Ren-Xiang Mou

Simultaneous determination of 15 phenylurea herbicides in rice and corn using HPLC with fluorescence detection combined with UV decomposition and post-column derivatization.

A method was developed for the simultaneous determination of 15 phenylurea herbicides (fenuron, tebuthiuron, metoxuron, monuron, chlortoluron, fluometuron, isoproturon, diuron, monolinuron, metobromuron, buturon, siduron, linuron, chlorbromuron, and neburon) in rice and corn samples by HPLC with fluorescence detection combined with UV decomposition and post-column derivatization. After extraction with acetonitrile and evaporation, the herbicides were redissolved in n-hexane and purified on a Florisil solid-phase extraction column. HPLC separation was carried out on a C18 column with water-acetonitrile gradient elution. UV decomposition was carried out under a 254-nm UV lamp. The method was evaluated in terms of the limits of detection and quantification. The linearity was satisfactory, with a correlation coefficient of >0.9980. Precision and recovery studies were evaluated at three concentration levels for each matrix. Good precision was obtained, with relative standard deviation in the range 1.5-9.6% for spiked rice samples and 0.9-9.9% for spiked corn samples. Recovery (n=6) ranged between 75.3% and 104.3% for rice and between 75.0% and 105.1% for corn. The intra-day precision (n=5) for the 15 herbicides in rice and corn samples spiked at an intermediate level was between 1.5% and 7.1%, and the inter-day precision over 10 days (n=10) was between 6.4% and 15.6%.

Characterization of cadmium-resistant bacteria and their potential for reducing accumulation of cadmium in rice grains

Cadmium (Cd) pollution is a serious widespread environmental problem that not only destroys the microbial ecology of soil and decreases crop production, but also poses a serious risk to human health. Many methods have been used for the remediation of Cd pollution but none of these is totally satisfactory. Microbial remediation strategies have attracted increasing interest since they are environmentally friendly and cost-effective. In the present study, three Cd-resistant bacteria were isolated and evaluated for potential application in Cd bioremediation. Based on their morphological, physiological and biochemical characteristics, together with 16S rDNA gene sequence analyses, bacteria were identified as Stenotrophomonas acidaminiphila (2#), Pseudomonas aeruginosa (9#) and Delftia tsuruhatensis (12#). Pseudomonas aeruginosa showed very high tolerance to metals, especially Cd (2200mg/L), Zn (1800mg/L) and Pb (1200mg/L), and is thought to be a multi-metal-resistant bacterium. Pseudomonas aeruginosa was also sensitive to 13 different antibiotics. The effects of the bacterial strains on the growth of rice plants and their ability to reduce Cd accumulation from Cd-contaminated soils in pot experiments were also evaluated. For Oryza sativa L. A grown in contaminated soil (3mg/kg Cd), the accumulation of Cd was decreased by 31.2 and 25.5% in brown rice and polished rice, respectively, by strain 9#; Pseudomonas aeruginosa was more effective in reducing Cd accumulation in rice grains than a mixture of strains. For Oryza sativa L. B, a mixture of strains acting synergistically was more effective than a single strain in reducing Cd accumulation; treatment with mixed strains (strains+3mg/kg Cd) resulted in 41.3, 35.9, and 32.6% reductions in Cd accumulation in unhulled rice, brown rice and polished rice, respectively. Although different results were obtained for two rice varieties, it can still be concluded that Cd-resistant bacteria are suitable for reducing Cd accumulation in rice grains and show potential for bioremediation of Cd-contaminated soils.

Profiling of phytohormones and their major metabolites in rice using binary solid-phase extraction and liquid chromatography-triple quadrupole mass spectrometry

A high-throughput method was developed using liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) for the profiling and quantification of 43 phytohormones and their major metabolites, including auxins, abscisic acid, jasmonic acid, salicylic acid, cytokinins and gibberellins in a single sample extract. Considerable matrix effects (MEs) were observed (with most ME values in the range of 29%-84%, but maximum MEs of more than 115%, even up to 206%, existed) in sample extracts for most of the compounds studied. The application of the proposed binary solid-phase extraction using polymer anion and polymer cation exchange resins, was performed to purify 25 acidic and 18 alkaline phytohormones and their major metabolites prior to the LC-MS/MS analysis, which markedly reduced the MEs to acceptable levels, with ME values in the range of ±15%. Moreover, all of the isomers of cytokinins and their metabolites were fully separated on a sub-2μm particle C18 reverse-phase column with the optimized mobile phase consisting of methanol and 5mM ammonium formate. The method showed good linearity for all 43 analytes with regression coefficients (R(2))>0.991. Limits of detection ranged from 0.19 to 7.57 fmol for auxin, gibberellins, abscisic acid and their metabolites, 29.7 fmol for jasmonic acid, 18.1 fmol for salicylic acid, and from 0.03 to 0.31 fmol for cytokinins and their metabolites. The mean recoveries for all of the analytes were from 70.7 to 118.5%, and the inter-day precisions (n=6) were less than 18.7%, with intra-day precisions (n=6) within 25.4%. Finally, 20 compounds were successfully quantified in rice sample profiles using the proposed method, which will greatly facilitate the understanding of hormone-related regulatory networks that influence rice growth and development. To our knowledge, there are limited reports that measure this level of phytohormone species in rice samples using a single analysis.

A novel method for the simultaneous analysis of seven biothiols in rice (Oryza sativa L.) using hydrophilic interaction chromatography coupled with electrospray tandem mass spectrometry.

Analysis of biothiols is still problematic, due to their high polarity, oxidation sensitivity and time-consuming sample preparation. In this paper, a direct, rapid and sensitive method was developed for simultaneous quantification of unbound cysteine (Cys), glutathione (GSH) and phytochelatins (PCs) in rice leaf, stem and root samples by hydrophilic interaction chromatography coupled with electrospray tandem mass spectrometry (HILIC-MS/MS). Homogenized samples were extracted with water containing 50mM dithiothreitol, without derivatization and further clean-up, and the extracts were injected directly onto an Xbridge Amide-HILIC column (3.5μm, 150mm×2.1mm i.d.). The best chromatographic separation and MS sensitivity was achieved using a linear gradient elution with 10mM aqueous ammonium formate and acetonitrile as the mobile phase. In MS/MS mode the detection limit (S/N≥3) of seven biothiols was 3-105nM. Good linearities were observed (r>0.995) with linear dynamic range at least over three orders of magnitude. Recoveries for most analytes were within the range of 77-128%, with relative standard deviations less than 18.2%. The intra-day precision (n=7) was 6.1-11.7%, and the inter-day precision over 15 d (n=15) was 8.5-16.3% for all biothiols. The optimized HILIC-MS/MS method was applied to study the influence of different cadmium (Cd) concentrations (0, 1 and 50μM) on contents of Cys, GSH and PC2-6 in rice tissue. With increasing Cd concentrations in nutrient solutions, contents of PC2-4 in rice roots increased but contents of Cys and GSH decreased. Contents of PC2-4 in both rice leafs and stems increased markedly at high dose Cd (50μM) treatment compared with controls, compared with low Cd concentrations (1μM). However, both PC5 and PC6 were not detected throughout the stress experiment.

Analysis of ustiloxins in rice using polymer cation exchange cleanup followed by liquid chromatography–tandem mass spectrometry

Ustiloxins are cyclopeptide mycotoxins produced by the pathogenic fungus Ustilaginoidea virens of rice false smut. Quantification of ustiloxins is essential to assess the food safety of rice infected by rice false smut disease. This paper describes a sensitive method for the simultaneous quantification of ustiloxins A, B, C, D and F in rice grains using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Since notable matrix enhancement effects (21%-78%) occurred for all of the target analytes (except for ustiloxin A), several solid phase extraction materials were tested for their ability to retain ustiloxins from aqueous solutions prior to the LC-MS/MS analysis, including C18 sorbents, polymer anion exchange sorbents resin (PAX), and polymer cation exchange resin (PCX). The PCX resin was adopted due to its higher extraction capability and selectivity for all targets compared to others, and in this case, almost no matrix effects (-5% to 8%) were observed for all of the ustiloxins monitored. The developed method reached limits of quantification of 0.2-2ngg-1, and linearity was statistically verified over two orders of magnitude with regression coefficients (R2)>0.991. The mean recoveries were from 85% to 109%, and the inter-day precisions (n=11) were less than 16%, with intra-day precisions (n=6) within 12%. Analysis of samples showed that ustiloxin A was the dominant species, with the content ranging from 5.5 to 273.8ngg-1, followed by ustiloxin B (≤88.7ngg-1), while concentrations of ustiloxins C, D and F were slightly lower (≤43.2ngg-1). To our knowledge, this is the first report on the determination and analysis of five ustiloxins simultaneously in a single analysis.

Nickel in milled rice (Oryza sativa L.) from the three main rice-producing regions in China

ABSTRACT Nickel (Ni) concentrations in milled rice obtained from China and their variations among different provinces and varieties, as well as associated health risks, were investigated. Results showed that the mean Ni concentration in milled rice was 0.49 ± 0.51 mg/kg, which was much higher than reported in United Kingdom, French and Iranian cereals. There were significant variations (P < 0.05) of Ni concentrations in milled rice among different provinces and among varieties in the same province. According to the dietary risk assessment, the mean values of the target hazard quotient for chronic risk ranged from 1.24 to 1.46 for 2–4, 4–7 and 7–11-year-old children, and all values of margin of exposure for hypersensitivity risk were considerably below 10 for all age groups, indicating that the current dietary exposure to Ni in rice is of concern for 2–11-year-old children and Ni-sensitised individuals. It is essential to establish a continuous monitoring programme to control Ni contamination in rice.

Characterization of the phytochelatins and their derivatives in rice exposed to cadmium based on high-performance liquid chromatography coupled with data-dependent hybrid linear ion trap orbitrap mass spectrometry.

RATIONALE The identification and quantification of phytochelatins (PCs) and their derivatives are important to understand their roles in plant growth and development. A method couplling high-performance liquid chromatography with hybrid linear ion trap Orbitrap mass spectrometry (HPLC-LTQ/Orbitrap) was developed to screen PCs that have the same characteristic product ions. This approach was used for the fragmentation pattern analysis of glutathione (GSH) and PC standards, which allowed identification of the fragmentation pathways of their derivatives isolated from rice roots, stems and leaves. METHODS In this study, we developed a method to detect and identify PCs and their derivatives in rice based on HPLC/LTQ-Orbitrap. Spectrum interpretation and MS/MS fragmentation patterns of PCs provide sufficient information to discover the novel PC derivatives. This approach includes precursor ion scan and product ion scan to detect and character the novel PC derivatives. RESULTS Based on HCD-MS/MS fragmentation patterns, four PCs and 18 PC derivatives were identified. Among them, seven PC derivatives, i.e., iso-PC2 (Asn), iso-PC3 (Asn), iso-PC2 (Cys), des-γGlu-iso-PC3 (Ser), des-Cys-iso-PC2 (Glu), des-Cys-iso-PC3 (Glu) and des-Cys-iso-PC4 (Glu), have not been previously reported. This method was validated by profiling GSH, PCs and PC derivatives in rice. Preliminary results revealed that PCs and their derivatives, except GSH, are markedly induced by Cd treatment. CONCLUSIONS The HPLC/LTQ-Orbitrap method was successfully developed for the identification of PCs and their derivatives. The C-terminal linked to Gly is replaced with Glu, Ser, Asn, Gln or Cys, thereby creating a family of chemicals that share several structural properties. This technique could be particularly useful for investigators studying plant metabolomics. Copyright

Isolation, identification, and characterization of Ustilaginoidea virens from rice false smut balls with high ustilotoxin production potential

Ustilaginoidea (U.) virens grows on rice grains and leads to significant rice yield losses in most of the major rice producing areas. Meanwhile, ustiloxins produced by U. virens are a serious hazard to human health and ecological safety of farmlands. The other key point is that ustiloxins have been regarded as a novel resource with their potential in the treatment of cancers. There is no better way to extract ustiloxins than from pure culture of the high ustilotoxin‐producing strains. U. virens has become a key research organism. However, due to the presence of some interference components, it is a certain difficulty in the successful isolation of the strain from the false smut balls. We present here a detailed study based on the separation, screening and identification of high ustiloxins‐producing strains of U. virens. Through this study, we got a satisfactory success rate of separation and provided a good solution to the problem of separation. At the same time, this study provides quality resources for researchers interested in ustiloxins as anticancer agents.