Determination of organochlorine pesticides in vegetable matrices by stir bar sorptive extraction with liquid desorption and large volume injection-gas chromatography-mass spectrometry towards compliance with European Union directives.

A novel analytical approach to determine trace levels of 20 organochlorine pesticides (OCPs) in nine vegetable matrices (lettuce, spinach, green bean, green pepper, tomato, broccoli, potato, carrot and onion) is proposed, based on stir bar sorptive extraction followed by liquid desorption and large volume injection-gas chromatography coupled to mass spectrometry using the selected-ion monitoring mode acquisition (SBSE-LD/LVI-GC-MS(SIM)). The experimental procedure consists of a previous ultrasonic extraction of the freeze-dried vegetable samples (100.0mg) with methanol (2mL) followed by centrifugation and dissolution in aqueous media prior to SBSE-LD/LVI-GC-MS(SIM) under optimised conditions. Assays were performed on 30mL aqueous samples using stir bars coated with 47microL of polydimethylsiloxane, an equilibrium time of 180min (1000rpm; 20 degrees C) and acetonitrile as back-extraction solvent, providing convenient analytical performance to monitor OCPs in vegetable matrices at the trace level. Besides the selectivity reached, the data obtained clearly demonstrate that the matrices involved have a strong effect on the recovery yields (10-110%) of the OCPs under study, in particular the green vegetables especially the leafy ones. By using the standard addition methodology, good linearity (r(2)>0.99) and convenient precisions (RSD<20%) were found for almost all cases, depending on the particular OCP and vegetable matrix involved. Furthermore enough sensitivity was also achieved (limit of detection <10microgkg(-1)) for all OCPs under study towards compliance with the European Union regulations for the maximum residue limits of pesticides in agricultural vegetables. The methodology showed to be easy of work-up, fast, almost solventless with low sample amount requirement, when compared with conventional methods of sample preparation to screen pesticides in vegetable matrices.

Use of experimental design in the optimization of stir bar sorptive extraction for the determination of polybrominated diphenyl ethers in environmental matrices.

Stir bar sorptive extraction and liquid desorption (LD) followed by large volume injection and capillary gas chromatography coupled to mass spectrometry (SBSE-LD-LVI-GC-MS), had been applied for the determination of ultra-traces of eleven polybrominated diphenylethers (PBDEs), from tetra to nona congeners (BDE-47, BDE-100, BDE-99, BDE-85, BDE-154, BDE-153, BDE-183, BDE-197, BDE-196, BDE-207 and BDE-206), in environmental matrices. Instrumental calibration under the selected-ion monitoring (SIM) mode acquisition and parameters that could affect the SBSE-LD efficiency are fully discussed. A complete randomized factorial design was established for the first time to optimize the main experimental parameters that affecting the SBSE-LD efficiency, including decisive interactions, which provides a more realistic picture of the sampling process. The analysis of variance (ANOVA) was the statistical method used to analyze data. From the data obtained, it can be emphasized that experimental parameters such as extraction time (240 min), agitation speed (1250 rpm), methanol content (40%) and desorption conditions (acetonitrile, 15 min), were the best analytical compromise for the simultaneous determination between tetra and nona congeners in aqueous media. A remarkable recovery (65.6-116.9%) and repeatability (<12.1%) were attained, whilst the experimental data allowed very good agreement with predict theoretical equilibrium described by the octanol-water partition coefficients (K(PDMS/W) approximately = K(O/W)), with the exception of nona congeners since slightly lower yields were measured. Furthermore, excellent linear dynamic ranges from 0.01 to 14.0 microg/L (r2>0.9917) and low detection limits (0.3-203.4 ng/L) were also achieved for the eleven congeners studied. The proposed methodology was applied for the determination of ultra-trace levels of PBDEs in waste water, sediments and printed board circuit matrices by the standard addition approach, showing to be reliable, sensitive and having a low sample amount requirement in compliance with the international regulatory bodies.

New strategies to screen for endocrine-disrupting chemicals in the Portuguese marine environment utilizing large volume injection-capillary gas chromatography-mass spectrometry combined with retention time locking libraries (LVI-GC-MS-RTL).

A new analytical strategy to screen for endocrine-disrupting chemicals (EDCs) in environmental matrices is presented. The strategy uses solid-phase extraction followed by large volume injection and capillary gas chromatography coupled to mass spectrometry combined with retention time locking libraries (SPE-LVI-GC-MS-RTL). Characterization of the proposed methodology (SPE-LVI-GC-MS) for selected classes of EDCs enabled high reproducibility and robustness at the ultratrace level. The RTL databases used allowed hundreds of non-target semivolatiles (i.e., pesticides, polycyclic aromatic hydrocarbons, polychlorinated biphenyls and other classes of suspected EDCs from a great number of unknown environmental matrices) to be simultaneously screened for in an easy, fast and remarkable manner. The application of the proposed methodology to real environmental samples demonstrated its remarkable selectivity and sensitivity at the ultratrace level. Screening assessments performed on water and sediment matrices from eight Portuguese estuaries and coastal waters identified EDC "hotspots." These EDCs mainly come from agricultural and a wide variety of industrial sources, and include pesticides and pesticide metabolites, phenolic derivatives and polycyclic aromatic hydrocarbons, which are included in the lists of priority substances published by international environmental agencies. The estuaries that contained relatively high levels of pesticides were Guadiana, Sado and Mondego, while Minho, Douro and Formosa showed enhanced levels of phenolic derivatives. Dibutyltin and tributyltin, selected as target compounds to be monitored by SPE-LVI-GC-MS in the selected ion monitoring mode, were shown to be widespread contaminants at trace levels in almost all of the sediment matrices assessed. The reliability of the proposed methodology undoubtedly makes it a valuable tool that could replace other analytical strategies currently used to screen for EDCs present in the environment at ultratrace levels.

Considerations on ultra-trace analysis of phthalates in drinking water.

Stir bar sorptive extraction with liquid desorption followed by large volume injection and capillary gas chromatography coupled to mass spectrometry (SBSE-LD/LVI-GC-MS), had been applied for the determination of ultra-traces of seven-phthalates (dimethyl phthalate, diethyl phthalate, di-n-butyl phthalate, butyl benzyl phthalate, bis(2-ethylhexyl) adipate, bis(2-ethylhexyl) phthalate and bis(1-octyl) phthalate) in drinking water samples, which are included in the priority lists set by several international regulatory organizations. Instrumental calibration under the selected-ion monitoring mode acquisition (LVI-GC-MS(SIM)), experimental parameters that could affect the SBSE-LD efficiency, as well as, the control of the contamination profile are fully discussed. Throughout systematic assays on 30 mL water samples spiked at the 0.40 microg/L level, it had been established that stir bars coated with 47 microL of polydimethylsiloxane, an equilibrium time of 60 min (1,000 rpm) and methanol as back extraction solvent, allowed the best analytical performance to monitor phthalates in water matrices. From the data obtained, good accuracy and a remarkable reproducibility (< 14.8%) were attained, providing experimental recovery data in agreement with the theoretical equilibrium described by the octanol-water partition coefficients (K(PDMS/W) approximately K(O/W)), with the exception of bis(2-ethylhexyl) adipate, bis(2-ethylhexyl) phthalate and bis(1-octyl) phthalate, for which lower yields were measured. Additionally, a remarkable linear dynamic range between 25 and 2,000 ng/L (r(2)>0.99) and low detection limits (3-40 ng/L) were also achieved for the seven-phthalates studied. The application of the present method to monitor phthalates in tap and bottled mineral water samples, allowed convenient selectivity and high sensitivity up to 1.0 microg/L level, using the standard addition methodology. The proposed method showed to be feasible and sensitive with a low sample volume requirement to monitor phthalates in drinking water matrices at the ultra-trace level, in compliance with international regulatory directives on water quality.

Development of a stir-bar-sorptive extraction-liquid desorption-large-volume injection capillary gas chromatographic-mass spectrometric method for pyrethroid pesticides in water samples.

Stir-bar-sorptive extraction followed by liquid desorption and large-volume injection capillary gas chromatography with mass spectrometric detection (SBSE-LD-LVI-GC-MS), had been applied for the determination of ultra-traces of eight pyrethroid pesticides (acrinathrin, cypermethrin, deltamethrin, esfenvalerate, fenpropathrin, fenvalerate, and permethrin cis and trans isomers) in water samples. Instrumental calibration for selected-ion monitoring acquisition and conditions that could affect the SBSE-LD efficiency are fully discussed. By performing systematic assays on 30-mL water samples spiked at the 0.10 microg L(-1) level it was established that stir-bars coated with 47 microL polydimethylsiloxane, an equilibrium time of 60 min (750 rpm), 5% methanol as organic modifier, and acetonitrile as back-extraction solvent, provided the best analytical performance to monitor pyrethroid pesticides in water matrices. Good accuracy (81.8-105.0%) and remarkable reproducibility (<11.7%) were obtained, and the experimental recovery data were in good agreement with the theoretical equilibrium described by octanol-water partition coefficients (log K(O/W)), with the exception of acrinathrin for which lower yields were measured. Excellent linear dynamic ranges between 25 and 400 ng L(-1) (r2>0.994), low quantification (3.0-7.5 ng L(-1)) and detection (1.0-2.5 ng L(-1)) limits were also achieved for the eight pyrethroid pesticides studied. The method was successfully used for analysis of tap-water and groundwater matrices spiked at the 0.10 microg L(-1), revealing the suitability of the method for determination of pyrethroid pesticides in real samples. The method was shown be reliable and sensitive and a small volume of sample was required to monitor pyrethroids at ultra-trace levels, in compliance with international regulatory directives on water quality.

CREB1 encodes a nuclear activator, a repressor, and a cytoplasmic modulator that form a regulatory unit critical for long-term facilitation.

Although CREB seems to be important for memory formation, it is not known which of the isoforms of CREB, CREM, or ATF1 are expressed in the neurons that undergo long-term synaptic changes and what roles they have in memory formation. We have found a single Aplysia CREB1 gene homologous to both mammalian CREB and CREM and have characterized in the sensory neurons that mediate gill-withdrawal reflex the expression and function of the three proteins that it encodes: CREB1a, CREB1b, and CREB1c. CREB1a is a transcriptional activator that is both necessary and, upon phosphorylation, sufficient for long-term facilitation. CREB1b is a repressor of long-term facilitation. Cytoplasmic CREB1c modulates both the short- and long-term facilitation. Thus, in the sensory neurons, CREB1 encodes a critical regulatory unit converting short- to long-term synaptic changes.

Differential expression of Kv4 K+ channel subunits mediating subthreshold transient K+ (A-type) currents in rat brain.

The mammalian Kv4 gene subfamily and its Drosophila Shal counterpart encode proteins that form fast inactivating K+ channels that activate and inactivate at subthreshold potentials and recover from inactivation at a faster rate than other inactivating Kv channels. Taken together, the properties of Kv4 channels compare best with those of low-voltage activating "A-currents" present in the neuronal somatodendritic compartment and widely reported across several types of central and peripheral neurons, as well as the (Ca2+-independent) transient outward potassium conductance of heart cells (Ito). Three distinct genes have been identified that encode mammalian Shal homologs (Kv4. 1, Kv4.2, and Kv4.3), of which the latter two are abundant in rat adult brain and heart tissues. The distribution in the adult rat brain of the mRNA transcripts encoding the three known Kv4 subunits was studied by in situ hybridization histochemistry. Kv4.1 signals are very faint, suggesting that Kv4.1 mRNAs are expressed at very low levels, but Kv4.2 and Kv4.3 transcripts appear to be abundant and each produces a unique pattern of expression. Although there is overlap expression of Kv4.2 and Kv4.3 transcripts in several neuronal populations, the dominant feature is one of differential, and sometimes reciprocal expression. For example, Kv4.2 transcripts are the predominant form in the caudate-putamen, pontine nucleus and several nuclei in the medula, whereas the substantia nigra pars compacta, the restrosplenial cortex, the superior colliculus, the raphe, and the amygdala express mainly Kv4.3. Some brain structures contain both Kv4.2 and Kv4.3 mRNAs but each dominates in distinct neuronal subpopulations. For example, in the olfactory bulb Kv4.2 dominates in granule cells and Kv4.3 in periglomerular cells. In the hippocampus Kv4.2 is the most abundant isoform in CA1 pyramidal cells, whereas only Kv4.3 is expressed in interneurons. Both are abundant in CA2-CA3 pyramidal cells and in granule cells of the dentate gyrus, which also express Kv4.1. In the dorsal thalamus strong Kv4.3 signals are seen in several lateral nuclei, whereas medial nuclei express Kv4.2 and Kv4.3 at moderate to low levels. In the cerebellum Kv4.3, but not Kv4.2, is expressed in Purkinje cells and molecular layer interneurons. In the cerebellar granule cell layer, the reciprocity between Kv4.2 and Kv4.3 is observed in subregions of the same neuronal population. In fact, the distribution of Kv4 channel transcripts in the cerebellum defines a new pattern of compartmentation of the cerebellar cortex and the first one involving molecules directly involved in signal processing.

Cloning of a novel component of A-type K+ channels operating at subthreshold potentials with unique expression in heart and brain.

1. Proteins of the Kv4 or Shal-related subfamily are key components of transient K+ channels (A channels) operating at subthreshold values of the membrane potential. We have cloned and characterized a new mammalian Kv4 or Shal-related cDNA (Kv4.3) that predicts a protein with strong sequence conservation with the other known members of this subfamily. 2. Injection of Kv4.3 transcripts into Xenopus oocytes generates an A type K+ current, with small but physiologically significant differences from the currents expressed by Kv4.2 and Kv4.1 mRNAs. Kv4.3 currents can be modified to resemble native A currents by coinjection with a low molecular weight mRNA fraction from rat brain which does not express detectable currents on its own. Particularly striking is a 7-to-10-fold increase in the rate of recovery from inactivation, a 5- to 10-fold increase in current magnitude and a 3- to 4-fold increase in sensitivity to 4-amino pyridine (4-AP). 3. In situ hybridization histochemistry was used to compare the expression of the three known Kv4 genes. Kv4.2 and Kv4.3 (but not Kv4.1) are abundant in the adult rat brain, with each displaying a specific, but sometimes overlapping pattern of expression. Moreover, a reciprocal gradient of expression of Kv4.2 and Kv4.3 transcripts is seen in some brain areas, such as in the pyramidal cell layers of the hippocampus and the granule cell layer of the cerebellum. Therefore Kv4 proteins may form heteromultimeric channels of distinct subunit composition in different neurons. Moreover, the results suggest that neurons such as pyramidal cells in the hippocampus and granule cells in the cerebellum represent heterogeneous cell populations in terms of their ISA, and hence in their firing patterns. Kv4.2 and Kv4.3 also display complementary expression in the heart, with Kv4.3 being more abundant in atria and Kv4.2 in ventricle. The existence of multiple Kv4 proteins forming channels of variable subunit combinations helps explain the diversity of ISA channels in neurons.

Identification of molecular components of A-type channels activating at subthreshold potentials.

1. Xenopus oocytes injected with rat brain mRNA express a transient K+ current similar to the A current that activates transiently near the threshold for Na+ action potential generation (ISA) seen in somatic recordings from neurons. We used hybrid arrest with antisense oligonucleotides to investigate which of the cloned K+ channel proteins might be components of the channels responsible for the ISA expressed from brain mRNA. An oligonucleotide complementary to a sequence common to all known mammalian Shal-related mRNAs [KV4.1, KV4.2, and KV4.3 (the nomenclature of Sh K+ channel genes of Chandy and colleagues was used in this paper)] blocked the expression of the ISA. An oligonucleotide complementary only to the KV4.2 mRNA, the most abundant Shal-related transcript in rat brain RNA preparations, was also quite efficient in arresting the expression of the ISA from brain. These experiments indicate that Shal-related proteins are important components of the channels carrying the ISA expressed in oocytes injected with brain mRNA. However, there are several significant differences between this ISA and the currents expressed in the same oocytes by in vitro transcribed KV4.1 or KV4.2 cRNA. Most of these differences are eliminated if KV4.1 or KV4.2 cRNA is coinjected with brain poly-(A) RNA treated with antisense oligonucleotides which arrest the expression of the ISA, or with a 2-4Kb rat brain poly-(A) RNA fraction which does not express detectable K+ currents under the same recording conditions. These data support the hypothesis that ISA channels such as those expressed from brain mRNA contain Shal proteins that can be modified by proteins encoded in RNAs that by themselves do not express K+ currents.

Region-specific expression of a K+ channel gene in brain.

Northern blot analysis and in situ hybridization studies reveal the highly localized expression in rat brain of transcripts from a gene (KShIIIA) encoding components for voltage-gated K+ channels. KShIIIA expression is particularly prominent throughout the dorsal thalamus. The expression of KShIIIA is compared to that of a closely related gene, here called NGK2-KV4. These two genes encode transcripts that induce currents in Xenopus oocytes that are as of yet indistinguishable, but they show very different patterns of expression in rat brain. NGK2-KV4 transcripts are particularly abundant in the cerebellar cortex, where KShIIIA expression is very weak. These results demonstrate the existence of cell-type-specific K+ channel components and suggest that one reason for the unusually large diversity of K+ channel proteins is the presence of subtypes that participate in specific brain functions.