Determination of an experimental plant growth regulator on wheat and cotton plants by reversed-phase ion-pair partition high-performance liquid chromatography

After extraction from spiked wheat and cotton samples, and preliminary clean-up by anion-exchange chromatography, the plant growth regulator, 2-benzoxazolinylidenma-lononitrile, was determined by reversed-phase ion-pair partition h.p.l.c. wih tetrabutyl-ammonium as the pairing ion. In extracts, the solute could be separated from interferences by varying the tetrabutylammonium concentration; the average recovery of the compound from wheat plants (0.13–55.6 ppm) was 96.5% and from cotton plants (0.15–134.6 ppm) 104.4% by the h.p.l.c. method. These recoveries were confirmed by the scintillation counting method with the radiolabelled compound. In a dissipation study, a biological half-life of 1.5–2.0 days was found for the compound on cotton plants. Reversed-phase ion-pair partition h.p.l.c. is a promising technique for the determination of trace levels of ionic (or ionizable) compounds in environmental samples.     

Evaluation of matrix solid-phase dispersion (MSPD) extraction for multi-mycotoxin determination in different flours using LC-MS/MS

An existing matrix solid-phase dispersion (MSPD) method for aflatoxins (AFs) and ochratoxin A (OTA) extraction was extended by further 14 mycotoxins. After it careful optimization, this method was applied to determine the occurrence of these mycotoxins on commercial flour samples (with different cereals composition) collected from local markets. In a total of 49 samples investigated, 9 mycotoxins were identified. Nivalenol (NIV) and Beauvericin (BEA) were the mycotoxins found most frequently. The samples that presented major contamination were wheat flours and bakery preparations. Despite of the great number of positives finding, only one wheat flour sample exceeded the maximum limits (ML) for OTA established by the European Union (EU). However, it would be interesting to calculate the total ingest of these mycotoxins along the years.     

STUDY ON MECHANISM OF ETHYLENE CONTROL OF SEX DIFFERENTIATION IN Lagenaria siceraria

The sex expression of Lagenaria siceraria can be modified by phytohormones. This modification is related to "a certain bisexual stage" during floral bud differentiation. ACC (ethylene precursor) and STS (ethylene antagonist) can change the direction of sex differentiation of the floral buds in vitro. STS induces maleness by means of inhibiting ethylene action, directly promoting the development of the primordia of stamens, and then suppressing that of pistils. Hence, it can be deduced that ethylene induces femaleness by directly suppressing the development of the primordia of stamens and then promoting that of pistils. Such a regulation of the development of sexual organs is due to a change in the direction of assimilates to the pistils by ACC and to the stamens by STS. Therefore, ethylene induces femaleness by first limiting the sink strength of the stamens and then increasing that of the pistils.     

Extraction and speciation of arsenic in plants grown on arsenic contaminated soils

A sequential arsenic extraction method was developed that yielded extraction efficiencies (EE) that were approximately double those using current methods for terrestrial plants. The method was applied to plants from two arsenic contaminated sites and showed potential for risk assessment studies. In the method, plants were extracted first by 1:1 water-methanol followed by 0.1 M hydrochloric (HCl) acid. Total arsenic in plant and soil samples collected from contaminated sites was mineralized by acid digestion and detected by inductively coupled plasma-atomic emission spectrometry (ICP-AES) and hydride generation-atomic absorption spectrometry (HG-AAS). Arsenic speciation was done by high performance liquid chromatography coupled with HG-AAS (HPLC-HGAAS) and by HPLC coupled with ICP-mass spectrometry (HPLC-ICP-MS). Spike recovery experiments with arsenite (As(III)), arsenate (As(V)), methylarsonic acid (MA) and dimethylarsinic acid (DMA) showed stability of the species in the extraction processes. Speciation analysis by X-ray absorption near edge spectroscopy (XANES) demonstrated that no transformation of As(III) and As(V) occurred due to sample handling. Dilute HCl was efficient in extracting arsenic from plants; however, extraction and determination of organic species were difficult in this medium. Sequential extraction with 1:1 water-methanol followed by 0.1 M-HCl was most useful in extracting and speciating both organic and inorganic arsenic from plants. Trace amounts of MA and DMA in plants could be detected by HPLC-HGAAS aided by the process of separation and preconcentration of the sequential extraction method. Both organic and inorganic arsenic compounds could be detected simultaneously in synthetic gastric fluid extracts (GFE) but EEs by this method were lower than those of the sequential method. The developed sequential method was shown to be reliable and applicable to various terrestrial plants for arsenic extraction and speciation.     

Investigation of free and conjugated seleno‐amino acids in wheat bran by hydrophilic interaction liquid chromatography with tandem mass spectrometry

An analytical method for determining seleno‐methionine, methyl‐seleno‐cysteine, and seleno‐cystine in wheat bran was developed and validated. Four different extraction procedures were evaluated to simultaneously extract endogenous free and conjugated seleno‐amino acids in wheat bran in order to select the best extraction protocol in terms of seleno amino acid quantitation. The extracted samples were subjected to a clean‐up by a reversed phase/strong cation exchange solid‐phase extraction and analyzed by chiral hydrophilic interaction liquid chromatography‐tandem mass spectrometry. The optimized extraction protocol was employed to validate the methodology. Process efficiency ranged from 58 to 112% and trueness from 73 to 98%. Limit of detection and limit of quantification were lower than 1 ng/g. Four wheat bran samples were analyzed for both total Se and single seleno‐amino acids determination. The results showed that Se‐ seleno‐methyl‐l selenocysteine was the major seleno‐amino acid in wheat bran while seleno‐methionine and seleno‐cysteine were both minor species.     

Evaluation of two methods for the extraction of antioxidants from medicinal plants

The efficiencies of two traditional extraction methods used in Chinese medicine (the decoction method and the maceration method) were evaluated for the extraction of antioxidants from medicinal plants. A group of medicinal plants possessing nutritious and tonic functions were chosen as model plants. A commonly used extraction method was used as a reference method. The antioxidant capacities and total phenolic contents of the extracts were measured by ferric-reducing antioxidant power and Trolox equivalent antioxidant capacity assays as well as the Folin–Ciocalteu method, respectively. The results obtained indicated that the two traditional extraction methods could effectively extract antioxidants from medicinal plants. These extraction methods can be applied to the analysis and purification of antioxidants in plants, respectively. At home, people can use these methods to extract antioxidants from plants for consumption. In the food industry, these methods could be utilized to prepare crude extracts from plants containing antioxidants for use as food additives. Figure Relation and comparison of extraction efficiencies of two traditional extraction methods with the reference method Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enhanced extraction efficiency of miRNA from cells by addition of Triton X-100

The determination of microRNA (miRNA) levels in biomaterials has become important for understanding their biological functions and for the diagnosis of various diseases. An effective extraction method is needed for maximizing the recovery of miRNAs from cells, while minimizing RNA degradation during the extraction because miRNAs present only approximately 0.01 % of total RNA. In this study, we used Triton X-100 (TX-100) to improve the extraction efficiency of miRNAs with TRIzol® reagent, which is a commonly used commercial microRNA isolation kit. The concentration of TX-100 and the incubation time after the addition of TX-100 were optimized to maximize the extraction efficiency. The extraction recovery by a combination of TX-100 and TRIzol® reagent was approximately 1.9-fold greater than that by the TRIzol® reagent alone. We have established a very effective extraction method for the extraction of low-abundance miRNAs in biological samples for the determination of miRNA levels in biomaterials.     

Fast wheat variety classification by capillary gel electrophoresis-on-a-chip after single-step one-grain high molecular weight glutenin extraction

Wheat variety identification based on one-step single-grain wheat extraction and fast capillary gel electrophoresis-on-a-chip (CGE-on-a-chip) analyses was evaluated for 15 different wheat varieties grown in Austria. The results of the capillary-based separation system were compared to the internationally accepted method from the International Union for the Protection of New Varieties of Plants which is based on time-consuming sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Comparable protein patterns were observed making the CGE-on-a-chip system a promising tool for high-throughput analysis in food control. For the development of a robust method protein extraction, shelf life of wheat extracts and the instrument’s variability were evaluated. It turned out that a one-step single-grain wheat extraction allowed the sample to be stored at 4 °C for up to 4 weeks without losing any valuable protein information. Furthermore, the technical variation of the whole method is very low making the biological variation of the selected wheat grains the only uncertain factor. Additionally, two unsupervised statistical methods (hierarchical cluster analysis and principal component analysis) were used for variety identification. Identification was successful for a reduced data set of 14 samples from five different wheat varieties making the combination of CGE-on-a-chip analysis of one-step single-grain extraction in combination with automatic data evaluation a promising tool for fast wheat differentiation (within a day).     

Rapid isolation method for lipopolysaccharide and lipid A from gram-negative bacteria

A fast, convenient extraction method for lipopolysaccharide (LPS), using a commercial RNA isolating reagent, allows the isolation of LPS or lipid A from low milligram (dry weight) quantities of bacterial cells. The method avoids the use of specialized equipment and has been used for processing relatively large numbers of samples. The major components of the commercial RNA isolating reagent, Tri-Reagent, are phenol and guanidinium thiocyanate in aqueous solution. The bacterial cell membranes are disrupted with guanidinium thiocyanate, which eliminates the need for mechanical cell disruption (e.g. French press) or heating. LPS and its degradation products, with particular attention paid to its bioactive lipid A portion, were measured and compared with those from the most common conventional extraction method, hot phenol-water. Negative ion quadrupole ion trap and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, fatty acid composition analysis by capillary gas chromatography, total and free phosphate by UV spectrophotometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that LPS and lipid A isolated using the Tri-Reagent approach were cleaner and suffered less degradation through loss of phosphate and (or) fatty acyl side chains from lipid A. The Tri-Reagent extraction method generated low free phosphate contamination, 11% of the total phosphate concentration, whereas the hot phenol-water extraction method gave approximately 58% as free, inorganic phosphate. Similar results were observed for the degradation of fatty acyl side chains. The time required by the new method is considerably shorter (two or three days) than that required by conventional hot phenol-water extraction (about two weeks).     

Determination of flavonoids in cultivated sugarcane leaves, bagasse, juice and in transgenic sugarcane by liquid chromatography-UV detection

A high-performance liquid chromatography (HPLC) method with photo-diode array (DAD) detection was developed to separate and quantify flavonoids in sugarcane leaves and bagasse (= the crushed sugarcane refuse from juice extraction), and in sugarcane juice. Sugarcane flavonoids consist of a complex mixture of aglycones and glycosides (including flavonolignan glycosides), and the HPLC-UV method herein proposed is suitable for their quantification as total flavonoids. This method was applied to analyze samples of cultivated sugarcane, commercial juice and transgenic sugarcane leaves. Sugarcane leaves proved a promising source of flavonoids: an average of 1.10 mg of total flavonoids/g plant material was found in fresh leaves. Moreover, the flavonoid content of sugarcane juice (0.6 mg/mL) is comparable to other food sources of flavonoids previously reported. Transgenic sugarcane leaves ("Bowman-Birk" and "Kunitz") were compared with non-modified ("control") plant samples using the proposed HPLC-UV method, which indicated that the content of total flavonoids in transgenic plants is different from that in non-modified sugarcane.     

顶空吸附萃取-气相色谱法分析小麦中部分风味物质

采用顶空吸附萃取法(headspace sorptive extraction, HSSE)建立了小麦中部分风味物质的分析方法。以硅橡胶为原料,采用溶胶-热空气硫化法,在模具中定型制得硅橡胶萃取棒。萃取棒的硅橡胶层体积约为87 μL,热稳定性好,耐热温度达到390 ℃。萃取棒吸附的风味物质经自制热解吸装置热脱附,被吹扫进入气相色谱仪进行分析。考察了萃取温度及时间、相比、热解吸条件对该方法萃取效率的影响。在优化条件下分析小麦粉标准样品,结果表明:方法的线性关系良好(r>0.9979),检出限为0.09～1.00 μg/kg,标准物质的平均添加回收率为95%～121%,相对标准偏差在2.2%～7.8%之间。对小麦粉实际样品进行分析,采用外标法得到了7种风味物质的绝对含量。该方法简便快捷,检出限低,适用于小麦中风味物质的快速定量分析。     

Enhanced spectrofluorimetric determination of aflatoxin B1 in wheat by second-order standard addition method

Determination of aflatoxin B1 (AFB1) in wheat has been accomplished by enhanced spectrofluorimetry in combination with second-order standard addition method (EF-SOSAM). The adopted strategy combined the use of parallel factor analysis (PARAFAC) for extraction of the pure analyte signal and the standard addition method, for a determination in the presence of matrix effect caused by wheat matrix. The method is based on the enhanced fluorescence of AFB1 by beta-cyclodextrin in 10% (w/w) methanol-water solution. After sample treatment and without any extended cleanup steps and derivatization process, four standard additions were performed for each sample. A specific PARAFAC model was built from three-way arrays formed by excitation-emission spectra and 5 measurements (sample plus 4 additions). The scores related to AFB1 were used for a linear regression in the standard addition method. Two naturally contaminated wheat and spiked wheat samples containing AFB1 in the range 0-18 microg kg(-1) were analyzed by EF-SOSAM and compared with HPLC results. EF-SOSAM analysis of spiked wheat samples gave a good correlation with spiked values (R(2)>0.990). The limit of detection of method was 0.9 microg kg(-1) for the determination of AFB1 in wheat samples.     

Solid‐phase extraction combined with dispersive liquid–liquid microextraction for the determination of pyrethroid pesticides in wheat and maize samples

A rapid, selective and sensitive sample preparation method based on solid‐phase extraction combined with the dispersive liquid–liquid microextration was developed for the determination of pyrethroid pesticides in wheat and maize samples. Initially, the samples were extracted with acetonitrile and water solution followed phase separation with the salt addition. The following sample preparation involves a solid‐phase extraction and dispersive liquid–liquid microextraction step, which effectively provide cleanup and enrichment effects. The main experimental factors affecting the performance both of solid‐phase extraction and dispersive liquid–liquid microextration were investigated. The validation results indicated the suitability of the proposed method for routine analyze of pyrethroid pesticides in wheat and maize samples. The fortified recoveries at three levels ranged between 76.4 and 109.8% with relative standard deviations of less than 10.7%. The limit of quantification of the proposed method was below 0.0125 mg/kg for the pyrethoroid pesticides. The proposed method was successfully used for the rapid determination of pyrethroid residues in real wheat and maize samples from crop field in Beijing, China.     

Comparison of Different Pretreatment Strategies for Enzymatic Hydrolysis of Wheat and Barley Straw

In biomass-to-ethanol processes a physico-chemical pretreatment of the lignocellulosic biomass is a critical requirement for enhancing the accessibility of the cellulose substrate to enzymatic attack. This report evaluates the efficacy on barley and wheat straw of three different pretreatment procedures: acid or water impregnation followed by steam explosion versus hot water extraction. The pretreatments were compared after enzyme treatment using a cellulase enzyme system, Celluclast 1.5 L from Trichoderma reesei, and a beta-glucosidase, Novozyme 188 from Aspergillus niger. Barley straw generally produced higher glucose concentrations after enzymatic hydrolysis than wheat straw. Acid or water impregnation followed by steam explosion of barley straw was the best pretreatment in terms of resulting glucose concentration in the liquid hydrolysate after enzymatic hydrolysis. When the glucose concentrations obtained after enzymatic hydrolyses were related to the potential glucose present in the pretreated residues, the highest yield, approximately 48% (g g-1), was obtained with hot water extraction pretreatment of barley straw; this pretreatment also produced highest yields for wheat straw, producing a glucose yield of approximately 39% (g g-1). Addition of extra enzyme (Celluclast 1.5 L+Novozyme 188) during enzymatic hydrolysis resulted in the highest total glucose concentrations from barley straw, 32-39 g L-1, but the relative increases in glucose yields were higher on wheat straw than on barley straw. Maldi-TOF MS analyses of supernatants of pretreated barley and wheat straw samples subjected to acid and water impregnation, respectively, and steam explosion, revealed that the water impregnated + steam-exploded samples gave a wider range of pentose oligomers than the corresponding acid-impregnated samples.     

基于真菌毒素污染差异的液相色谱-串联质谱法鉴别板栗粉中掺假小麦粉

建立了分散固相萃取-超快速液相色谱-串联质谱法同时测定板栗粉和小麦粉中43种真菌毒素的方法,对48份板栗粉和80份小麦粉样品的污染状况进行调查,筛选出5种专属于小麦粉的标志性真菌毒素.样品采用84％(v/v)乙腈水溶液提取,提取液采用C18结合增强型脂质去除净化剂(EMR-Lipid)净化,采用响应曲面-中心组合设计优...     

High-speed counter-current chromatography for the study of defense metabolites in wheat. Characterization of 5,6-O-methyl trans-aconitic acid

High-speed counter-current chromatography (HSCCC) methods were developed for the study of induced defense metabolites in wheat (Triticum aestivum) against powdery mildew (Blumeria graminis f. sp. tritici). A single HSCCC purification step afforded extraction of mg-quantities of an induced compound with antifungal activity. Subsequent LC-MS and NMR analyses have led to the characterization of 5,6-O-methyl trans-aconitic acid, the first such report of this compound in a plant species. The inducible nature of aconitic acid was evidenced by comparing the metabolite profiles of leaf extracts from plants treated or not with soluble silicon and infected or not with powdery mildew. In a second step, dual-mode HSCCC was used to enhance the separation of other forms of aconitic acid in wheat. Based on these results, it was concluded that 5,6-O-methyl trans-aconitic acid plays an important role as a defense molecule in wheat plants and that HSCCC is a powerful separation method for purifying such compounds from complex plant-pathogen interactions.     

Ultrasonic extraction and LC determination of linear alkylbenzene sulfonate in plant tissues

Summary A new method has been developed for the extraction and determination of linear alkylbenzene sulfonate (LAS) in plant tissues (rice stems and leaves). It consists of methanol-ultrasonic extraction followed by clean-up with aluminum oxide, enrichment with C₁₈ solid-phase extraction column and determination by HPLC. Both efficiency and accurracy of the overall method were high, i.e. mean recovery of: 89% (84 to 93% for LAS concentrations ranging from 1 to 100 mg kg⁻¹) and repeatability of: 3% relative standard deviation for 6 replicate analyses. With a 2 g sample for analysis, LAS levels of 0.5 mg kg⁻¹ in plants could be detected with the proposed method. Further advantages were: it was less time consuming (1 h for extraction), less solvent consumption, and smaller samples (2 to 3 g) required when compared with Soxhlet extraction.     

Influence of sample extraction solutions on the detection of wheat proteins by mass spectrometry

Routinely used methods for the detection of gluten mostly use denaturing agents and high salt concentrations to increase the extractability of the gluten fraction. These work well in combination with ELISA methods, but may have a negative effect on MS methods due to their influence on the ionization of the analyte leading to a significant reduction of signal intensities. A newly developed HPLC/MS/MS method was used to assess this influence. Four different extraction buffers were compared: 70% ethanol, TRIS-HCl, TRIS-HCl with dithiothreitol, and a commercially available cocktail solution. Unprocessed and processed wheat samples were analyzed. When analyzing unprocessed samples, a negative effect on ionization could be observed. Considering extraction capabilities and signal intensities, TRIS-HCl seemed to be the most suitable buffer in combination with the MS method. To assess whether the method was capable of detecting hidden wheat protein in different kinds of food, different food samples containing 0 to 34000 microg/g gluten were analyzed using the TRIS-HCI extraction buffer.     

Trace Determination of Benzalkonium Chlorides in River and Wastewater by Capillary Electrophoresis following Solid‐Phase Extraction Coupled with Salting‐Out Extraction

Trace levels analysisbenzalkonium chlorides (BAKs) in river water and wastewater treatment plants (WWTP) effluents were determined by capillary electrophoresis (CE) following solid‐phase extraction (SPE) and salting‐out extraction. Salting‐out extraction using an appropriate ratio of sodium chloride (NaCl) and acetonitrile (ACN) mixed with concentrated SPE elutant was capable of providing more than 500‐fold enhancement in detection sensitivity. The ratios of ACN and NaCl for salting‐out extraction were investigated and optimized. Matrix interference was eliminated by salting‐out extraction. Limits of quantitation of BAK homologues were achieved at 0.1 μg/L in 250 mL water samples. Recoveries of BAKs in various spiked water samples ranged from 70% to 84% with relative standard deviation (RSD) less than 9%. Trace amounts of total BAKs were detected in river water and WWTP effluent samples ranging from 27 to 145 μg/L at the first time by CE.