Determination of herbicides and a metabolite in human urine by liquid chromatography-electrospray ionization mass spectrometry

A method was developed to determine simazine, atrazine and their metabolite, 2-chloro-4,6-diamino-1,3,5-triazine, in urine. The presence of these herbicides in urine may reflect possible exposure to pesticides. Sample preparation involved protein precipitation and solid-phase extraction. The samples were analyzed by high-performance liquid chromatography-mass spectrometry. The detection limits were 0.4 mug/l and the analytes have a linear response in the interval 6-800 mug/l. The precision of the method was reflected in the RSD of <2.4% for the herbicides studied. Based on the detectable herbicide levels from spiked urine samples collected from unexposed volunteers, this method can be used to determine the low levels necessary for establishing reference values of the selected herbicides and the metabolite. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Correlation between the mobility of spin-labeled peptide chains and resin solvation: An approach to optimize the synthesis of aggregating sequences

Resin solvation properties affect the efficiency of the coupling reactions in solid-phase peptide synthesis. Here we report a novel approach to evaluate resin solvation properties, making use of spin label electron paramagnetic resonance (EPR) spectroscopy. The aggregating VVLGAAIV and ING sequences were assembled in benzhydrylamine-resin with different amino group contents (up to 2.6 mmol/g) to examine the extent of chain association within the beads. These model peptidyl-resins were first labeled at their N-terminus with the amino acid spin label 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac). Their solvation properties in different solvents were estimated, either by bead swelling measurement or by assessing the dynamics of their polymeric matrixes through the analysis of Toac EPR spectra, and were correlated with the yield of the acylation reaction. In most cases the coupling rate was found to depend on bead swelling. Comparatively, the EPR approach was more effective. Line shape analysis allowed the detection of more than one peptide chain population, which influenced the reaction. The results demonstrated the unique potential of EPR spectroscopy not only for improving the yield of peptide synthesis, even in challenging conditions, but also for other relevant polymer-supported methodologies in chemistry and biology.

Puberty and growth rate in thoroughbred fillies

We measured progesterone and estradiol levels from birth to the beginning of adult life in 10 Thoroughbred fillies from the Equilia Stud Farm in Avare SP, Brasil. The animals were measured and weighed monthly for the determination of body development and of a possible correlation between the rate of weight and height gain and the onset of detectable sex hormone levels. Jugular blood was collected twice a week and stored at -20 degrees C until assay of progesterone by a solid phase RIA with a sensitivity of 0.32 nmol/L and of estradiol by liquid phase RIA adapted to low levels (3.67 pmol/L). The fillies were born with high serum levels of both hormones,which fell to undetectable levels by the first week of life. A variation in growth rate was observed, with the highest levels occuring from birth to the 3rd month of life, followed by a reduction until 15 mo of life, when fast growth was resumed. The monthly weight gain was 1.5% when the fillies reached puberty and 5.4% during the next month, (P<0.05, Friedman test). During this second period of accelerated growth after the beginning of progesterone production at detectable levels (above 0.318 nmol/L), the parameters of skeletal growth did not differ (P>0.05). The month of onset of puberty was the month of lowest weight gain in the life of the fillies, and it coincided with the highest insolation period. In conclusion, horses, like all other developed vertebrates, have a double pattern of development, with the acceleration observed at puberty depending on sex steroids, which in turn coincides with the highest insolation period. Gonadal activity characterized by serum progesterone levels was low from birth to the onset of puberty. After puberty the progesterone cycles were similar to those of adult animals with a mature hypothalamic-gonadal axis. (C) 1997 by Elsevier B.V.

Níveis séricos de cortisol e 17-& estradiol durante o ciclo estral em marrãs (Sus domestica - Linnaeus 1758)

In the present study 21 young swine females, sexually mature, reared and kept under an industrial management system were assessed for cortisol and oestradiol-17 beta serum prolifes during the oestrus cycle. Blood sampling was performed always at the same interval, from 8:00 to 10:00 a.m. Each animal was submitted to 14 venal punctions at days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 21, 22 and 23 of the oestrous cycle. The first day of the oestrous phase was assumed to be day 0 and the 23(rd) as the first of the next cycle. Hormone determinations were performed using a solid phase radioimmunoassay (RIA). Regarding to cortisol concentrations, there was a variation from 3.5 to 8.0 mu g/dl and for oestradiol-17 beta the mean values detected varied from 3.5 to 14.9pg/ml.

Rapid degradation of propanil in rice crop fields

Propanil and its major degradation product, 3,4-dichloroaniline (DCA), were monitored in surface water and soil samples from two rice fields of the Ebre Delta area (Tarragona, Spain) following agricultural application. On-line solid-phase extraction (SPE) (water) and Soxhlet extraction (soil) followed by liquid chromatography/diode array detection (LC/DAD) were used for the trace determination of both compounds. Unequivocal confirmation/identification was conducted by using liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry, LC/APCI/MS (using negative and positive ionization modes). Concentrations of the herbicide propanil in water samples varied from 1.9 to 55.9 mu g/L. Propanil degraded very rapidly to DCA, and high concentrations of this product were found, varying from 16.5 to 470 mu g/L in water and 119 +/- 22 mu g/kg in soil samples. No detectable DCA (<0.001%) was found in the applied pesticide formulation, indicating that DCA formation took place after propanil application. These field results compared favorably with laboratory experiments showing that humic interactions had a strong influence on the pesticide degradation. The half-lifes under real conditions for propanil and DCA, calculated using a first-order decay, were 1.2 and 1.6 days, respectively.

Speciation analysis of Sn(II) and Sn(IV) using baker's yeast and inductively coupled plasma optical emission spectrometry

The use of Saccharomyces cerevisiae as a substrate to selectively retain Sn(II) and Sn(IV) has been investigated. Several factors affecting the retention of the analytes by yeast, such as pH, amount of biomass, temperature and time of contact were evaluated. Based on this study, a method for determination of Sn(II) and Sn(IV) combining inductively coupled plasma optical emission spectrometry (ICP OES) and solid phase extraction using Saccharomyces cerevisiae is proposed. The procedure consists of the selective retention of Sn(IV) by yeast at pH = 2.0 while Sn(II) remains in solution. Determination of tin in the solid phase was easily carried out by submitting a slurry of the yeast (0.5 g/40 mL) directly to ICP OES. The precision of the extraction procedure was characterized by an RSD lower than 4%. The detection limits of tin (3 sigma) in the solid phase and the liquid phase were 1.1 and 0.7 mu g L-1, respectively. The proposed approach was evaluated for determination of Sn(II) and Sn(IV) in spiked river water and real samples of industrial waste water (untreated and treated). For all samples, recoveries of spiked Sn(II) and Sn(IV) were between 85 and 112%.

Determination of Cd(II) and Cd-metallothioneins in biological extracts using baker's yeast and inductively coupled plasma optical emission spectrometry

The use of Saccharomyces cerevisiae as a sorbent material to separate Cd(II) and Cd-metallothionein complex (Cd-MT) has been explored. Solid-liquid phase extractions were carried out in batch mode and the main parameters of the process (pH, temperature, time of incubation, amount of biomass and analyte) were evaluated. Under optimized conditions, the yeast quantitatively retain (94 +/- 5%) the Cd(II) while 97 +/- 2% of the Cd-MT remain in the supernatant. on base of the findings of this study, a simple method is proposed to determine Cd(II) and Cd-MT in cytosols extracted from mouse kidney and crab hepatopancreas. Inductively coupled plasma optical emission spectrometry was used to quantify the analytes in solid and liquid phase. Determination of Cd in the solid phase was carried out by introducing a slurry of the yeast (0.0625 g/10 mL) directly to the inductively coupled plasma optical emission spectrometer. Mixed standards solutions, which also have been submitted to the extraction procedure, were used to quantify the analytes in the samples. Thus, matrix effects due to nebulization of the slurry were overcame. Limits of detection (3 sigma) for Cd(II) and Cd-MT were 1.5 and 1.2 mu g L-1, respectively. Relative standard deviations of signals were 4.2% for measurements in the slurry of solid phase and 2.1% for measurements in the liquid phase. Recoveries of the analytes in cytosol samples were between 76 and 114%. The concentrations of Cd(II) (2.4 +/- 0.5 mu g L-1) and Cd-MT (3.0 +/- 0.5 mu g L-1) found by using the proposed approach were close to those found by tangential-flow ultrafiltration technique (2.6 +/- 0.7 mu g L-1 for Cd(II) and 3.7 +/- 1.7 mu g L-1 for Cd-MT).

Differential pulse polarographic determination of ceftazidime in urine samples with and without prior extraction

Ceftazidime shows two main polarographic reduction peaks at pH 4.0, that at -0.45 V owing to reduction of the C=N bond in the methylethoxyimino group and that at -1.00 V owing to the reductive elimination of pyridine: the first peak is particularly suitable for the determination of ceftazidime. Ceftazidime can also be determined indirectly using the tensammetric peak at -0.60 V (in Britton-Robinson buffer pH 9.5) of pyridine liberated on hydrolysis. Ceftazidime can be determined in urine using the direct method only after Cls solid phase extraction, but it can be determined directly in the urine by hydrolysing it and using the pyridine peak. (C) 1997 Elsevier B.V. B.V.

Synthesis and pharmacological properties of TOAC-labeled angiotensin and bradykinin analogs

Angiotensin II (AngII) and bradykinin (BK) derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin label were synthesized by solid phase methodology. Ammonium hydroxide (pH 10, 50degreesC, 1 h) was the best means for reverting nitroxide protonation occurring during peptide cleavage. EPR spectra yielded rotational correlation times for internally labeled analogs that were nearly twice as large as those of N-terminally labeled analogs. Except for TOAC(1)-AngII and TOAC(0)-BK, which showed high intrinsic activities, other derivatives were inactive in smooth muscle preparations. These active paramagnetic analogs may be useful for conformational studies in solution and in the presence of model and biological membranes. (C) 2002 Elsevier B.V. All rights reserved.

A 4.2 kDa synthetic peptide as a potential probe to evaluate the antibacterial activity of coumarin drugs

The coumarin antibiotics are potent inhibitors of DNA replication whose target is the enzyme DNA gyrase, an ATP-dependent bacterial type II topoisomerase. The coumarin drugs inhibit gyrase action by competitive binding to the ATP-binding site of DNA gyrase B protein. The production of new biologically active products has stimulated additional studies on coumarin-gyrase interactions. In this regard, a 4.2 kDa peptide mimic of DNA gyrase B protein from Escherichia coli has been designed and synthesized. The peptide sequence includes the natural fragment 131-146 (coumarin resistance-determining region) and a segment containing the gyrase-DNA interaction region (positions 753-770). The peptide mimic binds to novobiocin (K-a = 1.4 +/- 0.3 x 10(5) m(-1)), plasmid (K-a = 1.6 +/- 0.5 x 10(6) m(-1)) and ATP (K-a = 1.9 f 0.4 x 10(3) m(-1)), results previously found with the intact B protein. on the other hand, the binding to novobiocin was reduced when a mutation of Arg-136 to Leu-136 was introduced, a change previously found in the DNA gyrase B protein from several coumarin-resistant clinical isolates of Escherichia coLi. In contrast, the binding to plasmid and to ATP was not altered. These results suggest that synthetic peptides designed in a similar way to that described here could be used as mimics of DNA gyrase in studies which seek a better understanding of the ATP, as well as coumarin, binding to the gyrase and also the mechanism of action of this class of antibacterial drugs. Copyright (C) 2004 European Peptide Society and John Wiley Sons, Ltd.

EPR investigation of the influence of side chain protecting groups on peptide-resin solvation of the Asx and Glx model containing peptides

In spite of all progressive efforts aiming to optimize SPPS, serious problems mainly affecting the assembly of aggregating sequences have persisted. Following the study intended to unravel the complex solvation phenomenon of peptide-resin beads, the XING and XAAAA model aggregating segments were labeled with a paramagnetic probe and studied via EPR spectroscopy. Low and high substituted resins were also comparatively used, with the X residue being Asx or Glx containing the main protecting groups used in the SPPS. Notably, the cyclo-hexyl group used for Asp and Glu residues in Boc-chemistry induced greater chain immobilization than its tert-butyl partner-protecting group of the Fmoc strategy. Otherwise, the most impressive peptide chain immobilization occurred when the large trytil group was used for Asn and Gln protection in Fmoc-chemistry. These surprising results thus seem to stress the possibility of the relevant influence of the amino-acid side chain protecting groups in the overall peptide synthesis yield. (C) 2007 Elsevier Ltd. All rights reserved.

Oxidative dissolution of research-grade minerals by Thiobacillus ferrooxidans and Thiobacillus thiooxidans

Solution- and solid-phase changes associated with galena (PbS) and sphalerite (ZnS) oxidation by T. ferrooxidans and T. thiooxidans, were determined. In experiments with galena, anglesite (PbSO4) was detected as a solid-phase product in biotic and abiotic experiments. In T. ferrooxidans cultures supplemented with FeSO4, jarosite [MFe3 (SO4)(2) (OH)(6)] was also detected as a new solid phase product, whereas SO was not detected in the residues. In sphalerite experiments, minor amounts of SO accumulated in FeSO4-amended sphalerite media with or without T. ferrooxidans or T. thiooxidans. Jarosite was only detected in T. ferrooxidans culture with FeSO4. By comparison with T. thiooxidans, T. ferrooxidans was more efficient in the oxidation of galena and sphalerite.

Isolation and sequence determination of peptides in the venom of the spider wasp (Cyphononyx dorsalis) guided by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry

Micro-scale (sub-pmol) isolation and sequence determination of three peptides from the venom of the solitary spider wasp Cyphononyx dorsalis is described. We isolated two novel peptides Cd-125 and Cd-146 and a known peptide Thr(6)-bradykinin from only two venom sacs of solitary spider wasp Cyphononyx dorsalis without bioassay-guided fractionation. but instead guided by MALDI-TOF MS. The MALDI-TOF MS analysis of each fraction showed the purity and molecular weight of the components, which led to the isolation of the peptides virtually without loss of sample amount. The sequences of the novel peptides Cd-125 (Asp-Thr-Ala-Arg-Leu-Lys-Trp-His) and Cd-146 (Ser-Glu-Thr-Gly-Asn-Thr-Val-Thr-Val-Lys-Gly-Phe-Ser-Pro-Leu-Arg) were determined by Edman degradation together with mass spectrometry. and finally corroborated by solid-phase synthesis. The known peptide Thr(6)-bradykinin (Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg) was identified by comparison with the synthetic authentic specimen. This is the first example for any kinins to be found in Pompilidae wasp venoms. The procedure reported here can be applicable to studies on many other components of solitary wasp venoms with limited sample availability. (C) 2001 Elsevier B.V. Ltd. All rights reserved.

Study of the effect of the peptide loading and solvent system in SPPS by HRMAS-NMR

The SPPS methodology has continuously been investigated as a valuable model to monitor the solvation properties of polymeric materials. In this connection, the present work applied HRMAS-NMR spectroscopy to examine the dynamics of an aggregating peptide sequence attached to a resin core with varying peptide loading (up to 80%) and solvent system. Low and high substituted BHAR were used for assembling the VQAAIDYING sequence and some of its minor fragments. The HRMAS-NMR results were in agreement with the swelling of each resin, i.e. there was an improved resolution of resonance peaks in the better solvated conditions. Moreover, the peptide loading and the attached peptide sequence also affected the spectra. Strong peptide chain aggregation was observed mainly in highly peptide loaded resins when solvated in CDCl3. Conversely, due to the better swelling of these highly loaded resins in DMSO, improved NMR spectra were acquired in this polar aprotic solvent, thus enabling the detection of relevant sequence-dependent conformational alterations. The more prominent aggregation was displayed by the VQAAIDYING segment and not by any of its intermediary fragments and these findings were also corroborated by EPR studies of these peptide-resins labelled properly with an amino acid-type spin probe. Copyright (c) 2005 European Peptide Society and John Wiley & Sons, Ltd.