Simultaneous confirmatory analysis of different transgenic maize (zea mays) lines using multiplex polymerase chain reaction-restriction analysis and capillary gel electrophoresis with laser induced fluorescence detection

A novel analytical procedure based on the combination of multiplex PCR, restriction analysis, and CGE-LIF to unambiguosly and simultaneously confirm the presence of multiple lines of genetically modified corn is proposed. This methodology is based on the amplification of event-specific DNA regions by multiplex PCR using 6-FAM-labeled primers. Subsequently, PCR products are digested by a mixture containing specific restriction endonucleases. Thus, restriction endonucleases selectively recognize DNA target sequences contained in the PCR products and cleave the double-stranded DNA at a given cleavage site. Next, the restriction digest is analyzed by CGE-LIF corroborating the length of the expected restriction fragments, confirming (or not) the existence of GMOs. For accurate size determination of the DNA fragments by CGE-LIF a special standard DNA mixture was produced in this laboratory for calibration. The suitability of this mixture for size determination of labeled DNA fragments is also demonstrated. The usefulness of the proposed methodology is demonstrated through the simultaneous detection and confirmatory analysis of samples containing 0.5% of GA21 and MON863 maize plus an endogenous gene of maize as control.     

Gas-liquid chromatographic determination of morphine, heroin, and cocaine.

Morphine, heroin, and cocaine are quantitatively determined with the same gas-liquid chromatographic system. The compounds are separated on a 6 ft X 2 mm id glass column packed with a 1:1 mixture of 5% SE-30 on 80--100 mesh Chromosorb W and 3% OV-17 on 80--100 mesh Varaport 30. The column is temperature-programmed. Flame ionization detector responses are measured with a computer-based data system. Heroin and cocaine are chromatographed directly; morphine is derivatized first. The procedure was evaluated with previously analyzed commercial and forensic samples. Accuracy and precision were 5 and 3%, respectively.     

Enhanced Removal of Micropollutants from Groundwater,Using pH Modification Coupled with Photolysis

Direct ultraviolet (UV) photolysis coupled with modification of solution pH was explored as a method for the removal of organic micropollutants from groundwater. Photodegradation rates of most of the investigated compounds were pH dependent, however, its correlation with photodegradation rate varied among compounds. The potential of the pH modification during photolysis was determined for removal of a mixture of two pharmaceuticals sulfamethoxazole (SMX) and triclosan (TCS) in groundwater. The treatment included initial photolysis of the mixture at the optimal pH for TCS (i.e., 7.5–7.9), followed by pH modification to the optimal pH for SMX (i.e., 5), prior to a second irradiation period. The described procedure dramatically increased the removal efficiency (up to threefold) of the treated mixture compared to UV treatment at constant pH.     

Multiresidue analysis of cotton defoliant, herbicide, and insecticide residues in water by solid-phase extraction and GC-NPD, GC-MS, and HPLC-diode array detection

A multiresidue procedure was developed for analysis of cotton pesticide and harvest-aid chemicals in water using solid-phase extraction and analysis by GC-NPD, GC-MS, and HPLC-DAD. Target compounds included the defoliants tribufos, dimethipin, thidiazuron; the herbicide diuron; and the insecticide methyl parathion. Three solid-phase extraction (SPE) media, octadecylsilyl (ODS), graphitized carbon black (GCB), and a divinylbenzene-N-vinyl pyrollidine copolymer (DVBVP), were evaluated. On GCB and ODS, recoveries varied depending on compound type. Recoveries were quantitative for all compounds on DVBVP, ranging from 87 to 115% in spiked deionized water and surface runoff. The method detection limit was less than 0.1 microg L(-)(1). SPE with DVBVP was applied to post-defoliation samples of surface runoff and tile drainage from a cotton research plot and surface runoff from a commercial field. The research plot was defoliated with a tank mixture of dimethipin and thidiazuron, and the commercial field, with tribufos. Dimethipin was detected (1.9-9.6 microg L(-)(1)) in all research plot samples. In the commercial field samples, tribufos concentration ranged from 0.1 to 135 microg L(-)(1). An exponentially decreasing concentration trend was observed with each successive storm event.     

Analysis of USP epinephrine injections for potency, impurities, degradation products, and d-enantiomer by liquid chromatography, using ultraviolet and electrochemical detectors

A liquid chromatographic (LC) method was adapted for the determination of epinephrine and related impurities in intravenous and cardiac injections; ultraviolet (UV) and electrochemical detectors (EC) were used in series. Epinephrine was determined and related impurities, i.e., adrenalone, epinephrine sulfonic acid, and norepinephrine, were detected directly in a small portion of the injection solution. Diastereoisomers of the epinephrine enantiomers were prepared by derivatization and determined by LC with a UV detector. The recovery of epinephrine added to epinephrine injection was 100%. The recovery of d-enantiomer from a d, l mixture was 100%. Impurities at levels less than 1% were easily detected. The LC method with UV detection is faster and more convenient than the USP XX method. In addition, impurities can be detected in the same portion of sample. The procedure is stability-indicating.     

Composting of the Solid Phase of Digestate from Biogas Production: Optimization of the Moisture,C/N Ratio,and pH Conditions

The management of biodegradable wastes is increasing, including the use of waste as a source of energy. Anaerobic digestion involves organic-matter decomposition under anoxic conditions by a microbial consortium, obtaining a source of renewable energy (biogas), mainly constituted of a mixture of carbon dioxide (CO₂, 25–45 percent), methane (CH₄, 55–75 percent), and the digested substrate (digestate). The direct application of digestate into agricultural soils presents several problems. These include agronomic (low concentration of nutrients, high salinity, etc.), economic (cost of transport and handling), and environmental issues (gaseous emissions, nutrient leaching, and pathogen spread). However, it is possible to obtain quality compost from the solid fraction of digestate, and the compost obtained can have good properties for use as container growing medium or for crop production. In this work, an optimized procedure has been developed for composting the solid phase of a digestate obtained from a continuous, anaerobic codigestion of cattle slurry with 84 percent of cattle manure, 7.4 percent of a mixture of maize/silage, and 8.6 percent of peach-juice pulp (fresh-mass basis). The experiment was designed to optimize pH, carbon (C) / nitrogen (N) ratio, and moisture values in order to maximize self-heating activity, using Dewar self-heating tests. A factorial optimization of moisture and C/N ratio was carried out. In the best moisture-C/N ratio treatment, pH optimization was also developed. To predict the optimum conditions of the studied residue related to the increase of temperature per dry matter, a multiple correlation analysis based on moisture, C/N ratio, and pH was developed, which explained 80 percent of the variance in this experiment.     

An automated anion-exchange chromatographic procedure for the estimation of saccharides in acid hydrolysates of soil

It is well known that the major neutral monosaccharide components released from soil by acid hydrolysis are glucose, galactose, mannose, arabinose, xylose, ribose, rhamnose and fucose. A colorimetric determination of the saccharide mixture released from soil is unsatisfactory for determining an accurate figure for soil saccharides. More precise information can be obtained by the determination of monosaccharides after separation by chromatography. Paper and thin-layer chromatography for quantitative analysis are rather time consuming and laborious. The gas chromatographic procedure was applied successfully for the analysis of sugars in soil hydrolysates by OADES et at. (7). Preparation of the various derivatives for gas chromatography still requires many steps, much handling of the sample, and considerable time, although final analysis of the product derivative is accomplished in an hour or two.     

Spectrophotometric determination of allylisothiocyanate in mustard seed oil.

Allylisothiocyanate is determined spectrophotometrically after reaction with 2,3-dichloro-1,4-naphthoquinone. For pure samples, the color intensity is proportional to allylisothiocyanate content in the range 0.8-3.0 mg/ml reaction mixture. A modified procedure is used to estimate allylisothiocyanate content of mustard seed oil. The reaction is linear for allylisothiocyanate concentrations in the range 40-240 mug/ml reaction mixture. Two mustard seed oil samples contained 0.995 not equal to 0.020 and 0.981 not equal to 0.019% allylisothiocyanate.     

Determination of novobiocin residues in milk, blood, and tissues by liquid chromatography

Residues of novobiocin in milk, blood, and tissues can be detected by microbiological tests but cannot be distinguished from other antibiotics. A simple liquid chromatographic (LC) method was developed for identification of residues. Tissues were blended and milk and blood serum were mixed with 0.2M NH4H2PO4. The mixture was deproteinized by adding aqueous methanol and filtering. The LC apparatus consisted of a variable wavelength detector, set at 340 nm, an automatic loop injector, and a C18 column with guard cartridge. The flow rate was 1 mL/min and the solvent mixture of 0.01M H3PO4-acetonitrile-methanol was programmed from 50 + 0 + 50 (0-1 min) to 20 + 80 + 0 (20 min). Novobiocin was concentrated directly by solid-phase extraction on the analytical column. Five or more 200 microL aliquots of the filtrate in water-methanol (1 + 1) (adjusted if necessary) were injected with the column solvent at 50 + 0 + 50. After the final injection, the program was run to completion. Recoveries were 90-100% with sensitivities of 0.05 ppm or less. The procedure should be adaptable for use with formulations and feeds.     

A manual colorimetric procedure for measuring ammonium nitrogen in soil and plant Kjeldahl digests

Abstract

The measurement of NH4⁺‐N in soil, and plant digests is one of the greatest needs in laboratories conducting agricultural and environmental research. Many laboratories do not have access to automated equipment for colorimetric analysis of soil and plant digests. The objective of this research was to modify an automated colorimetric analysis procedure for determining NH₄+‐N in soil and plant digests for manual use, and compare the proposed technique with the standard distillation‐titration technique. The modified procedure is based on the color reaction between NH₄ ⁺‐ and a weakly alkaline mixture of Na salicylate and a chlorine source in the presence of Na nitroprusside. Wavelength scans indicated a very well defined peak for determinations at 650 nm. Time scans showed that color development in the manual procedure was rapid, 12 to 40 minutes depending on temperature, and that the color development remained stable for at least 120 minutes. Regression analysis of the results from 18 soil and 20 plant tissue sample determinations by distillation‐titration and the proposed method indicated NH₄ ⁺ ‐N recoveries of 99% or higher. The results obtained using the colorimetric procedure were very similar to the values obtained by distil ling and titrating the digests for both soil and plant samples as indicated by the large coefficients of determination (R² = 0.99).     

Simplification of the standard method for determination of non-exchangeable NH4−N in soil

The widely-used Silva-Bremner method for determining non- exchangeable NH₄?N in the soil was modified so as to reduce reagent requirement and the time taken to carry out the analyses. In the modification, boiling the mixture of soil and KOBr on a hot plate was replaced with heating the soil/KOBr mixture, directly placed in centrifuge tubes, in a boiling water bath or a microwave oven. The best procedure is to keep the soil/KOBr mixture for 10 minutes in the water bath or for 10 minutes at 50% of full power in a microwave oven. The modified method gave similar values to the standard method on 4 quite different soils and also slightly increased reproducibility.     

Optimum conditions for formation of aflatoxin M1-trifluoroacetic acid derivative

Because thin-layer chromatographic (TLC) confirmation of identity and reverse-phase liquid chromatographic (LC) determination with fluorescence detection of aflatoxin M1 both require the derivative formed in the reaction of M1 and trifluoroacetic acid (TFA), various reaction conditions were studied to obtain complete derivative formation. Of the various organic solvents tested, the reaction between M1 and TFA proceeded best in the nonpolar solvents hexane and isooctane. Other parameters investigated were reaction temperature and time, aflatoxin M1 concentration, and solvent volume. The following procedure is considered optimum: 200 microL each of hexane and trifluoroacetic acid are mixed with M1 standard in a silylated glass vial or with milk residue in a regular glass vial with a Teflon-lined screw cap and heated 10 min at 40 degrees C. The mixture is evaporated to dryness under N2, and the derivative is saved for TLC or LC. No unreacted aflatoxin M1 was detected by reverse-phase LC after this procedure was incorporated for analysis of milk samples.     

Thin layer chromatographic confirmation of aflatoxin M1 extracted from milk

Aflatoxin M1 can be confirmed directly on a thin layer plate by reacting the toxin with a mixture of reagents containing p-anisaldehyde. This confirmatory procedure requires only 2 elutions in the same direction using 2 different solvents. The mixture containing p-anisaldehyde is overspotted on M1 after the plate has been developed in toluene-ethyl acetate-ethyl ether-formic acid (25 + 35 + 40 + 5). The plate is heated at 110 degrees C for 10 min and then developed in hexane-acetone-chloroform (15 + 50 + 35). The Rf value of the green fluorescent derivative is less than that of the M1 standard. This confirmatory procedure requires only one-dimensional TLC, so several sample extracts and the standard can be run simultaneously. The minimum detectable quantity of aflatoxin M1 on the TLC plate with this test is 0.3 ng. p-Anisaldehyde reagent solution may also be used as a spray reagent for the confirmation of aflatoxin M1. The procedures described were satisfactory for confirming the mycotoxin in spiked samples of powdered and liquid milk.     

Liquid chromatographic analysis of dehydroacetic acid and its application to wines

A simple liquid chromatographic procedure is described for determination of the preservative dehydroacetic acid (DHA) in wine. No cleanup procedure is necessary; the sample is injected onto an NH2 column without further pretreatment. The mobile phase is a 9 + 1 mixture of acetonitrile-sodium acetate buffer adjusted to pH 4 with acetic acid. Total run time is less than 10 min. Dehydroacetic acid is determined by using the absorbance at 307 nm; the detection limit is estimated to be 0.2 ppm. The method may also be suitable for detecting DHA in a variety of foodstuffs in low concentrations.     

An Evaluation of the Loss-on-Ignition Method for Determining the Soil Organic Matter Content of Calcareous Soils

The Loss-on-Ignition (LOI) method is widely employed for measuring the organic matter (OM) content of soil samples. There is a risk of carbonate losses when calcareous soil samples are analyzed through LOI, but this has never been investigated in detail. Moreover, a worldwide standard protocol for determining the carbonate content of soils is not available. The aims of this study were (i) to evaluate two commonly employed carbonate analysis procedures using calcareous and non-calcareous soil samples: the gravimetric method with (GMF) and without (GM) the addition of the antioxidant iron(II) chloride (FeCl₂) and the acetic acid dissolution procedure (AAD); (ii) to evaluate the effect of ignition temperature on losses of pure calcite, calcite-quartz and calcareous soil samples. We found that the average apparent carbonate content of the non-calcareous soils was greatest for the GMF method followed by the AAD procedure. The GM method showed the smallest apparent carbonate contents. For the calcite-quartz sand mixture, ignition losses started at 600°C and increased with temperature in a sigmoidal way. LOI values stabilized at 750°C when 80% of the carbon dioxide was released. We recommend the GM procedure for carbonate analysis because the apparent carbonate contents of the non-calcareous soil samples were smallest. Furthermore, we recommend an LOI temperature of 550°C because at this ignition temperature 99.8% of the total calcite fraction remains in the soil samples.     

Simultaneous determination of ochratoxin A and cyclopiazonic,mycophenolic, and tenuazonic acids in cornflakes by solid-phase microextraction coupled to high-performance liquid chromatography

A solid-phase microextraction (SPME) method, coupled to liquid chromatography with diode array UV detection (LC-UV/DAD), for the simultaneous determination of cyclopiazonic acid, mycophenolic acid, tenuazonic acid, and ochratoxin A is described. Chromatographic separation was achieved on a propylamino-bonded silica gel stationary phase using acetonitrile/methanol/ammonium acetate buffer mixture (78:2:20, v/v/v) as mobile phase. SPME adsorption and desorption conditions were optimized using a silica fiber coated with a 60 microm thick polydimethylsiloxane/divinylbenzene film. Estimated limits of detection and limits of quantitation ranged from 3 to 12 ng/mL and from 7 to 29 ng/mL, respectively. The method has been applied to cornflake samples. Samples were subjected to a preliminary short sonication in MeOH/2% KHCO(3) (70:30, v/v); the mixture was evaporated to near dryness and reconstituted in 1.5 mL of 5 mM phosphate buffer (pH 3) for SPME followed by LC-UV/DAD. The overall procedure had recoveries (evaluated on samples spiked at 200 ng/g level) ranging from 74 +/- 4 to 103 +/- 9%. Samples naturally contaminated with cyclopiazonic and tenuazonic acids were found; estimated concentrations were 72 +/- 9 and 25 +/- 6 ng/g, respectively.     

Determination of molybdenum in plant tissue by graphite furnace atomic absorption spectrophotometry (GFAAS)

Abstract

The analysis of molybdenum in plant tissue using graphite furnace atomic absorption spectrophotometry (GFAAS) is described. The method involved wet digestion using a mixture of sulphuric, nitric and perchloric acids followed by a solvent extraction procedure. Molybdenum was extracted into di‐iso butyl ketone (DIBK) as the iron‐thiocyanate complex. The extract was then analysed for molybdenum by GFAAS.

The results of analyses of the reference plant materials (orchard leaves and citrus leaves) of the National Bureau of Standards (NBS) compared very well with the certified values. Other types of plant tissue were also analysed and the results correlated well with those obtained by an alternative method.     

不同植被配置模式对福建紫金山金铜矿废弃地土壤质量的恢复效果

为了了解植物治理措施对福建紫金山金铜矿矿山废弃地土壤质量的恢复效果,设置不同植被治理模式：马尾松＋胡枝子（模式A）、马尾松＋胡枝子＋香根草＋本地河滩草（模式B）、马尾松＋本地河滩草（模式C）、枫香＋本地河滩草（模式D）、桉树＋本地河滩草（模式E）和马尾松＋桉树＋本地河滩草（模式F）,同时设置矿区周边未经开采的对照样地,通过对不同配置模式下土壤理化性质的分析测定,比较其对矿山废弃地土壤质量恢复效果的差异。结果表明：恢复5 a后,不同植被配置模式土壤理化性质均有较大改善,除模式B和模式C外,其他模式土壤含水率均超过了对照,模式A、模式D、模式E和模式F分别达到对照的1.99、1.78、1.34倍和1.66倍;模式B、模式C和模式F可较快改善土壤团聚体结构,提高土壤抗蚀性;模式D可较快提高土壤各养分含量,有利于土壤营养状况的改善,恢复5 a后全N、水解N、全P和速效K分别为对照的54.98%、78.17%、63.18%和85.19%;模式F对土壤重金属Pb、Cd、Cr、Ni、Zn、Mn和Cu均有较好的修复效果;在植被配置模式中加入本地河滩草,可较大提高废弃地土壤的抗蚀能力。结合聚类分析结果可以得出,马尾松＋胡枝子＋香根草＋本地河滩草（模式B）、枫香＋本地河滩草（模式D）和马尾松＋桉树＋本地河滩草（模式F）3种模式较适合应用于紫金山矿山废弃地的植被恢复。     

Glomalin-related soil protein contains non-mycorrhizal-related heat-stable proteins, lipids and humic materials

Glomalin is reportedly a stable and persistent protein produced in copious quantities by mycorrhizal fungi and may be an important pool of organic N in soil. Glomalin-related soil protein (GRSP), however, is only operationally defined by its extraction method, and has been only poorly characterized at best. The goal of this study was to characterize the molecular structures within GRSP. Synchrotron-based X-ray absorption near-edge structure (XANES) spectroscopy and pyrolysis field-ionization mass spectrometry (Py-FIMS) revealed that GRSP contains a consortium of proteins along with many impurities. Employing proteomic techniques, we found that glomalin itself may be a thioredoxin-containing chaperone; however, no homologies with proteins or DNA of mycorrhizal origin were detected. Proteomics techniques further revealed that this fraction contains large amounts of soil-related heat-stable proteins and proteins of non-mycorrhizal origin. Results of this research show that the current extraction procedure that defines GRSP yields a mixture of compounds and thereby overestimates glomalin stocks when quantified using the Bradford assay. The chemical nature of glomalin has yet to be conclusively determined; it is unlikely that the chemical structure of glomalin can be elucidated from the mixture extracted as GRSP. Instead, an investigation into the specific biochemistry of immunoreactive assays currently used to define GRSP, followed by proteomic characterization of monoxenic mycorrhizal cultures may be required to advance our understanding of the chemical nature and agronomic significance of GRSP in soils.     

Colorimetric and spectrophotometric determination of total and non-phenolic alkaloids in ipeca and its formulations.

A simple method, requiring no chromatographic separation, is presented for the determination of the total and non-phenolic alkaloids in ipeca and its preparations. The complex formed between the alkaloid and methyl orange at pH 5.0 is extracted with chloroform and treated with 0.1N NaOH. The liberated dye, determined at 460 nm, is a measure of the total alkaloids. The chloroform phase remaining is treated with 0.1N H2SO4, and the acid extract is measured at 283 nm for the non-phenolic alkaloids, calculated as emetine. The proposed method was successfully applied to samples of ipeca powder, ipeca tincture, and 3 British Pharmaceutical Codex mixtures containing ipeca tincture, namely, ipecacuanha mixture, pediatric; ipecacuanha and ammonia mixture, pediatric; and belladonna and ipecacuanha mixture, pediatric. The proposed method compares favorably with the Egyptian Pharmacopoeia, British Pharmacopoeia, and USP methods and has a relative standard deviation of 1.54%. The present procedure is less time-consuming and requires about 45 and 90 min for the assay of ipeca tincture and powder, respectively. Only a small sample (0.2 mL tincture of 1.0 g powder) is required.