Identification of rat urinary and fecal metabolites of a new herbicide, pyribenzoxim

Rats were treated with pyribenzoxim (O-[2,6-bis[(4,6-dimethoxy-2-pyrimidinyl)oxy]benzoyl]oxime), a new herbicide, to investigate the related metabolites in urine and feces. Metabolites were identified using LC/MS (electrospray ionization) and GC/MS (electron impact ionization) following the relatively simple and rapid extraction and purification procedures. Three metabolites were identified in urine either from oral gavage or intravenous (iv) injection. They were benzophenone oxime (BO), benzophenone oxime glucuronide (BOG), and 2-hydroxy-6-(4,6-dimethoxypyrimidin-2-yloxy)benzoic acid (HDB). Benzophenone oxime was present in larger quantity than BOG and HDB in urine from oral treatment, while the case was opposite in urine from iv treatment. Glucuronide conjugate was confirmed unambiguously by enzyme hydrolysis. 2,6-Bis(4,6-dimethoxypyrimidin-2-yloxy)benzoic acid (KIH-2023) and benzophenone were identified in feces. Benzophenone was confirmed by GC/MS and HPLC/DAD since LC/MS could not produce an ESI spectrum. On the basis of the results obtained, a metabolic map of pyribenzoxim is proposed.     

Pharmacokinetics and urine metabolite identification of dehydroevodiamine in the rat

This study investigates the oral bioavailability and characterizes urine metabolites of dehydroevodiamine (DeHE), one of the bioactive alkaloids isolated from the fruit of Evodia rutaecarpa . A freely moving rat model coupled with an automated blood sample system was used to evaluate the pharmacokinetics of DeHE. High-performance liquid chromatography (HPLC), mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectrometry were applied to determine DeHE and its metabolites. The averaged oral bioavailability of DeHE (100 and 500 mg/kg) in the freely moving rats was approximately 15.35%. Cumulative fecal and urinary excretions of unchanged DeHE were 6 and 0.5%, respectively, after a single oral dose (500 mg/kg) of DeHE. The protein binding of DeHE in rat plasma was 65.6 ± 6.5%. Six metabolites, including five DeHE-O-glucuronides and one DeHE-sulfate, were identified after oral administration. The structures of two glucuronide conjugates, DeHE-10-O-glucuronide (M3) and DeHE-11-O-glucuronide (M4), and one sulfate conjugate, DeHE-12-sulfate (M6), were assigned. The findings indicate that the oral bioavailability of DeHE was much higher than that of evodiamine, and hydroxylation and conjugative metabolism were essential for the urinary elimination of DeHE.     

Quantification of soy isoflavones, genistein and daidzein, and conjugates in rat blood using LC/ES-MS.

Genistein is the principal soy isoflavone to which the putative beneficial effects of soy consumption have been attributed; however, the possibility of adverse biological effects (e.g., estrogenic, antithyroid) has also been raised. This paper describes development and validation of a simple and sensitive analytical method for the determination of genistein in the blood of rats receiving dietary genistein (<0.5-1250 microg of genistein aglycone/g of chow). The method uses serum/plasma deproteination, liquid-liquid extraction, deuterated genistein and daidzein internal standards, isocratic LC separation, and electrospray mass spectrometric quantification using selected ion monitoring. Extraction efficiency is approximately 85%, the detection limits for genistein and daidzein from 50 microL of rat blood are approximately 5 nM, and the limit of quantification is approximately 15 nM. Interassay precision (relative standard deviation 4.5-4.6%) and intraassay precision (3.3-6.7%) were determined from replicate analysis of a spiked control and an incurred serum sample. The distribution of conjugated and unconjugated forms of genistein in the blood of rats was determined using selective enzyme hydrolysis. The glucuronide was the predominant metabolite (>90%), and only small amounts of the sulfate conjugate and the aglycone were observed at all dose levels. No evidence for additional metabolites was obtained. The 7- and 4'-glucuronide conjugates of genistein were identified using electrospray mass spectrometry and (1)H NMR. Total blood genistein ranged from <15 nM in animals fed soy-free control diet to as high as 8.9 microM in male rats fed 1250 microg of genistein/g of chow and encompasses blood isoflavone levels observed in humans consuming a typical Asian diet and nutritional supplements (0.1-1 microM) and infants consuming soy formulas (2-7 microM).     

Effect of alkali and enzymatic pretreatments of Eucommia ulmoides leaves and barks on the extraction of gutta percha

This paper exploited a novel method of single solvent recycling plus enzymatic pretreatment. Petroleum ether (60-90 degrees C) was an ideal solvent to extract Eucommia gum at high temperature (approximately 80 degrees C) and to precipitate the gum (flocculent) as the temperature of the solution fell to below 40 degrees C. The gutta percha was almost completely precipitated from solution as the temperature cooled down to below 0 degree C. After filtration and recovery of the precipitated gum, the filtrate, petroleum ether, was applied to an activated carbon column to remove dissolved impurities and reused for next gutta percha extraction. Because impurities were kept in solution, the precipitated gutta percha was highly pure. In an experiment of enzyme hydrolyzing the plant cell wall, cuticle layers on the surfaces of leaves prevented cellulase from approaching to and hydrolyzing the cellulose of the cell wall. NaOH (1%) at 70 degrees C efficiently degraded the cuticle layer, which greatly improved the enzymatic hydrolysis of cellulose within eucommia leaves. Gutta percha that was extracted from the alkali- and cellulase-treated leaves had a degree of polymerization as high as in the leaves, and the yield increased from 2.5% of milled leaves up to over 3.2%. The tensile strength of obtained gutta percha increased from 0.02 MPa of milled Eucommia leaves to 60.5 MPa, the breakage extensibility ranged from about 0 to 24%, and the tearing strength ranged from 0.5 to 36 kN/m.     

Metabolic profiling of flavonol metabolites in human urine by liquid chromatography and tandem mass spectrometry

Twenty-one flavonol metabolites have been identified by LC/ESI-MS/MS in human urine, including isomers, after the consumption of cooked onions. Metabolites identified include quercetin monoglucuronides, methyl quercetin monoglucuronides, a quercetin monoglucuronide sulfate, quercetin diglucuronides, a methyl quercetin diglucuronide, quercetin glucoside sulfates, methyl quercetin, quercetin, and kaempferol monoglucuronides. The fragmentation patterns of flavonol metabolites obtained by MS/MS were distinctive for some isomers, indicating that fragmentation patterns may be useful predictors of conjugation position. Two isomers of sulfate quercetin glucosides were also found in urine, suggesting that many of the quercetin glucosides in onion are absorbed intact and undergo metabolism to the sulfate conjugate. Additionally, the interindividual variation in urinary quercetin metabolite profiles was determined by comparing the relative level of six different quercetin metabolites excreted in the urine of healthy volunteers. The ranges of quercetin metabolites excreted were similar among volunteers, yet notable differences in the levels of metabolites among individuals were observed. This study demonstrates the potential of monitoring the range of quercetin metabolites to reveal information on interindividual biotransformation capacity in response to dietary manipulations and as a biomarker for flavonol consumption.     

Acid- and base-catalyzed hydrolysis of chloroacetamide herbicides

Despite the prevalence of chloroacetamides as herbicides, little is known about the rates or products of acid- or base-catalyzed hydrolysis of these compounds. Mechanisms of acid-catalyzed reactions may parallel those catalyzed by (hydr)oxide minerals, while base-catalyzed processes have as important counterparts reactions with environmental nucleophiles (such as reduced sulfur species). The current study systematically investigates how the structure of nine chloroacetamides affects their reactivity in 2 N NaOH, 2 N HCl, or 6 N HCl at 25 or 85 degrees C. Base-catalyzed hydrolysis proceeds either through an intermolecular SN2 reaction to hydroxy-substituted derivatives or (in a few cases) through amide cleavage, while both amide and ether group cleavages are observed under acidic conditions. Our results reveal that subtle differences in chloroacetamide structure [notably the type of (alkoxy)alkyl substituent] can dramatically influence reactivity and reaction mechanism. Hydroxy-substituted, morpholinone, and secondary aniline derivatives were identified upon reaction for several years in deionized water at circumneutral pH.     

病死猪酶解及超声波预处理工艺优化

病死畜禽资源化利用是解决病死畜禽污染的一条重要途径。为探索病死猪肉酶解工艺条件,该文以猪肉为原料,以胰蛋白酶为试验用酶,以水解度为指标,选取加酶量、底物浓度、pH值、温度作为试验因素,通过单因素试验初步确定了胰蛋白酶的水解条件,并分析了各因素对酶解反应的影响规律。应用Box-Behnken中心组合设计建立数学模型,以水解度为响应值,进行了四因素三水平的响应面优化试验,确定了最佳酶解工艺条件,并通过响应面模型的曲面图直观地分析了各影响因素之间的交互作用。在此基础上,探索频率为20 k Hz,功率为500 W的超声波预处理猪肉20 min对酶解效果的影响,并应用扫描电子显微镜在微观结构上对其原因进行了分析。结果表明,各因素对酶解反应的影响大小依次为:加酶量温度pH值底物浓度,在试验范围内得到的酶解最佳工艺条件为:加酶量为1.15%(质量分数)、底物质量浓度为80.5 g/L、pH值为7.96、温度为40.6℃,酶解1 h的预测水解度可达16.74%,验证试验水解度为16.77%,表明试验结果与软件分析结果相符,最佳水解时间为6 h,此时的水解度为28.91%。超声波预处理后,最佳水解时间为4 h,水解度达到32.86%,因此超声波预处理能缩短水解周期2 h,提高水解度4个百分点。由此可见,应用超声波预处理可以提高酶解效率,缩短工作时间。     

Development of a sensitive and specific solid phase extraction--gas chromatography-tandem mass spectrometry method for the determination of elenolic acid, hydroxytyrosol, and tyrosol in rat urine

A novel gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed, using an ion trap mass spectrometer, for the simultaneous determination of olive oil bioactive components, elenolic acid, hydroxytyrosol, and tyrosol, in rat urine. Samples were analyzed by GC-MS/MS prior to and after enzymatic treatment. A solid phase extraction sample pretreatment step with greater than 80% analytical recoveries for all compounds was performed followed by a derivatization reaction prior to GC-MS/MS analysis. The calibration curves were linear for all compounds studied for a dynamic range between 1 and 500 ng. The limit of detection was in the mid picogram level for tyrosol and elenolic acid (300 pg) and in the low picogram level for hydroxytyrosol (2.5 pg). The method was applied to the analysis of rat urine samples after sustained oral intake of oleuropein or extra virgin olive oil as a diet supplement.     

Comparison of HCl extraction versos total iron analysis for iron tissue analysis

Extraction of Fe from fresh leaves with 0.1 N HCl proved to be a better indicator of the Fe status of a variety of ornamental tropical foliage and flowering plants compared to total Fe, 0.1 N HCl‐ether, and IN HCl extraction. It consistently gave higher correlations (r = 0.73 to 0.95 depending on the species) with chlorophyll concentration than the other methods tested. However, even 0.1 N HCl extraction did not distinguish levels of Fe deficiency accurately when compared between species.     

Improving xanthophyll extraction from marigold flower using cellulolytic enzymes

In this work is studied the effect of a noncommercial enzyme preparation on xanthophyll extraction from marigold flower (Tagetes erecta). The enzymatic extract was synthesized by endogenous microorganisms previously isolated and identified as Flavobacterium IIb, Acinetobacter anitratus, and Rhizopus nigricans. The results show that the extraction yield depends directly on the extent of the enzymatic hydrolysis of cell walls in the flower petals and that it is possible to reach yields in excess of those previously reported for treatments with commercially available enzymes (29.3 g/kg of dry weight). HPLC analysis of the product indicates that the original xanthophyll profile is not altered. The enhanced extraction system appears to be very competitive when compared to the traditional process and current alternatives.     

Enzyme-assisted extraction of moniliformin from extruded corn grits

Water has been known to be the ideal solvent for moniliformin but is not suitable to extract this toxin from cooked matrices due to instant swelling upon addition of the solvent. In this study, an improved method to extract moniliformin from extruded corn grits using alpha-amylase was developed. In an effort to optimize the method, the efficacy of using a protease was also studied. Treatment with alpha-amylase resulted in a clear solution with decreased suspended solid content as measured by transmittance (%T), which improved from 0 to 96% in 10 min. The detected level of moniliformin from extruded corn grits was increased to 4.02 mug/g when extracted with 1% tetrabutylammonium hydrogen sulfate following alpha-amylase treatment compared to 2.56 microg/g when it was extracted with 90% acetonitrile without enzyme treatment. The average recovery of moniliformin from extruded corn grits was 96% when alpha-amylase was used in the extraction procedure. Overall, the amounts of moniliformin detected in extruded corn grits increased significantly by using enzyme hydrolysis. Chromatographic separation was also benefited by lesser interference and improved peaks.     

Analysis of pesticide residues by chemical derivatization. II. N-methylcarbamates in natural water and soils.

A method for the quantitative determination of several N-methylcarbamates in natural waters and the applicability of the derivative to soil samples using a previously published extraction procedure are described. After extraction of the carbamates from the substrate, the carbamates are hydrolyzed in a 10% methanol-potassium hydroxide solution to form the phenolic hydrolysis products, which are isolated and derivatized with pentafluorobenzyl (PFB) bromide to produce the PFB ether derivatives. The PFB derivatives are cleaned up and fractionated on a silica gel microcolumn and determined by electron capture gas-liquid chromatography (GLC). Eight organophosphate pesticides and 2 phthalate acid esters that hydrolyze to phenols or phthalic acid were evaluated as potential interferences and were found not to interfere with any of the carbamates tested. Quantitative determinations of 0.1 mug carbofuran and 3-ketocarbofuran and 0.5 mug carbaryl, metmercapturon, and Mobam in a 1 L water sample are possible. Propoxur was not determined at levels less than 1 mug/L due to the short GLC retention time of the derivative and interferences from the reagents at the lower levels.     

Changes in the content of bioactive polyphenolic compounds of olive mill wastewater by the action of exogenous enzymes

The aim behind the present research is to develop an enzymatic treatment for olive mill wastewater (OMW) to release high amounts of simple phenolics having high antioxidant value. OMW was hydrolyzed by a mixed enzyme preparation rich in β-glucosidase produced by Aspergillus niger . This research shows that A. niger β-glucosidase played a major role in the release of simple phenolic compounds from OMW. These compounds were recovered by ethyl acetate extraction and identified by HPLC and LC-MS. The main identified phenolic compound is hydroxytyrosol. The results of enzymatic hydrolysis of OMW under optimum conditions indicated a maximum hydroxytyrosol concentration of 2.9 g L(-1) compared to 0.015 g L(-1) contained in the control (test without added enzyme). The above results prove that OMW is a potential substrate for producing hydroxytyrosol through enzymatic hydrolysis of its glycosides.     

Effect of gelatinization and hydrolysis conditions on the selectivity of starch hydrolysis with alpha-amylase from Bacillus licheniformis

Enzymatic hydrolysis of starch can be used to obtain various valuable hydrolyzates with different compositions. The effects of starch pretreatment, enzyme addition point, and hydrolysis conditions on the hydrolyzate composition and reaction rate during wheat starch hydrolysis with alpha-amylase from Bacillus licheniformis were compared. Suspensions of native starch or starch gelatinized at different conditions either with or without enzyme were hydrolyzed. During hydrolysis, the oligosaccharide concentration, the dextrose equivalent, and the enzyme activity were determined. We found that the hydrolyzate composition was affected by the type of starch pretreatment and the enzyme addition point but that it was just minimally affected by the pressure applied during hydrolysis, as long as gelatinization was complete. The differences between hydrolysis of thermally gelatinized, high-pressure gelatinized, and native starch were explained by considering the granule structure and the specific surface area of the granules. These results show that the hydrolyzate composition can be influenced by choosing different process sequences and conditions.     

Hydrolysate preparation for analysis of amino acids in sorghum grains: effect of oxidative pretreatment

Sorghum samples were either untreated or oxidized with performic acid (PA) before hydrolysis, and their amino acid contents were determined by cation exchange chromatography using an amino acid analyzer. HCl was used to destroy excess PA. Oxidative pretreatment of the samples resulted in increased yields of Cys (as cysteic acid), Met (as Met dioxide), and His, destroyed Tyr and Phe, and resulted in the appearance of an extraneous peak which most likely consisted of halogenation by-products (HBP) of Tyr and Phe. The destruction of Tyr and Phe occurred despite the presence of phenol, a halogen scavenger, in both the PA and hydrolysis reagents. The higher His values observed in all oxidized samples most likely resulted from the co-elution of His with Tyr and Phe HBP. It was concluded that the complete (except Trp) amino acid content of a feedstuff cannot be accurately determined from only one oxidized hydrolysate preparation by using this particular procedure.     

Accurate and Precise Measurement of Organic Carbon Content in Carbonate-Rich Soils

Accurate measurement of soil organic carbon (SOC) is dependent on precise and fast methods for the separation of organic and inorganic carbon. The widely used methods involving thermal decomposition of soil samples at a specific temperature in an automated carbon (C) analyzer are susceptible to interference by carbonates and overestimation of organic C, and thus removal of carbonates by acid pretreatment of samples is recommended. Two carbonate-removal pretreatments including hydrochloric (HCl) acid addition and HCl fumigation are compared using the calcium carbonate (CaCO₃) standard and soil samples of varying SOC contents. Both pretreatment methods provided similar measurements of organic C, indicating that both methods are efficient in removal of carbonates present in the soil. However, the HCl fumigation method exhibited greater accuracy and precision compared to the HCl addition method. Hence, SOC measurement procedure involving HCl fumigation as a pretreatment for the removal of carbonates is recommended for carbonate-rich soils.     

Comparison of Extraction Procedures Used in Determination of Phosphorus Species by 31P‐NMR in Chilean Volcanic Soils

Abstract

Four procedures were employed to extract phosphorus (P) from volcanic soils for ³¹P‐NMR experiments. The procedures involve 0.5 M NaOH extraction, 0.5 M NaOH and Chelex 100 cation exchange resin extraction, NaOH‐EDTA extraction, and HCl‐NaOH two step sequential extraction with Chelex 100 clean up. Results showed that inorganic‐P, monoester‐P, diester‐P and pyrophosphate were present. Their detection was dependent on the extraction procedure used.

The NaOH procedure gives only a broad and vaguely defined signal with poor signal to noise ratio. The incorporation of Chelex 100 in the extraction enhanced the signal to noise ratio and allowed the distinction of inorganic‐P, monoester‐P, diester‐P and pyrophosphate. The two step sequential extraction involving HCl, NaOH, and Chelex 100 significantly improve the signal to noise ratio. The NaOH‐EDTA extraction procedure is efficient only in samples with low OC contents.

When soils have low OC content, any of the four extraction procedures can be successfully used. If the OC and the Fe concentration in the extracted solutions are high, the Chelex 100 became essential in clean up the metallic ions. Both the NaOH and Chelex 100 and the HCl‐NaOH‐Chelex produced satisfactory results and the later procedure by far the best resolved spectra.     

Identification of nosiheptide in feeds and detection of residues in animal tissues

Nosiheptic is determined in fermentation broths of Streptomyces actuosus either by a microbiological method using Staphylococcus aureus or, more easily, by an automated colorimetric method. The results obtained with both methods correspond well for concentrations greater than 100 microgram/mL with a standard deviation of 1-3%. For determination of nosiheptide as a feed additive, the microbiological assay is made more specific by pretreatment with petroleum ether and 1N HCl. Standard deviation is less than 4%, and the assay is sensitive to 1 ppm. Nosiheptide is identified in feed containing other frequently used antibiotics by thin layer chromatography with bioautography; sensitivity is 1 ppm. The absence of traces of nosiheptide in tissues of treated swine and broiler is confirmed by microbiological diffusion, sensitive to 0.025 ppm.     

Optimization of a liquid chromatography-tandem mass spectrometry method for quantification of the plant lignans secoisolariciresinol, matairesinol, lariciresinol, and pinoresinol in foods

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of the four major enterolignan precursors [secoisolariciresinol, matairesinol, lariciresinol, and pinoresinol] in foods. The method consists of alkaline methanolic extraction, followed by enzymatic hydrolysis using Helix pomatia (H. pomatia) beta-glucuronidase/sulfatase. H. pomatia was selected from several enzymes based on its ability to hydrolyze isolated lignan glucosides. After ether extraction samples were analyzed and quantified against secoisolariciresinol-d8 and matairesinol-d6. The method was optimized using model products: broccoli, bread, flaxseed, and tea. The yield of methanolic extraction increased up to 81%, when it was combined with alkaline hydrolysis. Detection limits were 4-10 microg/(100 g dry weight) for solid foods and 0.2-0.4 microg/(100 mL) for beverages. Within- and between-run coefficients of variation were 6-21 and 6-33%, respectively. Recovery of lignans added to model products was satisfactory (73-123%), except for matairesinol added to bread (51-55%).     

Purification and characterization of a new endoglucanase from Aspergillus aculeatus

Endoglucanase has been isolated from Aspergillus aculeatus. The purified enzyme showed a single band and had a molecular weight of 45,000 Da as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a specific activity of 1.4 units/mg. The purified enzyme was identified as endoglucanase, showing a high specific activity toward CM-cellulose and low specific activity toward Avicel. The activity of the isolated enzyme was optimum at a pH of 5.0 and temperature of 40 degrees C, respectively. The isoelectric point of the enzyme was 4.3. T(m) was found to be 57 degrees C. The treatment of the endoglucanase with diethylpyrocarbonate resulted in the modification of the histidine residues present in the enzyme, with a concomitant loss of 70% of the original enzymatic activity. However, carbodiimide completely inactivated the endoglucanase. The results show that the enzyme is able to sustain 50% of its activity even when heated at 90 degrees C for a period of 5 h. Endoglucanase can be used in the controlled hydrolysis of cellulose and other cellulose-rich foods. It can be used in the development of targeted functional foods from agrimaterials for value addition in the food chain.