Identification and quantitation of sulfamethazine metabolites by liquid chromatography and gas chromatography-mass spectrometry

Methods for the identification and quantitation of carbon-14 labeled sulfamethazine [4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzenesulfonamide], N4-acetylsulfamethazine, the N4-glucose conjugate of sulfamethazine, and desaminosulfamethazine in swine tissue are described. Tissues are ground and extracted with methanol, and the 14C-labeled compounds are purified by XAD-2 column chromatography and C-18 reverse phase liquid chromatography (LC) and the 14C-labeled compounds are then methylated and identified by gas chromatography-mass spectrometry analysis. Quantitation is accomplished by measuring the amount of 14C-activity that cochromatographs (C-18 reverse phase LC) with reference compounds.     

新型除草剂丙酯草醚的微量合成

从苯酚、对氨基苯甲酸和2-甲硫基-4,6-二氯嘧啶出发,建立了包含取代、氧化和缩合等7步反应的丙酯草醚除草剂微量制备方法。最终产物经制备HPLC纯化,其收率为53%,化学纯度大于98%,用MS(EI)、HPLC-MS1、H-NMR、UV等方法确认了丙酯草醚的结构。所建立的丙酯草醚微量制备方法,为放射性同位素标记丙酯草醚的合成奠定了基础。     

Formation of arginine modifications in a model system of Nα-tert-butoxycarbonyl (Boc)-arginine with methylglyoxal

The present study deals with the mechanistic reaction pathway of the α-dicarbonyl compound methylglyoxal with the guanidino group of arginine. Eight products were formed from the reaction of methylglyoxal with N(α)-tert-butoxycarbonyl (Boc)-arginine under physiological conditions (pH 7.4 and 37 °C). Isolation and purification of substances were achieved using cation-exchange chromatography and preparative high-performance liquid chromatography (HPLC). Structures were verified by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry. 2-Amino-5-(2-amino-4-hydro-4-methyl-5-imidazolinone-1-yl)pentanoic acid (3) was determined as the key intermediate precursor within the total reaction scheme. Kinetic studies identified N(δ)-(5-methyl-4-oxo-5-hydroimidazolinone-2-yl)-L-ornithine and N(7)-carboxyethylarginine as thermodynamically more stable products from compound 3. Further mechanistic investigations revealed an acidic hydrogen at C-8 of compound 3 to trigger aldol condensations. This reactivity of compound 3 allowed for the addition of another molecule of methylglyoxal to form products, such as N(δ)-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)-l-ornithine and argpyrimidine.     

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.     

Aroma extract dilution analysis of Cv. Marion (Rubus spp. hyb) and Cv. Evergreen (R. laciniatus L.) blackberries

Cultivar Marion and Evergreen blackberry aromas were analyzed by aroma extract dilution analysis. Sixty-three aromas were identified (some tentatively) by mass spectrometry and gas chromatography-retention time; 48 were common to both cultivars, and 27 have not been previously reported in blackberry fruit. A comparison of cultivars shows that both have comparable compound types and numbers but with widely differing aroma impacts, as measured by flavor dilution (FD) factors. Ethyl 2-methylbutanoate, ethyl 2-methylpropanoate, hexanal, furanones (2,5-dimethyl-4-hydroxy-3-(2H)-furanone, 2-ethyl-4-hydroxy-5-methyl-3-(2H)-furanone, 4-hydroxy-5-methyl-3-(2H)-furanone, 4,5-dimethyl-3-hydroxy-2-(5H)-furanone, and 5-ethyl-3-hydroxy-4-methyl-2-(5H)-furanone), and sulfur compounds (thiophene, dimethyl sulfide, dimethyl disulfide, dimethyl trisulfide, 2-methylthiophene, and methional) were prominent in Evergreen (FD 512-2048). Except for ethyl 2-methylpropanoate, these same compounds were also prominent in Marion, but the FD factors varied significantly (FD 8-256) from Evergreen. The aroma profile of blackberry is complex, as no single volatile was unanimously described as characteristically blackberry.     

Synthesis of dimethyl derivatives of imidazolinone herbicides: their use in efficient gas chromatographic methods for the determination of these herbicides

The dimethyl derivatives of imazaquin, imazapyr, imazmethapyr, imazethapyr, 2-[4,5 dihydro-1, 4-dimethyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-5-methoxymethyl- 3-pyridine carboxylic acid, 2-[4,5-dihydro-1,4 -dimethyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-4-methyl benzoic acid, and 2-[4,5-dihydro-1,4-dimethyl-4-(1-methyl ethyl)-5-oxo-1H-imidazol-2-yl]-5-methyl benzoic acid were prepared and fully characterized. The availability of these derivatives has led to the development of efficient and multiresidue gas chromatographic methods for trace level analysis of imidazolinone herbicides in matrixes such as water, soybean, and soil.     

Quantitative studies and sensory analyses on the influence of cultivar, spatial tissue distribution, and industrial processing on the bitter off-taste of carrots (Daucus carota l.) and carrot products

Although various reports pointed to 6-methoxymellein (1) as a key player imparting the bitter taste in carrots, activity-guided fractionation experiments recently gave evidence that not this isocoumarin but bisacetylenic oxylipins contribute mainly to the off-taste. Among these, (Z)-heptadeca-1,9-dien-4,6-diyn-3-ol (2), (Z)-3-acetoxy-heptadeca-1,9-dien-4,6-diyn-8-ol (3), and (Z)-heptadeca-1,9-dien-4,6-diyn-3,8-diol (falcarindiol, 4) have been successfully identified. In the present study, an analytical procedure was developed enabling an accurate quantitation of 1-4 in carrots and carrot products. To achieve this, (E)-heptadeca-1,9-dien-4,6-diyn-3,8-diol was synthesized as a suitable internal standard for the quantitative analysis of the bisacetylenes. On the basis of taste activity values, calculated as the ratio of the concentration and the human sensory threshold of a compound, a close relationship between the concentration of 4 and the intensity of the bitter off-taste in carrots, carrot puree, and carrot juice was demonstrated, thus showing that compound 4 might offer a new analytical measure for an objective evaluation of the quality of carrot products. Quantitative analysis on the intermediate products in industrial carrot processing revealed that removing the peel as well as green parts successfully decreased the concentrations in the final carrot puree by more than 50%.     

Characterization of the aroma-active compounds in pink guava (Psidium guajava, L.) by application of the aroma extract dilution analysis

The volatiles present in fresh, pink-fleshed Colombian guavas ( Psidium guajava, L.), variety regional rojo, were carefully isolated by solvent extraction followed by solvent-assisted flavor evaporation, and the aroma-active areas in the gas chromatogram were screened by application of the aroma extract dilution analysis. The results of the identification experiments in combination with the FD factors revealed 4-methoxy-2,5-dimethyl-3(2 H)-furanone, 4-hydroxy-2,5-dimethyl-3(2 H)-furanone, 3-sulfanylhexyl acetate, and 3-sulfanyl-1-hexanol followed by 3-hydroxy-4,5-dimethyl-2(5 H)-furanone, ( Z)-3-hexenal, trans-4,5-epoxy-( E)-2-decenal, cinnamyl alcohol, ethyl butanoate, hexanal, methional, and cinnamyl acetate as important aroma contributors. Enantioselective gas chromatography revealed an enantiomeric distribution close to the racemate in 3-sulfanylhexyl acetate as well as in 3-sulfanyl-1-hexanol. In addition, two fruity smelling diastereomeric methyl 2-hydroxy-3-methylpentanoates were identified as the ( R,S)- and the ( S,S)-isomers, whereas the ( S,R)- and ( R,R)-isomers were absent. Seven odorants were identified for the first time in guavas, among them 3-sulfanylhexyl acetate, 3-sulfanyl-1-hexanol, 3-hydroxy-4,5-dimethyl-2(5 H)-furanone, trans-4,5-epoxy-( E)-2-decenal, and methional were the most odor-active.     

Liquid chromatographic determination of three sulfonamides in animal tissue and egg

Sulfonamides are widely used as a feed additive in animal production in Japan. The present paper is a determination of 3 sulfonamides: sulfamethazine (SMZ), sulfamonomethoxine [SMX, 4-amino-N-(3-methoxypyrazinyl)-benzenesulfonamide], and sulfadimethoxine (SDX) in animal tissue and egg by liquid chromatography (LC). Tissues were extracted with acetonitrile and fat was removed by liquid/liquid partition. The sulfonamides were purified by an ODS cartridge column; then each compound was separated by an ODS LC column and detected at 268 nm. Quantification levels were 0.02 ppm for SMZ and SMX, and 0.04 ppm for SDX; detection limits were 0.01 ppm for SMZ and SMX, and 0.02 ppm for SDX. Calibration curves were linear between 2 and 40 ng for SMZ and SMX, and between 4 and 80 ng for SDX. Recoveries from muscle and egg samples spiked with 1-2 micrograms/10 g were 81-98%.     

Ultimate fate of halosulfuron methyl and its effects on enzymatic and microbial activities in three differently textured soils

Halosulfuron methyl is a sulfonylurea herbicide used worldwide for weed control in sugarcane, maize, wheat, and rice production. Considering its environmental impact, this study evaluated the effects of soil type, application rate, and temperature on the dynamics of halosulfuron methyl degradation.Additionally, as soil microbes and enzymes are reliable indicators of the impacts of anthropogenic activities on soil health, the effects of halosulfuron methyl on soil enzymatic and microbial activiti...     

Structural elucidation of phototransformation products of azimsulfuron in water

The photodegradation of the sulfonylurea herbicide azimsulfuron, N-[[(4,6-dimethoxypyrimidin-2-yl)amino]carbonyl]-1-methyl-4-(2-methyl-2H-tetrazole-5-yl)-1H-pyrazole-5-sulfonamide (AZS), was studied in water at different wavelengths and in the presence of photocatalysts. AZS was rapidly degraded by UV light, affording three photoproducts. The main product, accounting for about 70% of photodegraded herbicide, was identified as 6-amino-5-[(4,6-dimethoxypyrimidin-2-yl)methylamino]-1,5,6,8-tetrahydro-7-oxa-8lambda(6)-tia-1,2,5,6-tetraza-azulen-4-one (ADTA) by single-crystal X-ray diffraction. With simulated sunlight irradiation, the reaction was slower and 2-amino-4,6-dimethoxypyrimidine (DPA) and 1-methyl-4-(2-methyl-2H-tetrazole-5-yl)-1H-pyrazole-5-sulfonamide (MPS), arising from a photohydrolytic cleavage of the sulfonylurea bridge, were the only byproducts observed. The reactions followed first-order kinetics. The addition of dissolved organic matter (DOM) did not modify significantly the AZS photodegradation rate. The presence of Fe2O3 accelerated more than twice the reaction rate affording two major products, DPA and MPS, together with minor amounts of N-[[(5-hydroxy-4,6-dimethoxypyrimidin-2-yl)amino]carbonyl]-1-methyl-4-(2-methyl-2H-tetrazole-5-yl)-1H-pyrazole-5-sulfonamide (AZS-OH). The greatest degradation rate was detected in the presence of TiO2. Only the photohydroxylation product AZS-OH was observed, which was transformed rapidly into oxalic acid.     

Investigation of the change in the flavor of a coffee drink during heat processing

Heat processing is responsible for the change in the flavor of a coffee drink. In this study, the application of gas chromatography-olfactometry of headspace samples (GCO-H) using the vapor fraction before and after heat processing of the coffee samples resulted in the detection of 12 odor-active peaks for which the flavor dilution (FD) factors changed. Eight potent odorants were identified from these peaks by gas chromatography-mass spectrometry (GC-MS). Among these components, methanethiol (putrid), acetic acid (sour), 3-methylbutanoic acid (sour), 2-furfuryl methyl disulfide (meaty), and 4-hydroxy-2,5-dimethyl-3(2H)-furanone (caramel-like) increased after heating of the coffee sample, whereas 2-furfurylthiol (roasty), methional (potato-like), and 3-mercapto-3-methylbutyl formate (roasty) decreased compared with the coffee sample before heat treatment. In addition, extensive studies have been carried out on the pH effects on the change in the concentration of 2-furfurylthiol during heat processing and in the pH range of 5-7; it was found that the concentration of this compound in the model solutions had significantly changed.     

Metabolic fate of isotopes during the biological transformation of carbohydrates to 2,5-dimethyl-4-hydroxy-3(2h)-furanone in strawberry fruits

Isotopically labeled D-glucose, D-fructose, 1-deoxy-D-fructose, and 6-deoxyhexoses were applied to detached ripening strawberry (Fragaria x ananassa) fruits, and the incorporation of the isotopes into the key strawberry aroma compounds 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF, 1) and 2,5-dimethyl-4-methoxy-3(2H)-furanone (DMMF, 2) was determined by gas chromatography-mass spectrometry. In contrast to previous reports the data clearly showed that 6-deoxy-D-fructose/6-deoxy-D-glucose and 1-deoxy-D-fructose are not natural precursors of the furanones. However, isotopically labeled 1 and 2 were observed after the application of [1-(2)H]-, [2-(2)H]-, and [6,6-(2)H(2)]-D-glucose as well as [U-(13)C(6)]-, [1-(13)C]-, [1-(2)H]-, [6,6-(2)H(2)]-D-fructose. The isotope label of [4-(2)H]-D-glucose was not recovered in the furanones. In contrast, [2-(2)H]-D-glucose was converted to [1- or 6-(2)H]-1 and [1- or 6-(2)H]-2 by the strawberry fruits. The observed isotope shift can be explained by the catalysis of phosphohexose isomerase in the course of the biogenesis of the hydroxyfuranone (1) and the methoxyfuranone (2) from D-glucose. Thus, the applied D-glucose is metabolized to D-fructose-6-phosphate prior to the transformation into the furanones.     

Aroma components of acid-hydrolyzed vegetable protein made by partial hydrolysis of rice bran protein

Hydrolyzed vegetable protein (HVP) was prepared from rice bran protein concentrate (RBPc) by partial hydrolysis with aqueous 0.5 N HCl at 95 degrees C for 12 or 36 h (H-RBPc-12 and H-RBPc-36, respectively). Aroma components of the RBPc and the HVPs were characterized by gas chromatography-olfactometry, gas chromatography-mass spectrometry, aroma extract dilution analysis, and calculation of odor activity values (OAVs). The predominant odorants in RBPc were 3-methylbutanal, hexanal, 2-aminoacetophenone, (E)-2-nonenal, phenylacetaldehyde, and beta-damascenone. Among these, the odor of 2-aminoacetophenone, present at 59 ng/g in RBPc, was reminiscent of the typical odor of RBPc. Most of the predominant odorants had higher log3FD factors in the H-RBPc-36 as compared to H-RBPc-12. Aroma impact compounds of H-RBPc-12 and H-RBPc-36 were 2-methoxyphenol (guaiacol), 4-hydroxy-2,5-dimethyl-3(2H)furanone, 3-hydroxy-4,5-dimethyl-2(5H)furanone (sotolon), vanillin, 3-methylbutanal, (E)-2-nonenal, 4-vinyl-2-methoxyphenol (p-vinylguaiacol), and beta-damascenone. Guaiacol had the highest OAV values of 2770 and 17650 in H-RBPc-12 and H-RBPc-36, respectively.     

Characterization of the bound volatile extract from baby kiwi (Actinidia arguta)

The glycosidically bound volatile fraction of baby kiwi ( Actinidia arguta ) was studied. Glycosidic precursors were isolated from juice by adsorption onto an Amberlite XAD-2 column. After enzymatic hydrolysis with Rapidase AR2000, the released aglycones were analyzed by GC-MS. Alcohols, terpenoids, and benzenoids were the most abundant compound classes. Aromatic compounds and norisoprenoids showed the highest concentrations. Major compounds were 2,5-dimethyl-4-hydroxy-3(2H)-furanone (Furaneol), benzyl alcohol, 3-hydroxy-β-damascone, hexanal, and (Z)-3-hexen-1-ol. Precursors of aroma compounds including benzoic acid, cinnamic acid, and coniferyl alcohol were also found. Eugenol, raspberry ketone, and 4-vinylguaiacol were identified for the first time in the fruit of an Actinidia species. The high concentration of 2,5-dimethyl-4-hydroxy-3(2H)-furanone in bound form (95.36 μg/kg) is particularly interesting and justifies further investigation.     

Degradation of primisulfuron by a combination of chemical and microbiological processes

Microbial degradation of the herbicide primisulfuron was investigated using enrichment cultures from contaminated soils and 20 axenic cultures. At neutral pH, no disappearance of the herbicide was detected either in the enrichment cultures or in the growth media of the axenic microbial cultures. During the growth of some of the microbial strains, however, the pH of the medium dropped below 6, resulting in the hydrolysis of primisulfuron. The rate of primisulfuron hydrolysis was clearly pH dependent; primisulfuron was more persistent in neutral or weakly basic solutions than in acidic solutions. After hydrolysis of the herbicide, four products were observed. These were identified as methyl 2-(aminosulfonyl)benzoate, 2-amino-4,6-(difluoromethoxy)pyrimidine, 2-N-[[[[[4, 6-bis(difluoromethoxy)-2-pyrimidinyl]amino]carbonyl]amino]sulfonyl ]be nzoic acid, and 2-(aminosulfonyl)benzoic acid. After hydrolysis, it was found that the fungus Phanerochaete chrysosporium mineralized 27 and 24% of (14)C-phenyl- and (14)C-pyrimidine-labeled products, respectively, after 24 days of incubation. Similarly, Trametes versicolor mineralized 13 and 11% of (14)C-phenyl- and (14)C-pyrimidine-labeled hydrolysis products, respectively. In addition, primisulfuron in a hydrolytically stable solution, at pH 7. 0, was rapidly decomposed after ultraviolet irradiation, and two photolysis products were isolated [methylbenzoate and 4, 6-(difluoromethoxy)pyrimidin-2-ylurea]. When (14)C-phenyl-labeled primisulfuron was exposed to photolysis for 24 h, 32% of the initial radioactivity was recovered as (14)CO(2), whereas no (14)CO(2) was detected if the herbicide was labeled at the (14)C-pyrimidine position. Mineralization of (14)C-pyrimidine-labeled products of photolyzed primisulfuron by P. chrysosporium was approximately 25% after 24 days. These results clearly indicate that hydrolysis and photolysis of primisulfuron facilitated microbial degradation.     

In vitro sensitivity of Botrytis cinerea to anthraquinone and anthrahydroquinone derivatives

The effect on mycelial growth of the fungus Botrytis cinerea of a set of structurally related tricyclic hydroquinones [9,10-dihydroxy-4,4-dimethyl-2,3,5,8-tetrahydroantracen-1(4H)-one and 9,10-dihydroxy-4,4-dimethyl-5,8-dihydroanthracen-1(4H)-one derivatives] and tricyclic quinones [4,4-dimethylanthracen-1,9,10(4H)-trione derivatives] was studied. In general, the anthraquinones presented higher activity than the anthrahydroquinones. Anthraquinone and anthrahydroquinone derivatives with methyl groups on the A ring showed higher antifungal activity than the unsubstituted ones, 4,4,6,7-tetramethyl-(4H)-anthracene-1,9,10-trione being the most active compound of this set. The presence of a polar group such as hydroxymethyl reduced the activity. The effect of two anthrahydroquinones and two anthraquinones on the conidia germination of the fungus was also determined. Anthrahydroquinones did not affect the germination. The most active compound was 4,4-dimethylanthracene-1,9,10(4H)-trione, with 100% inhibition of germination at 7 h of incubation. These results again suggest that the structure of the anthraquinones is important in exerting an antifungal effect on B. cinerea. Furthermore, possible mechanisms of action of compound 4,4-dimethylanthracene-1,9,10(4H)-trione were studied. This compound did not produce lipoperoxidation of membrane and did not induce the formation of oxygen reactive species, but it was able to permeabilize the plasmatic membrane of B. cinerea, increasing the phosphorus concentration in the intracellular medium.     

Differences in odor-active compounds of trincadeira wines obtained from five different clones

Odorant compounds of five young clonal red wines made from cv. Trincadeira, a native grape variety of Vitis vinifera L. grown in Portugal, were studied using 2001 and 2003 vintages. The study was carried out using gas chromatography-mass spectrometry (GC-MS) for compound identification and the gas chromatography-olfactometry (GC-O) posterior intensity method to detect the potentially most important aroma compounds. Forty-one odorant peaks were detected by GC-O analysis, from which 31 were identified by GC-MS. The odorant compounds with the highest odorant average intensities are 3-methylbutanoic acid, 2-phenylethanol, 2,5-dimethyl-4-hydroxy-3(2H)-furanone, and 4-vinylguaiacol. The GC-O analysis showed odor intensity differences among compounds, which was confirmed by analysis of variance (ANOVA). Principal component analysis (PCA) and hierarchical cluster analysis (HCA) showed that the five clonal wines from the 2001 vintage were more similar than those from the 2003 vintage. Moreover, stepwise linear discriminant analysis (SLDA) demonstrated that the factor vintage has influence on the Trincadeira clonal red wine odorant profile differentiation.     

Analysis and detection of the herbicides dimethenamid and flufenacet and their sulfonic and oxanilic acid degradates in natural water

Dimethenamid [2-chloro-N-(2,4-dimethyl-3-thienyl)-N-(2-methoxy-1-methylethyl)acetamide] and flufenacet [N-(4-fluorophenyl)-N-(1-methylethyl)-2-(5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl)oxy] were isolated by C-18 solid-phase extraction and separated from their ethanesulfonic acid (ESA) and oxanilic acid (OXA) degradates during their elution using ethyl acetate for the parent compound, followed by methanol for the polar degradates. The parent compounds were detected using gas chromatography-mass spectrometry in selected-ion mode. The ESA and OXA degradates were detected using high-performance liquid chromatography--electrospray mass spectrometry (HPLC-ESPMS) in negative-ion mode. The method detection limits for a 123-mL sample ranged from 0.01 to 0.07 microg/L. These methods are compatible with existing methods and thus allow for analysis of 17 commonly used herbicides and 18 of their degradation compounds with one extraction. In a study of herbicide transport near the mouth of the Mississippi River during 1999 and 2000, dimethenamid and its ESA and OXA degradates were detected in surface water samples during the annual spring flushes. For flufenacet, the only detections at the study site were for the ESA degradates in samples collected at the peak of the herbicide spring flush in 2000. The low frequency of detections in surface water likely is due to dimethenamid and flufenacet being relatively new herbicides. In addition, detectable amounts of the stable degradates have not been detected in ground water.