Cryo-ultramicrotomy and Mass Spectrometry Imaging Analysis of Nudibranch Microstructures.

In this paper, we investigate the presence of latrunculin A in the outer rim of a nudibranch Chromodoris kuiteri and show that by combining ultrathin cryosection methods with MALDI MSI we can achieve improved lateral (x and y) resolution and very high resolution in the z dimension by virtue of the ultrathin 200 nm thin cryosections. We also demonstrate that a post ionization laser increases sensitivity. Recent advances in MALDI source design have improved the lateral resolution (x and y) and sensitivity during MSI. Taken together, very high z resolution, from ultrathin sections, and improved lateral (x and y) resolution will allow for subcellular molecular imaging with the potential for subcellular 3D volume reconstruction.

Synthesis of New Thiazole Derivatives Bearing Thiazolidin-4(5H)-One Structure and Evaluation of Their Antimicrobial Activity

The first report about antimicrobial resistance was published in the 1940s. And today, the antimicrobial resistance has become a worldwide problem. Because of this problem, there is a need to develop new drugs. That's why we synthesized some novel thiazolidine-4-one derivatives and evaluated their antimicrobial activity. The final compounds were obtained by reacting 2-[(4,5-diphenylthiazol-2-yl)imino]thiazolidin-4-one with some aryl aldehydes. The synthesized compounds were investigated for their antimicrobial activity against four Candida species, five gram-negative and four gram-positive bacterial species. The lead compounds (4a- h) were obtained with a yield of at least 70%. All compounds showed antimicrobial activity. Compound 4f (MIC: 31.25 µg/ml) exhibited more efficacy than the other compounds against C. glabrata (ATCC 24433). Compound 4b (MIC: 62.5 µg/ml) was the most active compound against all bacterial species, particularly K. pneumoniae (NCTC 9633). Whereas, compound 4c (MIC: <31.25 µg/ml) was observed as the most active compound against E. coli (ATCC 25922). In general, all compounds (4a-4h) showed antimicrobial activity against all fungi and bacterial species. Compounds 4b (2,6-dichlorobenzylidene), 4c (2,6-dihydroxybenzylidene), 4f (1H-pyrrol-2- yl)methylene), 4g (4-triflouromethylbenzylidene) and 4h (2,3,4-trimethoxybenzylidene) were determined as the most active compounds

Direct Enantiomeric Separation and Determination of Hexythiazox Enantiomers in Environment and Vegetable by Reverse-Phase High-Performance Liquid Chromatography.

In the present study, the direct enantiomeric separation of hexythiazox enantiomers on Lux cellulose-1, Lux cellulose-2, Lux cellulose-3, Lux cellulose-4, Lux amylose-1 and Chirapak IC chiral columns were carefully investigated by reverse-phase high-performance liquid chromatography (RP-HPLC). Acetonitrile/water and methanol/water were used as mobile phase at a flow rate of 0.8 mL·min-1. The effects of chiral stationary phase, temperature, thermodynamic parameters, mobile phase component and mobile phase ratio on hexythiazox enantiomers separation were fully evaluated. Hexythiazox enantiomers received a baseline separation on the Lux cellulose-3 column with a maximum resolution of Rs = 2.09 (methanol/water) and Rs = 2.74 (acetonitrile/water), respectively. Partial separations were achieved on other five chiral columns. Furthermore, Lux amylose-1 and Chirapak IC had no separation ability for hexythiazox enantiomers when methanol/water was used as mobile phase. Temperature study indicated that the capacity factor (k) and resolution factor (Rs) decreased with column temperature increasing from 10 °C to 40 °C. The enthalpy (ΔH) and entropy (ΔS) involved in hexythiazox separation were also calculated and demonstrated the lower temperature contributed to better separation resolution. Moreover, the residue analytical method for hexythiazox enantiomers in the environment (soil and water) and vegetable (cucumber, cabbage and tomato) were also established with reliable accuracy and precision under reverse-phase HPLC condition. Such results provided a baseline separation method for hexythiazox enantiomers under reverse-phase conditions and contributed to an environmental and health risk assessment of hexythiazox at enantiomer level.

Soil fumigation alters adsorption and degradation behavior of pesticides in soil.

Many crops are produced using soil fumigation and chemical pesticides to control soil-borne fungi and bacterial diseases, nematodes and weeds. Fumigation of soil, however, may alter its ability to adsorb, degrade and volatilize pesticides, which can then change the potential for pesticides to leach into groundwater. Soil adsorption kinetics, Freundlich isothermal adsorption and pesticide degradation techniques were used to determine the potential for pesticides to pollute groundwater in fumigated soil. The effect on soil pesticide adsorption in different types of chloropicrin (CP) fumigated soils was also examined. We observed that the equilibrium adsorption (qe) decreased significantly at 24 h. Soil fumigation decreased the Freundlich Kf and Kfoc values, and increased the Freundlich exponent 1/n values, for pesticides in fumigated Beijing soil. Soil fumigation influenced the Kf of pendimethalin, oxyfluorfen and abamectin the most, which themselves had a larger Kf in untreated soil. This indicated that the greater the soil pesticide adsorption the greater the influence of a fumigation treatment on that pesticide. The Kf was decreased more in the Heilongjiang and Beijing CP-fumigated soils that had high organic carbon content compared to Hunan soil. Fumigation of the soil with CP extended the half-life values of fosthiazate (from 34.3 to 43.1 days) and azoxystrobin (from 52.9 to 64.2 days), which increased their potential to leach into groundwater. Famers should minimize the quantity of some pesticides applied to fumigated soil, or apply some pesticides 60 days after fumigation, in order to avoid ground water pollution when crops are grown in fields or greenhouses.

Identification and Quantitation of Four New 2-Alkylthiazolidine-4-carboxylic Acids Formed in Orange Juice by a Reaction of Saturated Aldehydes with Cysteine.

Despite several technological efforts to maximize the quality and shelf life of chilled stored not-from-concentrate orange juice, changes in the overall aroma profile might occur during storage. Besides the degradation of terpenoids, a loss of the aroma-active aldehydes, hexanal, octanal, nonanal, and decanal as well-as of 1-penten-3-one were recently confirmed as a major cause for the changes in the aroma profile of orange juice even during storage under aseptic conditions at 0 °C. To unravel the fate of the aroma-active aldehydes, model experiments were carried out considering the oxidation into the corresponding acids as well as a reaction with free amino acids present in orange juice. The oxidation into the acids could be confirmed by isotope labeling experiments; additionally, the reaction of the four aldehydes mentioned above with l-cysteine yielded four new compounds identified as 2-alkylsubstituted thiazolidine-4-carboxylic acids. Their quantitation in orange juice samples by newly developed stable isotope dilution assays revealed that these acids were already present in the fresh samples but were considerably increased after storage. Labeling experiments in orange juice administered with either labeled octanal or labeled cysteine confirmed that the reaction quickly occurs in the juice. The data contribute another puzzle piece to the loss of aroma-active aldehydes during orange juice storage, which may also be relevant in other foods.

Lipophilicity Evaluation of Some N1-Arylidene-Thiosemicarbazones and 1,3,4-Thiadiazolines with Antimicrobial Activity.

A: The lipophilic character of 20 previously reported compounds-derivatives of N 1 -arylidene-thiosemicarbazone (series ) and their corresponding 1,3,4-thiadiazolines (series )-has been determined by reversed-phase thin-layer chromatography, using i -propanol-water mixtures as eluents. Principal component analysis (PCA) allowed an objective estimation of the retention behavior of the tested compounds and also afforded to obtain a 2D scatterplot, described by the first two principal components, which had the effect of separating the compounds from each other most effectively. With the use of clustering methods ( K -means clustering) based on PCA data, the studied compounds were grouped into two congeneric classes. When comparing the obtained lipophilicity parameters' values with the antibacterial properties of the tested compounds, we noticed that the lipophilic character had no significant influence on their growth inhibitory activity.

A robust LC-MS/MS method for the determination of pidotimod in different biological matrixes and its application to in vivo and in vitro pharmacokinetic studies.

Pidotimod, (R)-3-[(S)-(5-oxo-2-pyrrolidinyl) carbonyl]-thiazolidine-4-carboxylic acid, was frequently used to treat children with recurrent respiratory infections. Preclinical pharmacokinetics of pidotimod was still rarely reported to date. Herein, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to determine pidotimod in rat plasma, tissue homogenate and Caco-2 cells. In this process, phenacetin was chosen as the internal standard due to its similarity in chromatographic and mass spectrographic characteristics with pidotimod. The plasma calibration curves were established within the concentration range of 0.01-10.00µg/mL, and similar linear curves were built using tissue homogenate and Caco-2 cells. The calibration curves for all biological samples showed good linearity (r>0.99) over the concentration ranges tested. The intra- and inter-day precision (RSD, %) values were below 15% and accuracy (RE, %) was ranged from -15% to 15% at all quality control levels. For plasma, tissue homogenate and Caco-2 cells, no obvious matrix effect was found, and the average recoveries were all above 75%. Thus, the method demonstrated excellent accuracy, precision and robustness for high throughput applications, and was then successfully applied to the studies of absorption in rat plasma, distribution in rat tissues and intracellular uptake characteristics in Caco-2 cells for pidotimod.

Quantitation of Thioprolines in Grape Wine by Isotope Dilution-Liquid Chromatography-Tandem Mass Spectrometry.

Cysteine reacts with reactive carbonyls to form thioprolines, which have been demonstrated to possess various pharmaceutical properties. Therefore, thioproline formation is considered as a major detoxification pathway for carcinogenic reactive carbonyls. In this study, we report the initial identification of thiazolidine-4-carboxylic acid (1) and 2-methylthiazolidine-4-carboxylic acid (2), two very common thioprolines, formed by reacting formaldehyde and acetaldehyde with cysteine in grape wine samples. We have developed an isotope dilution-liquid chromatography-tandem mass spectrometry method featuring high sensitivity (limit of detection of ≤1.5 ng/mL) and selectivity to quantitate compounds 1 and 2. The method after validated to be highly accurate (recovery of ≥92%) and precise [intraday relative standard deviation (RSD) of ≤4.1% and interday RSD of ≤9.7%] was applied to determine the varying compound 1 and 2 contents in grape wine samples. Results revealed the grape type and storage duration-dependent formation of thioprolines in grape wines. Overall, the results are expected to facilitate compound-dependent investigations of the health benefits of grape wine, and our findings could be adopted to predict the age of grape wine.

Analysis of 2-oxothiazolidine-4-carboxylic acid by hydrophilic interaction liquid chromatography: application for ocular delivery using chitosan nanoparticles.

Oxidative damage due to low levels of glutathione (GSH) is one of the main causes of cataract formation. It has been reported that 2-oxothiazolidine-4-carboxylic acid (OTZ), a cysteine prodrug, can increase the cellular level of GSH. Currently, there is no analytical method to separate and quantify OTZ from aqueous humour samples for cataract research. The present study aims to develop and validate a hydrophilic interaction liquid chromatography (HILIC) method for the quantification of OTZ in simulated aqueous humour (SAH). The developed method was validated according to FDA guidelines. Accuracy, precision, selectivity, sensitivity, linearity, lower limit of quantification (LLOQ), lower limit of detection (LLOD) and stability were the parameters assessed in the method validation. The developed method was found to be accurate and precise with LLOQ and LLOD of 200 and 100 ng/mL, respectively; method selectivity was confirmed by the absence of any matrix interference with the analyte peak. The constructed calibration curve was linear in the range of 0.2-10 µg/mL, with a regression coefficient of 0.999. In addition, the OTZ was found to be stable in SAH after three freeze/thaw cycles. Chitosan nanoparticles loaded with OTZ were formulated by the ionic gelation method. The nanoparticles were found to be uniform in shape and well dispersed with average size of 153 nm. The in vitro release of OTZ from the nanoparticles was quantified using the developed analytical method over 96 h. Permeation of OTZ through excised bovine cornea was measured using HILIC. The lag time and the flux were 0.2 h and 3.05 µg/cm(2) h, respectively.

Sensitive assay of hexythiazox residue in citrus fruits using gold nanoparticles-catalysed luminol-H2O2 chemiluminescence.

A new sensitive chemiluminescence (CL) procedure for the detection of hexythiazox (HXTZ) is presented, based on the quenching effect of the HXTZ in the luminol-H2O2 system using gold nanoparticles (GNPs) as a catalyst. The Box-Behnken design matrix and response surface methodology (RSM) have been applied in designing the experiments for studying the interactive effects of the three most important variables pH, luminol, and H2O2 concentrations on the CL intensity of luminol catalysed by GNPs. Under the optimal conditions, the CL intensity was linear with HXTZ concentration in the range of 0.017-0.42 µg mL(-1), and the limit of detection (LoD) was 0.011 µg mL(-1). The procedure has been successfully applied to the detection of HXTZ residues in citrus fruits and water samples at trace levels. Mean recoveries obtained were between 84.0% and 95.3%, with a repeatability precision of <6%. Meanwhile, the possible mechanism of the inhibited CL intensity was discussed.

Identification and quantification of antitumor thioproline and methylthioproline in Korean traditional foods by a liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry.

A liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometric method (LC-APCI-MS/MS) has been developed for the sensitive determination of antitumor thioproline and methylthioproline from fermented foods. Thioproline and methylthioproline were derivatized in one step with ethyl chloroformate at room temperature. These compounds were identified and quantified in various traditional Korean fermented foods by LC-APCI-MS/MS. The concentration range of thioproline of each food was found for doenjang (0.011-0.032mg/kg), gochujang (0.010-0.038mg/kg), and ganjang (0.010-0.038mg/kg). Those of methylthioproline of each food was found for doenjang (0.098-0.632mg/kg), gochujang (0.015-0.112mg/kg), and ganjang (0.023-1.468mg/kg). A prolonged aging time leads to an increase in both the thioproline and methylthioproline contents, suggesting that the storage time plays a key role in the formation of thioproline and methylthioproline in Korean traditional foods. The results here suggest that thioproline and methylthioproline are related to the biological activities of traditional Korean fermented foods.

Dissipation of hexythiozox on beans pods by HPLC-DAD.

An effective analytical method for the residue analysis of a novel acaricide hexythiozox and its dissipation in beans pods were studied. Hexythiozox residues were extracted from beans pods samples and the extract was cleaned up according to QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method and determined by high-performance liquid chromatography with photodiode array detector (HPLC-DAD). At fortification levels of 0.1, 0.5, and 1.0 mg kg(-1) in Beans Pods, it was shown that recoveries ranged from 82.4 % to 89.6 % with relative standard deviation (RSD) of 6 %-9 %. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.02 and 0.06 mg kg(-1), respectively. The dissipation half-life time of hexythiozox residues in beans pods was 12.04 days. According to maximum residue limit (MRL) 0.5 mg kg(-1), the preharvest interval (PHI) of hexythiozox on beans pods was 10 days after the treatment. Based on the results of this study and the relevant residue regulation, hexythiozox residue levels will be acceptable when applied to beans pods in Egypt.

Persistence behavior of Fantac biostimulant in chili and soil under subtropical conditions.

The persistence of the biostimulant Fantac (N-ATCA) in chili and soil was evaluated at two application rates (0.25 and 0.50 microg g(-1)) by high-performance liquid chromatography. At 0.25 microg g(-1) Fantac persisted up to 3 days in soil and 5 days in chili. However, at 0.50 microg g(-1) Fantac residues persisted up to 5 days both in soil and chili. The dissipation of the biostimulant from soil and chili appeared to occur in a single phase and conformed to first-order kinetics. Half-lives of Fantac in soil and chili were calculated.

Symptoms and immunologic markers among vulcanization workers in rubber industries in southern Sweden.

OBJECTIVES: The aim of this study was to estimate the risk of symptoms and the possible derangement of levels of immunologic markers for contemporary Swedish rubber workers. Furthermore, the relation between exposure and these biomarkers of response was examined using urinary levels of 2-thiothiazolidine-4-carboxylic acid (TTCA), which reflect the exposure. METHODS: Included in the study were 166 exposed workers and 117 controls. Medical and occupational histories were obtained in structured interviews. Symptoms were recorded, and immunologic markers were analyzed in blood. Urinary levels of TTCA were determined by liquid chromatography tandem mass spectrometry. RESULTS: Compared with the controls, the exposed workers had increased risks of eye symptoms [odds ratio (OR) 3.0], nose bleeds (OR 4.0), burning and dry throat (OR 3.0), hoarseness (OR 2.4), severe dry cough (OR 3.8), nausea (OR 4.3), and headache (OR 2.5). When the exposed workers were divided into three groups according to the TTCA levels, the highest risks were observed among the exposed workers with intermediate TTCA levels. Furthermore, the exposed workers in all of the TTCA subgroups had elevated concentrations of total immunoglobulin G when compared with the controls. Elevated concentrations of leukocytes, neutrophils, and eosinophils were observed in the group with high TTCA levels. CONCLUSIONS: This work shows an increased risk of several symptoms and elevated levels of some immunologic markers among exposed workers in Swedish rubber industries. In addition, relationships between urinary levels of TTCA and some biomarkers of response were reported.

Leaching of the organophosphorus nematicide fosthiazate.

Fosthiazate is an organophosphorus nematicide which was recently included in Annex I of the Directive 91/414/EEC under the clause that it should be used with special care in soils vulnerable to leaching. Thus, the leaching of fosthiazate was investigated in columns packed with three different soils which represented situations of high (site 2), intermediate (site 1) and low (site 3) leaching potential. The recommended dose of fosthiazate was applied at the surface of the soil columns and fosthiazate fate and transport was investigated for the next two months. Fosthiazate concentrations in the leachate collected from the bottom of the columns packed with soil from site 2 exceeded 0.1 microgl(-1) in most cases. This soil was characterized as acidic, indicating longer fosthiazate persistence, with low organic matter content, indicating weak adsorption, thus representing a situation vulnerable to leaching. In contrast, the lowest concentrations of fosthiazate in the leachate were evident in the columns packed with soil from site 3. This soil was characterized as alkaline, indicating faster degradation, with higher organic matter content, indicating stronger adsorption, thus representing a situation not favoring leaching of fosthiazate. The highest concentration of fosthiazate in the leachate from the columns packed with soil from site 2 was 3.44 microgl(-1) compared to 1.17 and 0.16 microgl(-1), which were the corresponding maximum values measured in columns packed with soil from sites 1 and 3, respectively. The results of the current study further suggest that fosthiazate is mobile in soil and can leach under conducive soil conditions like acidic soils with low organic matter content.

Pesticide residues in heterogeneous plant populations, a model-based approach applied to nematicides in banana (Musa spp.).

Nematicides are widely used to control plant-parasitic nematodes in intensive export banana (Musa spp.) cropping systems. Data show that the concentration of fosthiazate in banana fruits varies from zero to 0.035 g kg-1, under the maximal residue limit (MRL=0.05 mg kg-1). The fosthiazate concentration in fruit is described by a Gaussian envelope curve function of the interval between pesticide application and fruit harvest (preharvest interval). The heterogeneity of phenological stages in a banana population increases over time, and thus the preharvest interval of fruits harvested after a pesticide application varies over time. A phenological model was used to simulate the long-term harvest dynamics of banana at field scale. Simulations show that the mean fosthiazate concentration in fruits varies according to nematicide application program, climate (temperature), and planting date of the banana field. This method is used to assess the percentage of harvested bunches that exceed a residue threshold and to help farmers minimize fosthiazate residues in bananas.

[Structures of leucogen and its related substances in solution state].

AIM: To study the four diastereomers of leucogen and structure of the related substances. METHODS: LC-DAD, LC-MS/MS and LC- 1H NMR were used. LC was carried out with a Phenomnex Luna C18 (250 mm x 4.60 mm ID, 5 microm) column and a mobile phase of water-acetonitrile-glacial acetic acid (58:42: 0.3). RESULTS: The structures of leucogen and its related substances were identified. Leucogen and the related substances were found to have four diastereomers in solution state separately. The stability and transformation of the four diastereomers were analyzed and 3R4S5R was found to be more stable than the others according to quantum calculations. CONCLUSION: Leucogen have four diastereomers in solution state and it can transform from one diastereomer to the others, and the 3R4S5R is more stable than the others.

Direct trapping of formaldehyde formed via oxidative N-demethylation of N,N-dialkylarylamines by Bacillus megaterium using cysteamine derivatization.

Oxidative N-demethylation was measured by incubation experiments using Bacillus megaterium isolated from topsoil as a biocatalyst for the N-demethylation of the N,N-dialkylarylamines N,N-dimethylaniline and N-ethyl-N-methylaniline. Formed formaldehyde, normally difficult to analyse in biological systems because of further metabolization, was successfully trapped and converted into thiazolidine by addition of cysteamine into the incubation media. Studies using N,N-di-(trideutero-methyl)-aniline and N-ethyl-N-(trideuteromethyl)-aniline as well as N,N-di-[methyl-(13)C]-aniline and N-ethyl-N-[methyl-(13)C]-aniline were performed to confirm that the N-demethylation proceeds via formaldehyde.

Multiresidue determination of pesticides in plants by high-performance liquid chromatography following gel permeation chromatographic clean-up.

Gel permeation chromatography was applied as a clean-up step in a HPLC multiresidue method for the determination of several pesticides in plants, not amenable to analysis by GC. The pesticides investigated were diflubenzuron, triflumuron, clofentezine, hexythiazox and flufenoxuron. The clean-up technique resulted in a good separation of analytes from co-extractive matrix compounds. Complete HPLC separation of all pesticides was achieved under the conditions selected. The analytical procedure was characterized with high accuracy and precision and acceptable sensitivity to meet requirements for monitoring these pesticides in crops.