regarding uptake values by organisms and geographical location. Toxin amounts were significant, showing the importance and the need for continuity of these studies to gain more knowledge about the prevalence of these toxins, in order to better access human health risk. In addition, we suggest monitoring of these toxins should be extended to other vectors, starfish being a good alternative for protecting and accessing human health risk.

Quantification of free auxins in semi-hardwood plant cuttings and microshoots by dispersive liquid–liquid microextraction/microwave derivatization and GC/MS analysis

Several studies have suggested that differences in the natural rooting ability of plant cuttings could be attributed to differences in endogenous auxin levels. Hence, during rooting experiments, it is important to be able to routinely monitor the evolution of endogenous levels of plant hormones. This work reports the development of a new method for the quantification of free auxins in auxin-treated Olea europaea (L.) explants, using dispersive liquid–liquid microextraction (DLLME) and microwave assisted derivatization (MAD) followed by gas chromatography/mass spectrometry (GC/MS) analysis. Linear ranges of 0.5–500 ng mL 1 and 1–500 mg mL 1 were used for the quantification of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA), respectively. Determined by serial dilutions, the limits of detection (LOD) and quantification (LOQ) were 0.05 ng mL 1 and 0.25 ng mL 1, respectively for both compounds. When using the calibration curve for determination, the LOQ corresponded to 0.5 ng mL 1 (IAA) and 0.5 mg mL 1 (IBA). The proposed method proved to be substantially faster than other alternatives, and allowed free auxin quantification in real samples of semi-hardwood cuttings and microshoots of two olive cultivars. The concentrations found in the analyzed samples are in the range of 0.131–0.342 mg g 1 (IAA) and 20–264 mg g 1 (IBA).

Thermogravimetric analysis of selected coal-bearing strata kaolinite

Two kinds of coal-bearing kaolinite from China were analysed by X-ray diffraction (XRD), Thermogravimetric analysis-mass spectrometry (TG-MS), infrared emission spectroscopy. Thermal decomposition occurs in a series of steps attributed to (a) desorption of water at 68 °C for Datong coal bearing strata kaolinite and 56 °C for Xiaoxian with mass losses of 0.36 % and 0.51 % (b) decarbonization at 456 °C for Datong coal bearing strata kaolinite and 431 °C for Xiaoxian kaolinite, (c) dehydroxylation takes place in two steps at 589 and 633 °C for Datong coal bearing strata kaolinite and at 507 °C and 579 °C for Xiaoxian kaolinite. This mineral were further characterised by infrared emission spectroscopy (IES). Well defined hydroxyl stretching bands at around 3695, 3679, 3652 and 3625 cm-1 are observed. At 650 °C all intensity in these bands is lost in harmony with the thermal analysis results. Characteristic functional groups from coal are observed at 1918, 1724 and 1459 cm-1. The intensity of these bands decrease by thermal treatment and is lost by 700 °C.

Characterisation and source identification of selected pollutants in house dust

House dust is a heterogeneous matrix, which contains a number of biological materials and particulate matter gathered from several sources. It is the accumulation of a number of semi-volatile and non-volatile contaminants. The contaminants are trapped and preserved. Therefore, house dust can be viewed as an archive of both the indoor and outdoor air pollution. There is evidence to show that on average, people tend to stay indoors most of the time and this increases exposure to house dust. The aims of this investigation were to: " assess the levels of Polycyclic Aromatic Hydrocarbons (PAHs), elements and pesticides in the indoor environment of the Brisbane area; " identify and characterise the possible sources of elemental constituents (inorganic elements), PAHs and pesticides by means of Positive Matrix Factorisation (PMF); and " establish the correlations between the levels of indoor air pollutants (PAHs, elements and pesticides) with the external and internal characteristics or attributes of the buildings and indoor activities by means of multivariate data analysis techniques. The dust samples were collected during the period of 2005-2007 from homes located in different suburbs of Brisbane, Ipswich and Toowoomba, in South East Queensland, Australia. A vacuum cleaner fitted with a paper bag was used as a sampler for collecting the house dust. A survey questionnaire was filled by the house residents which contained information about the indoor and outdoor characteristics of their residences. House dust samples were analysed for three different pollutants: Pesticides, Elements and PAHs. The analyses were carried-out for samples of particle size less than 250 µm. The chemical analyses for both pesticides and PAHs were performed using a Gas Chromatography Mass Spectrometry (GC-MS), while elemental analysis was carried-out by using Inductively-Coupled Plasma-Mass Spectroscopy (ICP-MS). The data was subjected to multivariate data analysis techniques such as multi-criteria decision-making procedures, Preference Ranking Organisation Method for Enrichment Evaluations (PROMETHEE), coupled with Geometrical Analysis for Interactive Aid (GAIA) in order to rank the samples and to examine data display. This study showed that compared to the results from previous works, which were carried-out in Australia and overseas, the concentrations of pollutants in house dusts in Brisbane and the surrounding areas were relatively very high. The results of this work also showed significant correlations between some of the physical parameters (types of building material, floor level, distance from industrial areas and major road, and smoking) and the concentrations of pollutants. Types of building materials and the age of houses were found to be two of the primary factors that affect the concentrations of pesticides and elements in house dust. The concentrations of these two types of pollutant appear to be higher in old houses (timber houses) than in the brick ones. In contrast, the concentrations of PAHs were noticed to be higher in brick houses than in the timber ones. Other factors such as floor level, and distance from the main street and industrial area, also affected the concentrations of pollutants in the house dust samples. To apportion the sources and to understand mechanisms of pollutants, Positive Matrix Factorisation (PMF) receptor model was applied. The results showed that there were significant correlations between the degree of concentration of contaminants in house dust and the physical characteristics of houses, such as the age and the type of the house, the distance from the main road and industrial areas, and smoking. Sources of pollutants were identified. For PAHs, the sources were cooking activities, vehicle emissions, smoking, oil fumes, natural gas combustion and traces of diesel exhaust emissions; for pesticides the sources were application of pesticides for controlling termites in buildings and fences, treating indoor furniture and in gardens for controlling pests attacking horticultural and ornamental plants; for elements the sources were soil, cooking, smoking, paints, pesticides, combustion of motor fuels, residual fuel oil, motor vehicle emissions, wearing down of brake linings and industrial activities.

A mass spectrometric assay for the quantification of neuropeptide PYY in plasma

A multiple reaction monitoring mass spectrometric assay for the quantification of PYY in human plasma has been developed. A two stage sample preparation protocol was employed in which plasma containing the full length neuropeptide was first digested using trypsin, followed by solid-phase extraction to extract the digested peptide from the complex plasma matrix. The peptide extracts were analysed by LC-MS using multiple reaction monitoring to detect and quantify PYY. The method has been validated for plasma samples, yielding linear responses over the range 5–1,000 ng mL−1. The method is rapid, robust and specific for plasma PYY detection.

Identification of bifunctional GA and AG intrastrand crosslinks formed between cisplatin and DNA

A combination of enzymatic digestion and electrospray ionisation mass spectrometry (ESI-MS) was used to characterise bifunctional adducts in which cisplatin is bound to GA base sequences in 8mer and 16mer oligonucleotides that do not contain other, higher affinity binding sites. The extent of formation of bifunctional adducts with GA base sequences was significant, but less than that seen with similar oligonucleotides containing either AG or GG sequences.