Rapid profiling and quantification of phospholipid molecular species in human plasma based on chemical derivatization coupled with electrospray ionization tandem mass spectrometry.

In this study, we developed a novel strategy using solid-phase extraction (SPE) coupled with shotgun mass spectrometry (MS) based on trimethylsilyldiazomethane (TMSCHN2) stable-isotope derivatization for rapid profiling and accurate quantification of phospholipids (PLs) in human plasma. HybridSPE-Phospholipid (HybridSPE-PL, zirconia coated silica stationary phase) was used for sample pretreatment via the Lewis acid-base interaction between zirconia and phosphate moiety of PLs. This step allows rapid enrichment and recovery of PLs from human plasma. Afterward, PLs were derivatized with TMSCHN2, which leads to methylation of hydroxyl and amino groups in PLs and allows highly sensitive PL analysis by shotgun MS in positive ionization mode (limit of detection decreased up to 116.67 fold compared to underived PLs). We developed an accuracy quantification method for determination of PL molecular species in biological samples. Two or more PL standards were selected for each PL class and derivatized with TMSCHN2 without stable-isotope coding. They were then used as the internal standards. PLs in biological samples were isotopic derivatized via acid-catalyzed H/D exchange and methanolysis of TMSCHN2. For accurate quantification, a calibration curve for each class of PLs was typically constructed by using the internal standards to normalize the non-uniformity response caused by the differential fragmentation kinetics resulting from the distinct chemical constitution of individual PL species in the biological samples. This newly developed method was used to comprehensively analyze PL molecular species in human plasma samples. It is a promising methodology for rapid profiling and accurate quantification of complex lipid molecules in biological samples.

Research advances based on mass spectrometry for profiling of triacylglycerols in oils and fats and their applications.

Vegetable oils and animal fats are dietary source of lipids that play critical and multiple roles in biological function. Triacylglycerols (TAGs) are the principal component of oils and fats with significant difference in profile among different oils and fats. TAG profiling is essential for nutritional evaluation, quality control and assurance of safety in oils and fats. However, analysis of TAGs is a challenging task because of the complicated composition of TAGs and their similar physicochemical properties in oils and fats. The rapid development of mass spectrometry (MS) technology in recent years makes it possible to analyze the composition, content and structure of TAGs in the study of the physical, chemical and nutritional properties of oils, fats and related products. This review described the research advancement based on MS for profiling of TAGs in oil, fat and their applications in food. The application of MS, including direct infusion strategies, and its combination with chromatography, gas chromatography-MS (GC-MS) and liquid chromatography-MS (LC-MS), in the analysis of TAGs were reviewed. The advantages and disadvantages of these analytical methods with relevant applications for TAGs analysis in food were also described.

Rapid and sensitive detection of free fatty acids in edible oils based on chemical derivatization coupled with electrospray ionization tandem mass spectrometry.

In this study, a strategy based on chemical derivatization coupled with electrospray ionizationtandem quadrupole mass spectrometry (ESI-MS/MS) for rapid and sensitive detection of FFAs in edible oils was developed. A derivative reagent (N,N-diethyl-1,2-ethanediamine, DEEA) was employed to selectively label carboxyl groups of FFAs to form an amino compound with a tertiary amino group. The DEEA derivative products could lose a characteristic neutral loss fragment of 73Da in collision-induced dissociation (CID), which enabled to discriminate and analyze the DEEA derived FFAs with neutral loss scan (NLS 73Da)under the positive ion mode of mass spectrometry. The assay was linear over the concentration range 0.5-200nmol/L with satisfactory correlation coefficients (R2≥0.9942), whilst the limit of detection and quantitation were 0.1-0.3nmol/L and 0.3-1.0nmol/L, respectively. Finally, the established method was applied to determine dynamic FFA formation in seven types of edible oils subjected to a microwave heating treatment test.

Profiling and relative quantification of phosphatidylethanolamine based on acetone stable isotope derivatization.

Phosphatidylethanolamine (PE) is considered to be one of the pivotal lipids for normal cellular function as well as disease initiation and progression. In this study, a simple, efficient, reliable, and inexpensive method for the qualitative analysis and relative quantification of PE, based on acetone stable isotope derivatization combined with double neutral loss scan-shotgun electrospray ionization tandem-quadrupole mass spectrometry analysis (ASID-DNLS-Shotgun ESI-MS/MS), was developed. The ASID method led to alkylation of the primary amino groups of PE with an isopropyl moiety. The use of acetone (d0-acetone) and deuterium-labeled acetone (d6-acetone) introduced a 6 Da mass shift that was ideally suited for relative quantitative analysis, and enhanced sensitivity for mass analysis. The DNLS model was introduced to simultaneously analyze the differential derivatized PEs by shotgun ESI-MS/MS with high selectivity and accuracy. The reaction specificity, labeling efficiency, and linearity of the ASID method were thoroughly evaluated in this study. Its excellent applicability was validated by qualitative and relative quantitative analysis of PE species presented in liver samples from rats fed different diets. Using the ASID-DNLS-Shotgun ESI-MS/MS method, 45 PE species from rat livers have been identified and quantified in an efficient manner. The level of total PEs tended to decrease in the livers of rats on high fat diets compared with controls. The levels of PE 32:1, 34:3, 34:2, 36:3, 36:2, 42:10, plasmalogen PE 36:1 and lyso PE 22:6 were significantly reduced, while levels of PE 36:1 and lyso PE 16:0 increased.

Quantitation of triacylglycerols in edible oils by off-line comprehensive two-dimensional liquid chromatography-atmospheric pressure chemical ionization mass spectrometry using a single column.

In this investigation, off-line comprehensive two-dimensional liquid chromatography-atmospheric pressure chemical ionization mass spectrometry using a single column has been applied for the identification and quantification of triacylglycerols in edible oils. A novel mixed-mode phenyl-hexyl chromatographic column was employed in this off-line two-dimensional separation system. The phenyl-hexyl column combined the features of traditional C18 and silver-ion columns, which could provide hydrophobic interactions with triacylglycerols under acetonitrile conditions and can offer π-π interactions with triacylglycerols under methanol conditions. When compared with traditional off-line comprehensive two-dimensional liquid chromatography employing two different chromatographic columns (C18 and silver-ion column) and using elution solvents comprised of two phases (reversed-phase/normal-phase) for triacylglycerols separation, the novel off-line comprehensive two-dimensional liquid chromatography using a single column can be achieved by simply altering the mobile phase between acetonitrile and methanol, which exhibited a much higher selectivity for the separation of triacylglycerols with great efficiency and rapid speed. In addition, an approach based on the use of response factor with atmospheric pressure chemical ionization mass spectrometry has been developed for triacylglycerols quantification. Due to the differences between saturated and unsaturated acyl chains, the use of response factors significantly improves the quantitation of triacylglycerols. This two-dimensional liquid chromatography-mass spectrometry system was successfully applied for the profiling of triacylglycerols in soybean oils, peanut oils and lord oils. A total of 68 triacylglycerols including 40 triacylglycerols in soybean oils, 50 triacylglycerols in peanut oils and 44 triacylglycerols in lord oils have been identified and quantified. The liquid chromatography-mass spectrometry data were analyzed using principal component analysis. The results of the principal component analysis enabled a clear identification of different plant oils. By using this two-dimensional liquid chromatography-mass spectrometry system coupled with principal component analysis, adulterated soybean oils with 5% added lord oil and peanut oils with 5% added soybean oil can be clearly identified.

Ultrasonic pretreatment in lipase-catalyzed synthesis of structured lipids with high 1,3-dioleoyl-2-palmitoylglycerol content.

Production of structured lipid 1,3-dioleoyl-2-palmitoylglycerol (OPO), from tripalmitin (PPP) and oleic acid (OA) using lipases and ultrasonic pretreatment was conducted. Factors influencing both the ultrasonic conditions and enzymatic reaction were investigated. Optimum conditions could be attained with 6 min pretreatment time, 50% ultrasonic power, 3 s/9 s (work/pause) cycle of ultrasonic pulse, 1:8 PPP/OA molar ratio, 12% enzyme dosage and 50 °C temperature of. At the optimum conditions, the OPO yield of 51.8% could be achieved in 4h. Studies showed that the OPO content increased to 35.9% in 1h with ultrasonic pretreatment, in comparison to 4h without ultrasonic pretreatment. Reuse of Lipozyme RM IM for 10 cycles under ultrasonic irradiation did not cause essential damage to its lipase activity. Reaction kinetic model fitted well with the proposed Ping-Pong mechanism. The apparent kinetic constant (Vm'/K2) of ultrasound pretreatment reaction was 2.52 times higher than the conventional mechanical stirring, indicating that ultrasound pretreatment enhanced the substrates affinity to the enzyme. This study confirmed that ultrasonic pretreatment was more efficient in OPO production than conventional mechanical agitation.

Profiling of triacylglycerols in plant oils by high-performance liquid chromatography-atmosphere pressure chemical ionization mass spectrometry using a novel mixed-mode column.

In this investigation, a rapid and high-throughput method for profiling of TAGs in plant oils by liquid chromatography using a single column coupled with atmospheric pressure chemical ionization (APCI) mass spectrometry was reported. A novel mixed-mode phenyl-hexyl chromatographic column was employed in this separation system. The phenyl-hexyl column could provide hydrophobic interactions as well as π-π interactions. Compared with two traditionally columns used in TAG separation - the C18 column and silver-ion column, this column exhibited much higher selectivity for the separation of TAGs with great efficiency and rapid speed. By comparison with a novel mix-mode column (Ag-HiSep OTS column), which can also provide both hydrophobic interactions as well as π-π interactions for the separation of TAGs, phenyl-hexyl column exhibited excellent stability. LC method using phenyl-hexyl column coupled with APCI-MS was successfully applied for the profiling of TAGs in soybean oils, peanut oils, corn oils, and sesame oils. 29 TAGs in peanut oils, 22 TAGs in soybean oils, 19 TAGs in corn oils, and 19 TAGs in sesame oils were determined and quantified. The LC-MS data was analyzed by barcodes and principal component analysis (PCA). The resulting barcodes constitute a simple tool to display differences between different plant oils. Results of PCA also enabled a clear identification of different plant oils. This method provided an efficient and convenient chromatographic technology for the fast characterization and quantification of complex TAGs in plant oils at high selectivity. It has great potential as a routine analytical method for analysis of edible oil quality and authenticity control.

Preparation and characterization of novel lipid carriers containing microalgae oil for food applications.

This work investigated the suitability of lipid carriers as potential encapsulation method to improve the physical and chemical stability of microalgae oil high in docosahexaenoic acid (DHA). Lipid carriers with various oil contents were successfully prepared by a microfluidization method using stearic acid as solid lipid, microalgae oil as liquid lipid, and poloxamer 188 as surfactant. Results show that the mean particle diameter of the lipid carriers was in the range of 300 to 350 nm with the polydispersity index below 0.2. The lipid carriers were found to have spherical shape when examined under the transmission electron microscope. Data from the encapsulation efficiency and loading capacity indicate high distribution of microalgae oil throughout the lipid carriers and good physical stability as reflected by the particle size and size distribution during storage. Furthermore, the lower DPPH scavenging activity of lipid carriers compared with that of free microalgae oil suggests better chemical stability of microalgae oil encapsulated in lipid carriers. The addition of microalgae oil into lipid phase could disturb the crystalline order and form lattice defects to enable encapsulation of DHA as revealed by the results from differential scanning calorimetery. Current results suggest that this type of novel lipid carriers could be an efficient and promising carrier system for delivery of microalgae oil.

Online profiling of triacylglycerols in plant oils by two-dimensional liquid chromatography using a single column coupled with atmospheric pressure chemical ionization mass spectrometry.

The complexity of natural triacylglycerols (TAGs) in various edible oils is high because of the hundreds of TAG compositions, which makes the profiling of TAGs quite difficult. In this investigation, a rapid and high-throughput method for online profiling of TAGs in plant oils by two-dimensional (2D) liquid chromatography using a single column coupled with atmospheric pressure chemical ionization (APCI) mass spectrometry was reported. A novel mixed-mode 2D chromatographic column packed with silver-ion-modified octyl and sulfonic co-bonded silica was employed in this online 2D separation system. This novel 2D column combined the features of C8 column and silver-ion. In comparison with the traditional C18 column and silver-ion column, which are the two main columns used for the separation of complex TAGs in natural oil samples, this novel 2D column, could provide hydrophobic interactions as well as π-complexation interactions. It exhibited much higher selectivity for the separation of TAGs, and the separation was rapid. This online 2D separation system was successful in the separation of a large number of TAG solutes, and the TAG structures were evaluated by analyzing their APCI mass spectra information. This system was applied for the profiling of TAGs in peanut oils, corn oils, and soybean oils. 30 TAGs in peanut oils, 18 TAGs in corn oils, and 21 TAGs in soybean oils were determined and quantified. The highest relative content of TAGs was LLL, which was found in corn oil with the relative content up to 45.43 (%, w/w), and the lowest relative content of TAGs was LLS and OSS, which was found in soybean oil and corn oil respectively, with the relative content only 0.01 (%, w/w). In addition, the TAG data were analyzed by principal component analysis (PCA). Results of PCA enabled a clear identification of different plant oils. This method provided an efficient and convenient chromatographic technology for the fast characterization and quantification of complex TAGs in plant oils at high selectivity. It has great potential as a routine analytical method for analysis of edible oil quality and authenticity control.

Characterization and quantification of triacylglycerols in peanut oil by off-line comprehensive two-dimensional liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry.

The complexity of natural triacylglycerols (TAGs) in various edible oils is prodigious due to the hundreds of set is of TAG compositions, which makes the identification of TAGs quite difficult. In this investigation, the off-line 2D system coupling of nonaqueous RP and silver-ion HPLC with atmospheric pressure chemical ionization MS detection has been applied to the identification and quantification of TAGs in peanut oil. The method was successful in the separation of a high number of TAG solutes, and the TAG structures were evaluated by analyzing their atmospheric pressure chemical ionization mass spectra information. HPLC and MS conditions have been optimized and the fragmentation mechanisms of isomers have been validated. In addition, an internal standard approach has been developed for TAG quantification. Then this system was applied in peanut oil samples and there was a total of 48 TAGs including regioisomers that have been determined and quantified.

Rapid magnetic solid-phase extraction based on monodisperse magnetic single-crystal ferrite nanoparticles for the determination of free fatty acid content in edible oils.

This study proposes a rapid magnetic solid-phase extraction (MSPE) based on monodisperse magnetic single-crystal ferrite (Fe(3)O(4)) nanoparticles (NPs) for determining the quantities of eight free fatty acids (FFAs), including palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), arachidic acid (C20:0), eicosenoic acid (C20:1), and behenic acid (C22:0) in oil. The amine-functionalized mesoporous Fe(3)O(4) magnetic NPs were applied as a sorbent for MSPE of FFAs from oil samples in a process that is based on hydrophilic interaction. The extraction can be completed rapidly in a dispersive mode with the aid of vigorous vortex. Additional tedious processing steps such as centrifugation and evaporation of organic solvent were not necessary with this procedure. Furthermore, esterification of FFAs can be accomplished during the desorption procedure by using methanol/sulfuric acid (99:1, v/v) as the desorption solvent. Several parameters affecting the extraction efficiency were investigated, including the matrix solvent for extraction, the desorption solvent and desorption time, and the amount of sorbent and extraction time. The pretreatment process was rapid under optimal conditions, being accomplished within 15 min. When coupled with gas chromatography-flame ionization detection (GC-FID), a rapid, simple, and convenient MSPE-GC-FID method for the determination of FFAs in oil samples was established with a total analysis time within 25 min. The limits of detection for the target FFAs were found to be 7.22-26.26 ng/mL. Recoveries in oil samples were in the range of 81.33-117.75%, with RSDs of <6.4% (intraday) and <6.9% (interday). This method was applied successfully to the analysis of dynamic FFA formation in four types of edible oils subjected to an accelerated storage test. The simple, rapid, and cost-effective method developed in the current study offers a potential application for the extraction and preconcentration of FFAs from hydrophobic sample matrices, including edible fats and oils, fatty foods, and biological samples with high amounts of lipid.

Optimisation of preparation conditions and properties of phytosterol liposome-encapsulating nattokinase.

Phytosterol liposomes were prepared using the thin film method and used to encapsulate nattokinase (NK). In order to obtain a high encapsulation efficiency within the liposome, an orthogonal experiment (L9 (3)(4)) was applied to optimise the preparation conditions. The molar ratio of lecithin to phytosterols, NK activity and mass ratio of mannite to lecithin were the main factors that influenced the encapsulation efficiency of the liposomes. Based on the results of a single-factor test, these three factors were chosen for this study. We determined the optimum extraction conditions to be as follows: a molar ratio of lecithin to phytosterol of 2 : 1, NK activity of 2500 U mL⁻¹ and a mass ratio of mannite to lecithin of 3 : 1. Under these optimised conditions, an encapsulation efficiency of 65.25% was achieved, which agreed closely with the predicted result. Moreover, the zeta potential, size distribution and microstructure of the liposomes prepared were measured, and we found that the zeta potential was -51 ± 3 mV and the mean diameter was 194.1 nm. From the results of the scanning electron microscopy, we observed that the phytosterol liposomes were round and regular in shape and showed no aggregation.

Simultaneous analysis of multiple endogenous plant hormones in leaf tissue of oilseed rape by solid-phase extraction coupled with high-performance liquid chromatography-electrospray ionisation tandem mass spectrometry.

INTRODUCTION: Plant hormones are important signalling molecules that act at lower concentrations to regulate numerous plant physiological and developmental processes. In order to study the functions of plant hormones, it is necessary to develop a high-throughput and highly selective and sensitive method for determination of plant hormones. OBJECTIVE: Based on SPE-HPLC-ESI-MS/MS method, a highly selective and sensitive method for determination of six plant hormones in leaf tissue of oilseed rape was developed. METHODOLOGY: The extraction was performed with C18 solid-phase extraction cartridge and the sample was subsequently analysed by HPLC-ESI-MS/MS. Methanol and H2O with 0.05% formic acid was selected as the mobile phase for HPLC, using a gradient of increasing methanol content. The plant hormones were quantified in MRM mode and identified in IDA mode by the hybrid triple quadrupole/linear ion trap mass spectrometer with high sensitivity and selectivity. RESULTS: Under the optimal conditions, good linearities were obtained for six plant hormones with the correlation coefficients above 0.9924. The detection limits of the target compounds were in the range of 0.005-0.2 ng/mL. Reproducibility of the method was obtained with intra-day and inter-day relative standard deviations. The extraction recovery yields of plant hormones under SPE conditions ranged from 67.03 to 119.83%. Compared with previous methods, sample preparation time and amount of sample required for analysis of plant hormones were reduced, and more classes of hormones were quantitatively measured.

Some characteristics and functional properties of rapeseed protein prepared by ultrasonication, ultrafiltration and isoelectric precipitation.

BACKGROUND: The presence of complex protein constituents and difficulties in extracting protein from rapeseed meal limit the application of rapeseed protein in food processing. However, double-low rapeseed (low erucic acid, low glucosinolate) protein is a type of complete protein that is of potential use in the food industry. In this study the characteristics and functional properties of rapeseed protein prepared by ultrasonic-assisted extraction, ultrafiltration and isoelectric precipitation were analysed and compared with those of soybean protein. RESULTS: The extraction efficiency with the ultrasonic-assisted method was significantly higher than that obtained with the traditional method. Ultrafiltration and isoelectric precipitation yielded three different proteins: ultrafiltered protein RPs and precipitated proteins RP5.8 and RP3.6. Chromatographic separation of RPs resulted in four fractions: RPsI, RPsII, RPsIII and RPsIV. The distribution of the isoelectric point of rapeseed protein was investigated by two-dimensional electrophoresis. The amino acid composition of RPs renders it suitable for human consumption. The hydrophobic/hydrophilic amino acid ratio of rapeseed protein was higher than that of soybean protein. The functional properties (oil adsorption ability, emulsifying capacity, foaming capacity and foam stability) of RPs, RP5.8 and RP3.6 were found to be better than those of soybean protein. CONCLUSION: Ultrasonication and ultrafiltration were significantly better than the traditional method of rapeseed protein extraction. The ultrafiltered rapeseed protein RPs had superior functional properties. The results of this study provide useful indicators for rapeseed protein as a potential replacement for other proteins.

Preparation of novel polydimethylsiloxane solid-phase microextraction film and its application in liquid sample pretreatment.

In this paper, an extraction approach based on the use of a novel polydimethylsiloxane (PDMS) film as the extraction medium was described. Two kinds of PDMS films with smooth surface and frosted surface were prepared and were practically evaluated for extraction. A model analytical problem, the determination of organochlorine pesticides in water samples, was selected for practical evaluation of the film extraction procedure by direct extraction and solvent desorption, followed by gas chromatography (GC) analysis with electron capture detection (ECD). The main variables affecting the extraction process such as the extraction time, the extraction temperature, the elution conditions, etc. were studied. The method was characterized on the basis of its linearity, precision, and limits of detection. The novel approach was sensitive and precise enough for the detection of the target analytes in the low nanogram per liter range using 5 mL of sample. In fact, limits of detection ranging from 0.77 to 10.25 ng/L were obtained. Compared with the solid-phase microextraction (SPME) fiber, the robust extraction film has a large extraction capacity, low cost of preparation. Besides, owing to the simplicity of the extraction procedures, in-site sample preparation for environmental monitor may be realized.

Electrocatalytic oxidation of phytohormone salicylic acid at copper nanoparticles-modified gold electrode and its detection in oilseed rape infected with fungal pathogen Sclerotinia sclerotiorum.

Salicylic acid (SA) is a biological substance that acts as a phytohormone and plays an important role in signal transduction in plants. It is important to accurately and sensitively detect SA levels. A gold electrode modified with copper nanoparticles was used to assay the electrocatalytic oxidation of salicylic acid. It was found that the electrochemical behavior of salicylic acid was greatly improved at copper nanoparticles, indicating that anodic oxidation could be catalyzed at copper nanoparticles. And the pH had remarkable effect on the electrochemical process, a very well-defined oxidation peak appeared at pH 13.3 (0.2M NaOH). The kinetics parameters of this process were calculated and the heterogeneous electron transfer rate constant (k) was determined to be 1.34x10(-3)cms(-1), and (1-alpha)n(alpha) was 1.22. The gold electrode modified with copper nanoparticles could detect SA at a higher sensitivity than common electrodes. The electrode was used to detect the SA levels in oilseed rape infected with the fungal pathogen Sclerotinia sclerotiorum. The results showed that the SA concentration reached a maximum during the 10th-25th hours after infection. This result was very similar to that determined by HPLC, indicating that the gold electrodes modified with copper nanoparticles could be used as salicylic acid sensors.

A robust sampling approach for identification and quantification of methyl jasmonate in leaf tissue of oilseed rape for analysis of early signaling in Sclerotinia sclerotiorum resistance.

INTRODUCTION: Methyl jasmonate (MJA), which is a natrual hormonal regulator, is thought to be essential for the regulation of systemic defence responses. The information about MJA levels in plant tissues is helpful for the study of the disease resistance mechanism and genetically engineered cultivars with increased resistance. Therefore, the quantification of MJA levels in plant tissues by means of a sensitive and reliable method is of interest. OBJECTIVE: Development of a film extraction method coupled with GC for determination of methyl jasmonate in leaf tissue of oilseed rape for analysis of early signalling in sclerotinia sclerotiorum resistance. METHODOLOGY: A robust polydimethylsiloxane film was prepared and used for extraction of MJA in leaf tissues. By using in-solution extraction mode, optimum extraction efficiency was achieved with methanol-water (1 : 5, v/v) as extraction medium at 40 degrees C for 60 min. RESULTS: Under the optimal conditions, a detection limit of 0.2 ng/mL was achieved. Excellent reproducibility was found over a linear range of 1-1000 ng/mL. MJA in leaves infected by sclerotinia sclerotiorum was determined, with the results showing that basal levels of MJA (15 ng/g) were present in noninfested controls, but increased to 313 ng/g 10 h after fungal attack. CONCLUSION: The film extraction method is a simple, rapid and inexpensive sampling technique for determination of endogenous MJA in plant tissues that can be applied to most plants.

Real-time monitoring of oxidative burst from single plant protoplasts using microelectrochemical sensors modified by platinum nanoparticles.

Oxidative bursts from plants play significant roles in plant disease defense and signal transduction; however, it has not hitherto been investigated on individual living plant cells. In this article, we fabricated a novel sensitive electrochemical sensor based on electrochemical deposition of Pt nanoparticles on the surface of carbon fiber microdisk electrodes via a nanopores containing polymer matrix, Nafion. The numerous hydrophilic nanochannels in the Nafion clusters coated on the electrode surface served as the molecular template for the deposition and dispersion of Pt, which resulted in the uniform construction of small Pt nanoparticles. The novel sensor displayed a high sensitivity for detection of H(2)O(2) with a detection limit of 5.0 x 10(-9) M. With the use of this microelectrochemical sensor, the oxidative burst from individual living plant protoplasts have been real-time monitored for the first time. The results showed that oxidative burst from single protoplasts triggered by a pathogen analogue were characterized by quanta release with a large number of "transient oxidative microburst" events, and protoplasts from the transgenic plants biologically displayed better disease-resistance and showed a distinguished elevation and longer-lasting oxidative burst.

Microsensor in vivo monitoring of oxidative burst in oilseed rape (Brassica napus L.) leaves infected by Sclerotinia sclerotiorum.

Oxidative burst is the rapid and transient production of large amounts of reactive oxygen species, including superoxide anion, hydrogen peroxide (H(2)O(2)), and hydroxyl radical. A rapid and simple technique was employed for in vivo detection of oxidative burst in oilseed rape (Brassica napus L.) leaves, using a modified electrode. Platinum (Pt) micro-particles were dispersed on a Pt electrode, coated with a poly (o-phenylenediamine) film. This exhibited high sensitivity, selectivity and stability in H(2)O(2) detection. Amperometry was used to obtain satisfactory linear relationships between reductive current intensities and H(2)O(2) concentrations at -0.1 V potential in different electrolytes. This electrode was used in vivo to detect oxidative burst in oilseed rape following fungal infection. Oxidative bursts induced by infection of the fungal pathogen Sclerotinia sclerotiorum (Lib.) de Bary exhibited notably different mechanisms between a susceptible and a resistant glucose oxidase-transgenic genotype.

Quantitative determination of oil content in small quantity of oilseed rape by ultrasound-assisted extraction combined with gas chromatography.

Accurately quantitative determination of oil content in oilseed rape plays an important role in varieties breeding for improving oil content in seeds. However, large quantity of oilseeds were needed in order to obtain accuracy and precision results by using standard Soxhlet extraction method, which may be a handicap in analysis of small, rare and precious samples in plant breeding. In the present work, ultrasound-assisted extraction was evaluated as a simpler and more effective alternative to conventional extraction method for the isolation of oil from small quantity of oilseed rape (<20 mg). The oil of oilseed rape samples was extracted by ultrasound-assisted method, and then the fatty acids and total oil content of the seeds were qualitatively and quantitatively determined by gas chromatography (GC). Extraction efficiency of total oil obtained by ultrasound-assisted extraction through an orthogonal experiment (L(9) (3(4))) were investigated to get the best extraction conditions. Statistical analysis showed that the variable with the largest effect was the ultrasound-assisted extraction time which was followed by the ultrasound-assisted extraction power, and the liquid:solid ratio. A liquid:solid ratio of 1:4 (L:g), an ultrasound-assisted extraction time of 60 min and an ultrasound-assisted extraction power of 500 W were found to be optimal for oil extraction from oilseed rape. By comparing with the conventional method, it was found that the ultrasound-assisted extraction of oil from oilseed rape was about five times faster than the traditional extraction method. By the use of ultrasound-assisted extraction combined with GC analysis, the fatty acids and total oil content in small quantity of seeds (<20 mg) were successfully qualitatively determined and the results are in agreement with that obtained by traditional standard method.