Development of a specific and sensitive method for the detection of glyphosate pesticide and its metabolite in tea using dummy molecularly imprinted solid-phase extraction coupled with liquid chromatography-tandem quadrupole mass spectrometry.

Glyphosate, a widely used herbicide, and its primary metabolite aminomethyl phosphonic acid have been found to cause environmental and ecological issues and threaten human health. The conventional pretreatment method was insufficient for the extraction, concentration, and enrichment of trace substances, resulting in poor specificity. Thus, our objective was to develop a method for glyphosate pesticide detection using dummy molecularly imprinted solid-phase extraction (DMI-SPE) combined with liquid chromatography-tandem quadrupole mass spectrometry (DMI-SPE-LC/MS/MS). The sol-gel method was used to prepare the molecularly imprinted material, using glyphosine as the dummy template molecule, to achieve specific adsorption to glyphosate and reduce costs. The optimized polymerization conditions achieved maximum adsorption of 28.6 µg/mg glyphosate by the molecularly imprinted material. The established DMI-SPE-LC/MS/MS method was used to detect glyphosate and its metabolite (aminomethyl)phosphonic acid in tea. The concentration ranges of glyphosate and (aminomethyl)phosphonic acid (from 0.05 to 4 µg/mL) were linear with correlation coefficients of 0.999 and 0.991, respectively. The recoveries of (aminomethyl)phosphonic acid at three spiked levels ranged from 79.95% to 83.74%, with RSDs between 6.40% and 7.45%, while the recoveries of glyphosate ranged from 98.69% to 106.26%, with RSDs between 0.91% and 1.18%. Our results demonstrate that the developed DMI-SPE-LC/MS/MS method achieves high sensitivity and specific detection of glyphosate and its metabolite (aminomethyl)phosphonic acid in tea matrices.

Evaluation of pyraoxystrobin bioconcentration in zebrafish (Danio rerio) using modified QuEChERS extraction.

Pyraoxystrobin is a novel strobilurin fungicide that is widely used on many crops. The high log Kow of pyraoxystrobin implies that it tends to accumulate in aquatic organisms. This study optimized the sorbents of QuEChERS (quick, easy, cheap, effective, rugged, and safe) using 13C-labelled pyraoxystrobin as the internal standard (IS). It has been established a QuEChERS-LC-MS/MS IS method to study the bioconcentration and elimination of pyraoxystrobin in zebrafish (Danio rerio). The results indicated that the method had satisfactory linearity between 0.234 and 15 µg L-1 (R2 = 0.9996). The limits of detection (LOD) and quantification (LOQ) for pyraoxystrobin were 0.01 and 0.03 µg L-1, respectively. The LOQs of the method for water and zebrafish were 0.05 µg L-1 and 0.01 mg/kg, respectively. The mean recovery of pyraoxystrobin in zebrafish and water at fortification levels of 0.01-0.3 mg kg-1 and 0.05-1.5 µg L-1 ranged from 98.31 to 105.61% and 101.87 to 108.48%, respectively, with a % RSD (relative standard deviation) of 0.94-3.57%. The bioconcentration has been evaluated. The bioconcentration factors for pyraoxystrobin in zebrafish were 1,792 and 3,505 after exposure to 0.5 µg L-1 for 168 h and 0.05 µg L-1 for 216 h, respectively. The half-life of pyraoxystrobin in zebrafish was 9.0-9.5 d.

Euonymus japonicus phyllosphere microbiome is significantly changed by powdery mildew.

Euonymus japonicus Thunb. is a woody and ornamental plant popular in China, Europe and North America. Powdery mildew is one of the most serious diseases that affect E. japonicus growth. In this study, the diseased and apparently healthy leaves were collected from E. japonicus planted in a greenbelt in Beijing, and the effect of powdery mildew on the epiphytic microbial community was investigated by using Illumina sequencing. The results showed that the healthy leaves (HL) harbored greater bacterial and fungal diversity than diseased leaves (DL). Furthermore, both bacterial and fungal communities in DL exhibited significantly different structures from those in HL. The relative abundance of several bacterial phyla (Proteobacteria and Firmicutes) and fungal phyla (Ascomycota and Basidiomycota) were altered by powdery mildew. At the genus level, most genera decreased as powdery mildew pathogen Erysiphe increased, while the genera Kocuria and Exiguobacterium markedly increased. Leaf properties, especially protein content was found to significantly affect beta-diversity of the bacterial and fungal community. Network analysis revealed that positive bacterial interactions in DL were stronger than those in HL samples. Insights into the underlying the indigenous microbial phyllosphere populations of E. japonicus response to powdery mildew will help in the development of methods for controlling plant diseases.

Dibutyl phthalate contamination remolded the fungal community in agro-environmental system.

Dibutyl phthalate (DBP) is a typical soil contaminant that is widely used as plasticizer in modern agricultural production. In this study, an experiment was conducted to evaluate fungal community succession in a soil-vegetable ecosystem under different DBP concentrations. By using high-throughput sequencing of the ribosomal internal transcribed spacer (ITS) region, it was shown that DBP contamination caused significant changes to the soil fungal community, in terms of both α and ß diversities. The largest changes in fungal α and ß diversities were detected under 50 mg/kg DBP concentration at the first day of addition. The bulk soils, rhizosphere soils and the phyllosphere harbored different fungal communities, while the abundance of saprotrophs and plant pathogens in the phyllosphere have been increased under DBP contamination. From correlation analysis and partial Mantel test, the change in fungal community α diversity was the result of multiple factors (DBP concentration, bacterial community and soil properties) while the ß diversity of fungal community was mainly co-varied with the bacterial community after DBP contamination. Moreover, molecular ecological network analysis demonstrated that DBP contamination was detrimental to mutualistic relationships among fungal species and destabilized the network structure. Overall, the fungal communities in soils and around vegetables were largely remolded by DBP contamination that provides new insight into DBP contamination impacts on agricultural ecosystems.

Synthesis of matrinic amide derivatives containing 1,3,4-thiadiazole scaffold as insecticidal/acaricidal agents.

In continuation of our program aimed at the development of natural product-based pesticidal agents, a series of matrinic amide derivatives containing 1,3,4-thiadiazole scaffold were prepared, and their insecticidal and acaricidal activities were evaluated against Mythimna separata and Tetranychus cinnabarinus. Some compounds exhibited potent insecticidal and acaricidal activities. It suggested that R1 as a nitro group and R2 as a fluorine atom, were important for the insecticidal activity; R1 as the electron-donating groups and R2 as the methyl group, were necessary for the acaricidal activity.

Gordonia phthalatica sp. nov., a di-n-butyl phthalate-degrading bacterium isolated from activated sludge.

A phthalate esters-degrading bacterial strain, designated QH-11T, was isolated from an activated sludge wastewater treatment plant in Beijing, PR China. The cells were aerobic, Gram-stain-positive, non-motile, catalase-positive, oxidase-negative, short rods and formed white colonies on trypticase soy agar. This isolate contained meso-diaminopimelic acid as the diagnostic diamino acid and whole-cell hydrolysates contained arabinose and ribose. Diphosphatidylglycerol and phosphatidylethanolamine were the predominant polar lipids. According to the results of full-length of 16S rRNA gene sequence analysis, QH-11T represented a member of the genus Gordonia and showed the highest sequence similarity to Gordonia hydrophobica DSM 44015T (99.2 %), but was distinguishable by a low level of DNA-DNA relatedness (37.8 %). Genome-based comparisons indicated a clear distinction from the top ten most similar type strains (16S rRNA gene sequence) with pairwise average nucleotide identities (ANI) between 74.6 and 83.4 %. The predominant respiratory quinone was MK-9(H2), the mycolic acids present had 56 to 62 carbon atoms, and the major fatty acids were C16 : 0 (33.3 %), C17 : 1ω8c (23.4 %) and C18 : 1ω9c (17.9 %). The DNA G+C content was 68.0 mol%. On the basis of the results of DNA-DNA hybridization, ANI and physiological and biochemical tests, it is proposed that QH-11T represents a novel species of the genus Gordonia, for which the name Gordonia phthalatica sp. nov. is proposed. The type strain is QH-11T (CICC 24107T =KCTC 39933T).

Draft Genome Sequence of Sphingobium yanoikuyae TJ, a Halotolerant Di-n-Butyl-Phthalate-Degrading Bacterium.

Sphingobium yanoikuyae TJ is a halotolerant di-n-butyl-phthalate-degrading bacterium, isolated from the Haihe estuary in Bohai Bay, Tianjin, China. Here, we report the 5.1-Mb draft genome sequence of this strain, which will provide insights into the diversity of Sphingobium spp. and the mechanism of phthalate ester degradation in the estuary.

Characterization and Genomic Analysis of a Highly Efficient Dibutyl Phthalate-Degrading Bacterium Gordonia sp. Strain QH-12.

A bacterial strain QH-12 isolated from activated sludge was identified as Gordonia sp. based on analysis of 16S rRNA gene sequence and was found to be capable of utilizing dibutyl phthalate (DBP) and other common phthalate esters (PAEs) as the sole carbon and energy source. The degradation kinetics of DBP under different concentrations by the strain QH-12 fit well with the modified Gompertz model (R² > 0.98). However, strain QH-12 could not utilize the major intermediate product phthalate (phthalic acid; PA) as the sole carbon and energy source, and only a little amount of PA was detected. The QH-12 genome analysis revealed the presence of putative hydrolase/esterase genes involved in PAEs-degradation but no phthalic acid catabolic gene cluster was found, suggesting that a novel degradation pathway of PAEs was present in Gordonia sp. QH-12. This information will be valuable for obtaining a more holistic understanding on diverse genetic mechanisms of PAEs-degrading Gordonia sp. strains.

Analysis of the urine proteome of human contrast-induced kidney injury using two-dimensional fluorescence differential gel electrophoresis/matrix-assisted laser desorption time-of-flight mass spectrometry/liquid chromatography mass spectrometry.

BACKGROUND: The pathogenesis of contrast-induced nephropathy (CIN) remains unclear and is defined by changes in serum creatinine which is not a sensitive biomarker for acute kidney injury. Search for differentially expressed urinary protein or peptide could contribute to further understanding of the disease and may provide new biomarkers. METHODS: This is a small sample research. Urine samples were obtained from patients who underwent percutaneous coronary intervention and were labeled with 3 different fluorescent dyes. After 2-dimensional electrophoresis was run, the differentially regulated spots were picked out and identified by mass spectrometry. Another 31 patients were used as validation group. RESULTS: Among 56 significantly changed spots, mannose-binding lectin (MBL) and MBL-associated serine protease 2 were both significantly upregulated. Compared to the baseline value, urinary MBL was significantly increased in the CIN group (2.08, 1.42-5.72, vs. 1.09, 0.516-1.411; p < 0.01). Postprocedure urinary MBL in the CIN group was also significantly higher than that in the non-CIN group (2.08, 1.42-5.72, vs. 1.057, 0.738-1.885; p < 0.05). CONCLUSION: The studies suggested that MBL may be associated with contrast-induced acute kidney injury. It leads to an attempt to define a new pathogenesis and a novel biomarker for CIN.

The antigen for Hep Par 1 antibody is the urea cycle enzyme carbamoyl phosphate synthetase 1.

Hepatocyte paraffin 1 (Hep Par 1), a murine monoclonal antibody, is widely used in surgical pathology practice to determine the hepatocellular origin of neoplasms. However, identity of the antigen for Hep Par 1 is unknown. The aim of this study was to characterize the Hep Par 1 antigen. To identify the antigen, immunoprecipitation was used to isolate the protein from human liver tissue, and a distinct protein band was detected at approximately 165 kDa. The protein band was also present in small intestinal tissue, but was not present in several other non-liver tissues nor in three human hepatocellular carcinoma cell lines, Huh-7, HepG2, and LH86. The protein was purified and analyzed by mass spectrometry. It was identified as carbamoyl phosphate synthetase 1 (CPS1). CPS1 is a rate-limiting enzyme in urea cycle and is located in mitochondria. We demonstrated that hepatoid tumors (gastric and yolk sac) were immunoreactive with both Hep Par 1 antibody and anti-CPS1 antibody, further confirming the results of mass spectrometric analysis. We found that the three human hepatocellular carcinoma cell lines do not express either CPS1 RNA or protein. We confirmed that the gene was present in these cell lines, suggesting that suppression of CPS1 expression occurs at the transcriptional level. This finding may have relevance to liver carcinogenesis, since poorly differentiated hepatocellular carcinomas exhibit poor to absent immunoreactivity to Hep Par 1. In conclusion, we have identified the antigen for Hep Par 1 antibody as a urea cycle enzyme CPS1. Our results should encourage further investigation of potential role that CPS1 expression plays in liver pathobiology and carcinogenesis.

Isotopologue distributions of peptide product ions by tandem mass spectrometry: quantitation of low levels of deuterium incorporation.

Protonated molecular peptide ions and their product ions generated by tandem mass spectrometry appear as isotopologue clusters due to the natural isotopic variations of carbon, hydrogen, nitrogen, oxygen, and sulfur. Quantitation of the isotopic composition of peptides can be employed in experiments involving isotope effects, isotope exchange, and isotopic labeling by chemical reactions and in studies of metabolism by stable isotope incorporation. Both ion trap and quadrupole-time of flight mass spectrometry are shown to be capable of determining the isotopic composition of peptide product ions obtained by tandem mass spectrometry with both precision and accuracy. Tandem mass spectra of clusters of isotopologue ions obtained in profile mode are fit by nonlinear least squares to a series of Gaussian peaks which quantify the Mn/M0 values which define the isotopologue distribution (ID). To determine the isotopic composition of product ions from their ID, a new algorithm that predicts the Mn/M0 ratios and obviates the need to determine the intensity of all of the ions of an ID is developed. Consequently a precise and accurate determination of the isotopic composition of a product ion may be obtained from only the initial values of the ID, however, the entire isotopologue cluster must be isolated prior to fragmentation. Following optimization of the molecular ion isolation width, fragmentation energy, and detector sensitivity, the presence of isotopic excess (2H, 13C, 15N, 18O) is readily determined within 1%. The ability to determine the isotopic composition of sequential product ions permits the isotopic composition of individual amino acid residues in the precursor ion to be determined.

Human kinetics of orally and intravenously administered low-dose 1,2-(13)C-dichloroacetate.

Dichloroacetate (DCA) is a putative environmental hazard, owing to its ubiquitous presence in the biosphere and its association with animal and human toxicity. We sought to determine the kinetics of environmentally relevant concentrations of 1,2-(13)C-DCA administered to healthy adults. Subjects received an oral or intravenous dose of 2.5 microg/kg of 1,2-(13)C-DCA. Plasma and urine concentrations of 1,2-(13)C-DCA were measured by a modified gas chromatography-tandem mass spectrometry method. 1,2-(13)C-DCA kinetics was determined by modeling using WinNonlin 4.1 software. Plasma concentrations of 1,2-(13)C-DCA peaked 10 minutes and 30 minutes after intravenous or oral administration, respectively. Plasma kinetic parameters varied as a function of dose and duration. Very little unchanged 1,2-(13)C-DCA was excreted in urine. Trace amounts of DCA alter its own kinetics after short-term exposure. These findings have important implications for interpreting the impact of this xenobiotic on human health.

A GC-MS/MS method for the quantitative analysis of low levels of the tyrosine metabolites maleylacetone, succinylacetone, and the tyrosine metabolism inhibitor dichloroacetate in biological fluids and tissues.

We developed a sensitive method to quantitate the tyrosine metabolites maleylacetone (MA) and succinylacetone (SA) and the tyrosine metabolism inhibitor dichloroacetate (DCA) in biological specimens. Accumulation of these metabolites may be responsible for the toxicity observed when exposed to DCA. Detection limits of previous methods are 200 ng/mL (1.2 pmol/microL) (MA) and 2.6 microg/mL (16.5 pmol/microL) (SA) but the metabolites are likely present in lower levels in biological specimens. To increase sensitivity, analytes were extracted from liver, urine, plasma and cultured nerve cells before and after dosing with DCA, derivatized to their pentafluorobenzyl esters, and analyzed via GC-MS/MS.

Simultaneous determination of trace levels of nine haloacetic acids in biological samples as their pentafluorobenzyl derivatives by gas chromatography/tandem mass spectrometry in electron capture negative ion chemical ionization mode.

Haloacetic acids (HAAs) are environmentally and medically important chemicals. No analytical method is currently available to analyze EPA-regulated HAAs in biological samples at environmentally relevant low concentrations. Clinical studies of this class of chemicals are also limited by the lack of analytical techniques of high sensitivity and precision. We now report a new analytical method using gas chromatography/ion trap mass spectrometry for quantifying nine HAAs present inplasma, urine, and water at picogram per milliliter levels. The derivatization reactions of HAAs with pentafluorobenzyl bromide were optimized and detection with an electron capture negative ion chemical ionization mode was employed to enhance the sensitivity. Selected ion monitoring and selected reaction monitoring methods were utilized for quantitation. The detection limits of HAAs in plasma, urine, and water were 25-1000 pg/mL. Accuracies varied from 86.6 to 118.1% (intraday) and 81.7 to 119.6% (interday). Precisions (CV) varied from 0.9 to 19.9% (intraday) and 0.8 to 19.8% (interday), and linearities (r2) varied from 0.9732 to 0.9998 (intraday) and 0.9422 to 0.9987 (interday), respectively. Methyl tertbutyl ether and diethyl ether provided the highest extraction recoveries for the HAAs (74.9-107.2%). The method was applied successfully to a kinetic investigation of low levels of HAAs in humans consuming chlorinated drinking water.