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Quinoline Yellow (QY, Colour Index No. 47005) is internationally used as a colour additive in foods, drugs, and cosmetics. The manufacture of QY requires sulphonating quinophthalone, and depending on the degree of sulphonation, various forms of QY result, containing different proportions of quinophthalone mono-, di-, and trisulfonic acid sodium salts (monoSA, diSA, and triSA, respectively). Regulations on the specific composition and uses of QY differ across countries with associated differences in names for QY. The QY form certified for use in the U.S. in drugs and cosmetics is known as D&C Yellow No. 10 (Y10). The Code of Federal Regulations (CFR) specifies that Y10 and its lakes consist of predominantly monoSA's, the sum of whose levels is ≥ 75%, and that the sum level of diSA's is ≤ 15%, with one of them (6'8'diSA) at ≤ 3%. The present work reports the development of an HPLC method for determining those CFR-specified values and the level of a non-CFR-specified component, 6'8'5triSA. The selected analytes, 6'SA, 6'5diSA, 6'8'diSA, and 6'8'5triSA, were quantified by using five-point-calibration curves (R 2  > 0.999) with data-point ranges of 9.96-96.53%, 0.54-21.69%, 0.10-5.00%, and 0.11-5.53% by weight, respectively. The method was found to be precise (relative standard deviation values, 0.55-0.80%) and accurate (recovery values, 91.07-99.45%). LOD and LOQ values, respectively, were as follows: 1.23 and 3.70%, 6'SA; 0.42 and 1.26%, 6'5diSA; 0.11 and 0.34%, 6'8'diSA; and 0.01 and 0.04%, 6'8'5triSA. The HPLC method was applied successfully to the analysis of 20 Y10 and eight Y10 lake samples. It can be extended to other QY forms such as E104 and Yellow 203 because it enables analysis of 6'8'5triSA. This paper also addresses the implications of the varying structure depictions and CAS numbers of the QY components that are due to the existence of three tautomeric forms of quinophthalone.

A simple and efficient multiresidue method using dispersive solid phase extraction and liquid chromatography coupled with tandem mass spectrometry was developed for the targeted analysis of indaziflam and its five metabolites (indaziflam-diaminotriazine, indaziflam-carboxylic acid, indaziflam-triazine indanone, indaziflam-hydroxyethyl, and indaziflam-olefin) in pitaya samples (including roots, plants, flowers, peels, pulp, and whole fruit). The analytes were extracted with acetonitrile, and the extracts were purified using multiwalled carbon nanotubes. The method was validated using pitaya samples spiked at 0.5, 5, and 50 µg/kg, and the average recoveries varied from 61.1 to 103.7% with relative standard deviations lower than 12.7% (n = 5). This method exhibited sufficient linearity within the concentration range of 0.1-100 µg/L. The limits of detection and quantification were in the ranges of 0.001-0.1 and 0.003-0.3 µg/kg, respectively. The method was successfully applied to analyze pitaya samples in Nanning, and no indaziflam or its metabolites were detected in the samples analyzed., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Point of care testing (POCT) must comply with regulatory requirements according to standard EN ISO 22870, which identify biologists as responsible for POCT. INR for vitamin K antagonists (VKAs) monitoring is a test frequently performed in haemostasis laboratories. Bedside INR is useful in emergency room, in particular in case of VKAs overdosage but also for specific populations of patients like paediatrics or geriatrics. INR POCT devices are widely used at home by the patients for self-testing, but their use in the hospital by the clinical staff for bedside measurement is growing, with devices which now comply with standard for POCT accreditation for hospital use. The majority of point of care devices for INR monitoring has shown a good precision and accuracy with results similar to those obtained in laboratory. With the aim to help the multidisciplinary groups for POCT supervision, the medical departments and the biologists to be in accordance with the standard, we present the guidelines of the GFHT (Groupe français d'étude sur l'hémostase et la thrombose, subcommittee "CEC et biologie délocalisée") for the certification of POCT INR. These guidelines are based on the SFBC guidelines for the certification of POCT and on the analysis of the literature to ascertain the justification of clinical need and assess the analytical performance of main analysers used in France, as well as on a survey conducted with biologists.

We previously reported that coronatine, a virulence factor of plant bacteria, facilitates bacterial infection through an ER (endoplasmic reticulum)-mediated, non-canonical mechanism in the model dicot plant, Arabidopsis thaliana. Here, we report that this same ER-mechanism is ubiquitous among dicots and monocots, and works by affecting the ethylene signaling pathway widely found in plants. The subcellular localization of coronatine by the alkyne-tag Raman imaging (ATRI) approach provided a convincing clue.

(Acetoxy-)valerenic acid and total essential oil content are important quality attributes of pharmacy grade valerian root (Valerianae radix). Traditional analysis of these quantities is time-consuming and necessitates (harmful) solvents. Here we investigated an application of attenuated total reflection Fourier transform infrared spectroscopy for extractionless analysis of these quality attributes on a representative sample comprising 260 wild-crafted individuals covering the Central European taxonomic diversity of the Valeriana officinalis L. s. l. species aggregate with its three major ploidy cytotypes (i.e., di-, tetra- and octoploid). Calibration models were built by orthogonal partial least squares regression for quantitative analysis of (acetoxy-)valerenic acid and total essential oil content. For the latter, we propose a simplistic protocol involving apolar extraction followed by gas chromatography as a reference method for multivariate calibration in order to handle the analysis of samples taken from individual plants. We found good predictive ability of chemometric models for quantification of valerenic acid, acetoxyvalerenic acid, total sesquiterpenoid acid, and essential oil content with a root mean squared error of cross-validation of 0.064, 0.043, and 0.09 and root mean squared error of prediction of 0.066, 0.057, and 0.09 (% content), respectively. Orthogonal partial least squares discriminant analysis revealed good discriminability between the most productive phenotype (i.e., the octoploid cytotype) in terms of sesquiterpenoid acids, and the less productive ones (i.e., di- and tetraploid). All in all, our results demonstrate the application of attenuated total reflection Fourier transform infrared spectroscopy for rapid, extractionless estimation of the most important quality attributes of valerian root and minimally invasive identification of the most productive phenotype in terms of sesquiterpenoid acids., Competing Interests: The authors declare no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

1-Phenyl-2-propanone (P2P) is an internationally monitored precursor that has become increasingly difficult for illicit amphetamine producers to source, which means that alternative routes to its preparation have become increasingly important. One such approach includes the hydrolysis of alpha-phenylacetoacetonitrile (APAAN) with sulfuric acid. Previously, we reported the identification of 4,6-dimethyl-3,5-diphenylpryid-2-one following implementation of hydrolysis conditions and it was proposed that this compound might serve as one route specific by-product in the APAAN to P2P conversion. This study continued to explore the presence of impurities formed during this conversion and expanded also into a second route of P2P synthesis starting from alpha-methylstyrene (AMS). All P2P products underwent the Leuckart procedure to probe the presence of P2P-related impurities that might have carried through to the final product. Two by-products associated with the APAAN hydrolysis route to P2P were identified as 2,3-diacetyl-2,3-diphenylsuccinonitrile (1) and 2-methyl-1-phenyl-1,3-dicarbonitrile-1H-indene (2), respectively. Two by-products associated with the AMS route to P2P and subsequent Leuckart reaction were 1,1,3-trimethyl-3-phenyl-2,3-dihydro-1H-indene (3) and 1-phenyl-N-(phenylethyl)propan-2-amine (4), respectively. The two indenes (2 and 3) identified in synthesized amphetamine originating from P2P suggested that it might be possible to differentiate between the two synthetic routes regarding the use of APAAN and AMS. Furthermore, the association of these compounds with amphetamine production appears to have been reported for the first time. The presence of compounds 1 - 4 in seized amphetamine samples and waste products could facilitate the suggestion whether APAAN or AMS were employed in the synthesis route to the P2P. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Indaziflam is a new preemergence herbicide for the control of annual grass and broadleaf weeds in various cropping systems including pecan orchards. The objectives of this study were to (1) determine the mobility and dissipation of indaziflam and (2) evaluate herbicide efficacy in a flood-irrigated pecan orchard in southern New Mexico, USA. Indaziflam was applied at 0, 36.5, and 73.1g/ha in areas with (impacted) and without (unimpacted) tree injury symptoms. Soil samples were collected at 0-15, 15-30, and 30-46cm depths 26, 63, 90, and 126days after the first herbicide application. Additional soil samples were collected 4, 30, and 56days after the second application. Indaziflam was detected in soil samples collected at each depth, suggesting movement with irrigation water. Indaziflam concentrations decreased with increasing soil depth and time. Indaziflam mass recoveries were greater in the unimpacted area than in the impacted area after the first and second applications. Dissipation half-lives of indaziflam in the soil ranged from 30 to 86days for total indaziflam recovered from the entire soil profile after the first and second applications in both areas. The percent weed control was similar in the impacted and unimpacted areas for both rates of indaziflam on 26 and 63days after application; however, on 90days after the application, percent weed control was lower in the impacted than unimpacted area., (Copyright © 2016. Published by Elsevier B.V.)

Quantitative detection of the biological properties of living cells is essential for a wide range of purposes, from the understanding of cellular characteristics to the development of novel drugs in nanomedicine. Here, we demonstrate that analysis of cell biological properties within a microfluidic dielectrophoresis device enables quantitative detection of cellular biological properties and simultaneously allows large-scale measurement in a noise-robust and probeless manner. Applying this technique, the static and dynamic biological responses of live B16F10 melanoma cells to the small-molecule drugs such as N-ethylmaleimide (NEM) and [(dihydronindenyl)oxy]alkanoic acid (DIOA) were quantitatively and statistically examined by investigating changes in movement of the cells. Measurement was achieved using subtle variations in dielectrophoresis (DEP) properties of the cells, which were attributed to activation or deactivation of K(+)/Cl(-) cotransporter channels on the cell membrane by the small-molecule drugs, in a microfluidic device. On the basis of quantitative analysis data, we also provide the first report of the shift of the complex permittivity of a cell induced by the small-molecule drugs. In addition, we demonstrate interesting quantifiable parameters including the drug effectiveness coefficient, antagonistic interaction coefficient, kinetic rate, and full width at half-maximum, which corresponded to changes in biological properties of B16F10 cells over time when NEM and DIOA were introduced alone or in combination. Those demonstrated parameters represent very useful tools for evaluating the effect of small-molecule drugs on the biological properties of cells.

Indaziflam is a relatively new herbicide for which sorption-desorption information is lacking, and nothing is available on its metabolites. Information is needed on the multiple soil and pesticide characteristics known to influence these processes. For four soils, the order of sorption was indaziflam (N-[1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-[(1R)-1-fluoroethyl]-1,3,5-triazine-2,4-diamine) (sandy clay loam: Kf = 5.9, 1/nf = 0.7, Kfoc = 447; sandy loam: Kf = 3.9, 1/nf = 0.9, Kfoc = 276) > triazine indanone metabolite (N-[(1R,2S)-2,3-dihydro-2,6-dimethyl-3-oxo-1H-inden-1-yl]-6-[(1R)-1-fluoroethyl]-1,3,5-triazine-2,4-diamine) (sandy clay loam: Kf = 2.1, 1/nf = 0.8, Kfoc = 177; sandy loam: Kf = 1.7, 1/nf = 0.9, Kfoc = 118) > fluoroethyldiaminotriazine metabolite (6-[(1R-1-Fluoroethyl]-1,3,5-triazine-2,4-diamine) (sandy clay loam: Kf = 0.3, 1/nf = 0.9, Kfoc = 28; sandy loam: Kf = 0.3, 1/nf = 0.9, Kfoc = 22) = indaziflam carboxylic acid metabolite (2S,3R)-3-[[4-amino-6-[(1R)-1-fluoroethyl]-1,3,5-triazin-2-yl]amino]-2,3-dihydro-2-methyl-1H-indene-5-carboxylic acid) (sandy clay loam: Kf = 0.3, 1/nf = 0.9, Kfoc = 22; sandy loam: Kf = 0.5, 1/nf = 0.8, Kfoc = 32). The metabolites being more polar than the parent compound showed lower sorption. Desorption was hysteretic for indaziflam and triazine indanone metabolite, but not for the other two metabolites. Unsaturated transient flow Kd's were lower than batch Kd's for indaziflam, but similar for fluoroethyldiaminotriazine metabolite. Batch Kd's would overpredict potential offsite transport if desorption hysteresis is not taken into account.

Sappanwood (Caesalpinia sappan Linn.) is used as an herbal medicine. It is sometimes used to treat skin damage or as a facial cleanser. In the present study, the methanol (MeOH) extract of sappanwood was found to inhibit melanin synthesis in cultured human melanoma HMV-II cells stimulated with forskolin, and six active compounds (1-5 and 7) were isolated from the extract along with a non-active compound (6). Compounds 2-7 were identified as sappanchalcone (2), 3'-deoxy-4-O-methylsappanol (3), brazilein, (4), brazilin (5), sappanol (6), and 4-O-methylsappanol (7). Compound 1 was a new compound, and its structure was determined to be (6aS,11bR)-7,11b-dihydro-6H-indeno[2,1-c]chromene-3,6a,10,11-tetrol by spectroscopic analyses. Among the six active compounds, brazilin (5) (EC50: 3.0 ± 0.5 µM) and 4-O-methylsappanol (7) (EC50: 4.6 ± 0.7 µM) strongly suppressed melanin synthesis in HMV-II cells. Bioactive compounds showed moderate cytotoxicities against HMV-II cells with IC50 values of 83.1 ± 4.0 µM (for 2), 72.0 µM ± 2.4 (for 3), 33.8 ± 1.1 µM (for 4), 18.4 ± 0.8 µM (for 5), and 20.2 ± 0.8 (for 7), respectively. Brazilin (5) selectively suppressed the expression of mRNAs for tyrosinase-related protein (TYRP) 2 and tyrosinase but did not influence the expression of TYRP1. These results suggest that brazilin (5) is a new class of melanin inhibitor and that sappanwood could be used as a cosmetic material., (Georg Thieme Verlag KG Stuttgart · New York.)

Indaziflam {N-[(1R, 2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-[(1RS)-1fluoroethyl]-1,3,5-triazine-2,4-diamine} is a new pre-emergence herbicide recently registered for a broad spectrum weed control in Florida citrus. Experiments were conducted to evaluate leaching of indaziflam applied at 73 and 145 g ai ha(-1) in Florida Candler soil under simulated rainfall of 5, 10, and 15 cm ha(-1). Indaziflam leached the least (12.6 ± 0.6 cm) when applied at 73 g ai ha(-1) under 5 cm ha(-1) rainfall. Indaziflam leached furthest (30.2 ± 0.9 cm) when applied at 145 g ai ha(-1) under 15 cm ha(-1) rainfall. The visual control ratings of a bio-indicator species ryegrass (Lolium multiflorum L.) was 97% at 15 cm ha(-1) rainfall when indaziflam applied at 145 g ai ha(-1) in the 26 to 30 cm horizon indicating the maximum movement and activity of indaziflam. A dose response experiment was conducted to determine the sensitivity of ryegrass to various doses of indaziflam that confirmed that application of indaziflam at 29.20 g ai ha(-1) was sufficient to prevent germination of ryegrass. There was no mortality of ryegrass plants beyond the 30 cm and the biomass of ryegrass was comparable with untreated control indicating that indaziflam did not leach beyond this distance even under 15 cm ha(-1) rainfall.

Polycyclic aromatic hydrocarbons (PAHs) are regarded as key intermediates in the molecular growth process that forms soot from incomplete fossil fuel combustion. Although heavily researched, the reaction mechanisms for PAH formation have only been investigated through bulk experiments; therefore, current models remain conjectural. We report the first observation of a directed synthesis of a PAH under single-collision conditions. By using a crossed-molecular-beam apparatus, phenyl radicals react with C(3)H(4) isomers, methylacetylene and allene, to form indene at collision energies of 45 kJ mol(-1). The reaction dynamics supported by theoretical calculations show that both isomers decay through the same collision complex, are indirect, have long lifetimes, and form indene in high yields. Through the use of deuterium-substituted reactants, we were able to identify the reaction pathway to indene., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the present work, the applicability of hollow fiber-based liquid phase microextraction (HF-LPME) was evaluated for the extraction and preconcentration of valerenic acid prior to its determination by reversed-phase HPLC/UV. The target drug was extracted from 5.0 mL of aqueous solution with pH 3.5 into an organic extracting solvent (dihexyl ether) impregnated in the pores of a hollow fiber and finally back extracted into 10 μ L of aqueous solution with pH 9.5 located inside the lumen of the hollow fiber. In order to obtain high extraction efficiency, the parameters affecting the HF-LPME, including pH of the donor and acceptor phases, type of organic phase, ionic strength, the volume ratio of donor to acceptor phase, stirring rate and extraction time were studied and optimized. Under the optimized conditions, enrichment factor up to 446 was achieved and the relative standard deviation (RSD) of the method was 4.36% (n = 9). The linear range was 7.5-850 μg L⁻¹ with correlation coefficient (r²=0.999), detection limits was 2.5 μg L⁻¹ and the LOQ was 7.5 μg L⁻¹. The proposed method was evaluated by extraction and determination of valerenic acid in some Iranian wild species of Valerianaceae., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A novel peroxisome proliferator-activated receptor γ (PPARγ) agonist, KR-62980, was determined by liquid-liquid extraction with ethyl acetate and liquid chromatography-tandem mass spectrometry (LC/MS/MS) in rat plasma. In order to evaluate the pharmacokinetics of KR-62980, a reliable, selective and sensitive high-performance liquid chromatographic method with electrospray ionization tandem mass spectrometry was developed for the quantification of KR-62980 in rat plasma. KR-62980 and imipramine (IS) were separated on Hypersil GOLD C18 column with a mixture of acetonitrile-ammonium formate (10mM) (80:20, v/v) as mobile phase. The ion transitions monitored were m/z 437.2 → 114.2 for KR-62980, m/z 281.3 → 86.1 for imipramine in multiple reaction monitoring (MRM) mode. The percent recoveries of KR-62980 and imipramine were 90.1 and 98.4% from rat plasma, respectively. The linear dynamic range extended from 0.01 to 10 μg/ml with a correlation coefficient (R(2)) greater than 0.99 and the lower limit of quantification was 0.01 μg/ml. The mean of intra- and inter-assay precisions was 2.1 and 9.3%. The method was validated and successfully applied to the pharmacokinetic study of KR-62980 in rat., (Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.)

The study was designed to investigate the hepatic metabolism and transport system of valerenic acid, a main active constituent of valerian, in isolated perfused livers from Wistar and Mrp2-deficient TR(-) rats. After administration of 20 microM valerenic acid, the formation of seven valerenic acid glucuronides (M1-M7), namely two glucuronides of valerenic acid (M6, M7), four glucuronides of hydroxylated valerenic acid (M1, M3, M4, M5), and one glucuronide of hydroxylated dehydro-valerenic acid (M2) in bile and perfusate was quantified by HPLC. The hepatic extraction ratio and clearance of valerenic acid were very high in Wistar and TR(-) rats (E: 0.983 +/- 0.006 vs. 0.981 +/- 0.004; Cl: 35.4 +/- 0.21 mL/min vs. 35.3 +/- 0.14 mL/min). However, biliary excretion and efflux of conjugates differed greatly in TR(-) rats. While cumulative biliary excretion of unconjugated valerenic acid and the glucuronides M1-M7 dropped dramatically to 1-9%, their efflux into perfusate increased 1.5- to 10-fold. This indicates that valerenic acid and its glucuronides are eliminated into bile by Mrp2. In summary, valerenic acid was metabolized to several conjugates, whereby the canalicular transporter Mrp2 mediated biliary excretion of the parent drug and its glucuronides.

Natural plant extracts containing taste modifier compounds will gain more commercial interest in the future. Black cardamom, Amomum tsao-ko Crevost et Lemarié, used as a spice in Asia, produces a nice refreshing effect in the mouth. Therefore, an ethyl acetate extract was prepared, and constituents were separated by liquid chromatography. Guided by the tasting of each fraction (LC tasting), a new pungent compound was discovered, (+/-)-trans-2,3,3a,7a-tetrahydro-1H-indene-4-carbaldehyde. To confirm this new structure, a synthesis was performed starting from cyclopentene-1-carbaldehyde. The Wittig conditions were determined to control the stereochemistry of the ring fusion to prepare (+/-)-trans-(2,3,3a,7a-tetrahydro-1 H-inden-4-yl) methanol and (+/-)-cis-(2,3,3a,7a-tetrahydro-1H-inden-4-yl) methanol. After oxidation, (+/-)-trans-2,3,3a,7a-tetrahydro-1H-indene-4-carbaldehyde and (+/-)-cis-2,3,3a,7a-tetrahydro-1H-indene-4-carbaldehyde were tasted in water and only the trans-2,3,3a,7a-tetrahydro-1H-indene-4-carbaldehyde, present in black cardamom, produced a trigeminal effect in the mouth.

A previously established mobile phase composition for a high-performance liquid chromatography (HPLC)-electrochemical detection (ED) method for the determination of Brazilein is further examined by HPLC-UV. Each component in the mobile phase is evaluated by the HPLC-UV system, and a convenient HPLC-UV method for the determination of Brazilein is developed. The mobile phase of this HPLC-UV method consists of sodium dihydrogen phosphate (NaH(2)PO(4)). The detection limit of Brazilein (signal-to-noise = 3) is 1 ng, and the calibration graph of Brazilein ranges from 1-120 ng per 20-microL injection. The validity of the proposed HPLC-UV method is demonstrated by the analysis of Brazilein in real plant samples. The differences between the HPLC-ED method and the proposed HPLC-UV method are given.

A new protocol for the simultaneous determination of methyl tert-butyl ether (MTBE); its main degradation products: tert-butyl alcohol (TBA) and tert-butyl formate (TBF); other gasoline additives, oxygenate dialkyl ethers: ethyl tert-butyl ether (ETBE), tert-amyl methyl ether (TAME) and diisopropyl ether (DIPE); aromatics: benzene, toluene, ethylbenzene and xylenes (BTEX) and other compounds causing odour events such as dicyclopentadiene (DCPD) and trichloroethylene (TCE) in soils has been developed. On the basis of US Environmental Protection Agency (EPA) method 5035A, a fully automated closed-system purge-and-trap coupled to gas chromatography/mass spectrometry (P&T-GC/MS) was optimised and permitted to detect microg/kg concentrations in solid matrices avoiding losses of volatile compounds during operation processes. Parameters optimised were the sampling procedure, sample preservation and storage, purging temperature, matrix effects and quantification mode. Using 5 g of sample, detection limits were between 0.02 and 1.63 microg/kg and acceptable method precision and accuracy was obtained provided quantification was performed using adequate internal standards. Soil samples should be analysed as soon as possible after collection, stored under -15 degrees C for not longer than 7 days if degradation products have to be analysed. The non-preservative alternative (empty vial) provided good recoveries of the most analytes when freezing the samples up to 7 day holding time, however, if biologically active soil are analysed the preservation with trisodium phosphate dodecahydrate (Na(3)PO(4).12H(2)O or TSP) is strongly recommended more than sodium bisulphate (NaHSO(4)). The method was finally applied to provide threshold and background levels of several gasoline additives in a point source and in sites not influenced by gasoline spills. The proposed method provides the directions for the future application on real samples in current monitoring programs at gasoline pollution risk sites where till now little monitoring data for MTBE in soils are available.

Previously, we conducted a mutant screen of Pseudomonas syringae pv. tomato strain DC3000 to identify genes that contribute to virulence on Arabidopsis thaliana plants. Here we describe the characterization of one mutant strain, DB4H2, which contains a single Tn5 insertion in PSPTO3576, an open reading frame that is predicted to encode a protein belonging to the TetR family of transcriptional regulators. We demonstrate that PSPTO3576 is necessary for virulence in DC3000 and designate the encoded protein TvrR (TetR-like virulence regulator). TvrR, like many other TetR-like transcriptional regulators, negatively regulates its own expression. Despite the presence of a putative HrpL binding site in the tvrR promoter region, tvrR is not regulated by HrpL, an alternative sigma factor that regulates the expression of many known DC3000 virulence genes. tvrR mutant strains grow comparably to wild-type DC3000 in culture and possess an intact type III secretion system. However, tvrR mutants do not cause disease symptoms on inoculated A. thaliana and tomato plants, and their growth within plant tissue is significantly impaired. We demonstrate that tvrR mutant strains are able to synthesize coronatine (COR), a phytotoxin required for virulence of DC3000 on A. thaliana. Given that tvrR mutant strains are not defective for type III secretion or COR production, tvrR appears to be a novel virulence factor required for a previously unexplored process that is necessary for pathogenesis.

The aim of this study was to establish an easy and accurate method for the determination of Brazilein in plant samples due to its potential pharmacological activities. High-performance liquid chromatography (HPLC) with electrochemical detection (ED) was used for the assay of Brazilein in this study for the first time. Crucial influence parameters including concentration of dodecane-1-sulfonic acid sodium salt (DSASS), inorganic modifier, tetrabutyl-ammonium hydroxide solution (TBAOH), and applied potential of proposed method were investigated. The proposed method is simple, rapid (analysis time: approximately 10 min), sensitive [(detection limit: 0.6 ng per injection (20 microl) at a signal-noise ratio 3:1)], highly selective and precise (intra- and inter-day precisions were within 5%, n = 7). The calibration graph of Brazilein was linear in the range 0.6-150 ng per injection 20 microl. Recovery of Brazilein was over 92% by standard addition method.

Purpose: Valerian root ( Valeriana officinalis L.) has been used since antiquity as a medicinal herb. Recent studies have found that certain herbal products used concomitantly with conventional therapeutic products can markedly affect drug disposition., Methods: The in vitro effect of aliquots from 14 commercially available single-entity and blended products containing valerian root on cytochrome P450 CYP3A4-mediated metabolism and P-glycoprotein transport has been determined with aqueous, ethanol and acetonitrile extracts., Results: Hydroxyvalerenic acid, acetoxyvalerenic acid and valerenic acid content was analyzed and wide variation was found between samples and compared to the concentrations noted on the product labels. Valerian extracts from the products tested also exhibited a marked capacity to inhibit cytochrome P450 3A4-mediated metabolism and P-glycoprotein transport based upon the ATPase assay., Conclusions: There is wide variation between commercially available samples of valerian root. The findings from this study suggest that valerian root may have an initial inhibitory effect when taken with therapeutic products. Further work is warranted to determine whether valerian root can affect other CYP450 isozymes and how the results of this in vitro investigation can be extrapolated to in vivo situations.

Cfa1 was overproduced in Escherichia coli and Pseudomonas syringae, and the degree of 4'-phosphopantetheinylation was determined. The malonyl-coenzyme A:acyl carrier protein transacylase (FabD) of P. syringae was overproduced and shown to catalyze malonylation of Cfa1, suggesting that FabD plays a role in coronatine biosynthesis. Highly purified Cfa1 did not exhibit self-malonylation activity.

Phytohormones regulate the protective responses of plants against both biotic and abiotic stresses by means of synergistic or antagonistic actions referred to as signaling crosstalk. A bottleneck in crosstalk research is the quantification of numerous interacting phytohormones and regulators. The chemical analysis of salicylic acid, jasmonic acid, indole-3-acetic acid, and abscisic acid is typically achieved by using separate and complex methodologies. Moreover, pathogen-produced phytohormone mimics, such as the phytotoxin coronatine (COR), have not been directly quantified in plant tissues. We address these problems by using a simple preparation and a GC-MS-based metabolic profiling approach. Plant tissue is extracted in aqueous 1-propanol and mixed with dichloromethane. Carboxylic acids present in the organic layer are methylated by using trimethylsilyldiazomethane; analytes are volatilized under heat, collected on a polymeric absorbent, and eluted with solvent into a sample vial. Analytes are separated by using gas chromatography and quantified by using chemical-ionization mass spectrometry that produces predominantly [M+H]+ parent ions. We use this technique to examine levels of COR, phytohormones, and volatile organic compounds in model systems, including Arabidopsis thaliana during infection with Pseudomonas syringae pv. tomato DC3000, corn (Zea mays) under herbivory by corn earworm (Helicoverpa zea), tobacco (Nicotiana tabacum) after mechanical damage, and tomato (Lycopersicon esculentum) during drought stress. Numerous complex changes induced by pathogen infection, including the accumulation of COR, salicylic acid, jasmonic acid, indole-3-acetic acid, and abscisic acid illustrate the potential and simplicity of this approach in quantifying signaling crosstalk interactions that occur at the level of synthesis and accumulation.

Although most commercial valerian formulations are coated tablets not any comparison study of their drug release profiles has been published so far. The main objective of this work is to establish a drug release test suitable for studying and comparing different valerian tablets. Thus, hydroxyvalerenic and valerenic acid concentrations were assayed by HPLC using a C18 Kromasil (200 x 4.6 mm, 5 microm) column and a mobile phase containing methanol and an orthophosphoric acid solution 0.5% v/v in water at a ratio of 75:25 at a constant flow rate of 1 ml/min. Saturation solubilities for hydroxyvalerenic and valerenic acid at pH 6.8 were 26 +/- 5.1 and 1 +/- 0.6 microg/ml, respectively. Usually for drugs with such low solubility values, their oral absorption and hence bioavailability are limited by their dissolution characteristics. A dissolution test was conducted according to the general method 2 (paddles) of USP 24 using 500 ml buffer medium (pH 6.8) at 50 rpm. Five different formulations were studied and compared: one uncoated tablet formulation and four marketed coated tablets. The uncoated tablet formulation had the fastest release profile, whereas the coated tablets manifested very different release patterns, depending on the type of formulation. Because of these differences in drug release pattern not every tablet formulation may be appropriate for the same clinical indications. Clinical data are required to confirm the correlation between drug release pattern and the therapeutically value of each formulation.

Thirty-one commercial valerian preparations available in Australia, including teas, tablets, capsules and liquids, were analysed by HPLC for valepotriates, valerenic acid and valerenic acid derivatives. The concentration of valerenic acid and its derivatives ranged from < 0.01 to 6.32 mg/g of product. Powder capsules, on average, contained the highest concentration of valerenic acids (2.46 mg/g) and liquids the lowest concentration (0.47 mg/ml). The mean concentration of valerenic acid in the five products standardized against valerenic acid (3.56 mg/g) was significantly higher than in the 26 non-standardized products (0.89 mg/g). There was a significant relationship between valerenic acids content and added valerian root for the standardized products but not for the non-standardised products. Valepotriates were found at low levels (< 1.0 mg/g) in some teas but were not detected in any of the finished products.

An attempt was made to establish a method for the simultaneous determination of ethylbenzene, indan, indene and acenaphthene by capillary gas chromatography with flame ionization detection. The air was sampled on charcoal tubes and extracted with carbon disulfide-methanol (60:1, v/v). The four analytes were separated by gas chromatography using a capillary column of cross-linked 5% phenylmethylsilicone. Under the applied conditions the method showed detection limits of 1.8 microg/m3 for ethylbenzene, 2.1 microg/m3 for indan, 2.8 microg/m3 for indene and 3.4 microg/m3 for acenaphthene. Relative standard deviations were as follows: ethylbenzene, 6.2%; indan, 9.9%; indene, 13.6%; and acenaphthene, 14.4%. The recoveries for these compounds were 98.6, 97.9, 55.7 and 52.1%, and the accuracies were 2.5, 3.0, 44.3 and 47.8%, a working range of 1.5-30 ng/microl for ethylbenzene and 0.75-15 ng/microl for indan, indene and acenaphthene. The method was found to be suitable for the determination of environmental and occupational analysed ethylbenzene, indan, indene and acenaphthene exposure.

Capillary gas chromatography-Fourier transform infrared spectrometry-gas chromatography retention indices (GC/FTIR-RI), combined with the linear relationship between retention value and boiling points for polycyclic aromatic hydrocarbons, performed the determination of the composition of heavy oil fractions in high-temperature coal tar. More than 70 and 61 compounds were separated and identified in heavy oil fractions F7(300-330 degrees C) and F8(330-360 degrees C) respectively. The results are useful in coal tar distillation processing and pollution monitoring when such compounds are present.

Coronatine (COR) is a nonhost-specific phytotoxin that substantially contributes to the virulence of several pathovars (pvs.) of Pseudomonas syringae. The COR gene cluster in P. syringae is generally plasmid-encoded in pvs. atropurpurea, glycinea, morsprunorum, and tomato but chromosomally encoded in pv. maculicola. In the present study, we investigated whether the COR plasmids in four pathovars shared other traits including self-transmissibility, conserved oriV/par loci, and insertion sequences (ISs) known to reside on other plasmids in P. syringae. Three COR plasmids were shown to be self-transmissible, and all COR plasmids shared a related oriV/par region. Two COR plasmids hybridized to IS801, an IS element widely distributed in P. syringae. Further analysis of p4180A, a 90-kb COR plasmid in P. syringae pv. glycinea, indicated that multiple copies of IS801 were present on this plasmid, and all copies mapped outside the COR gene cluster. Sequence analysis of the region adjacent to the COR gene cluster in p4180A indicated the presence of additional IS elements including IS870, IS51, and IS1240. The IS elements borne on p4180A may have contributed to horizontal transfer of the COR gene cluster and the evolution of the COR biosynthetic pathway., (Copyright 1999 Academic Press.)

75 strains of 21 various Pseudomonas syringae (P.) pathovars were investigated in different tests for their toxin production. Data from literature about the production of the known phytotoxins phaseolotoxin (pv. phaseolicola), tabtoxin (pv. coronafaciens, pv. tabaci), coronatine (pv. atropurpurea, pv. glycinea, pv. maculicola, pv. morsprunorum, pv. tomato), and toxins of the lipodepsipeptide group (pv. aptata, pv. atrofaciens, pv. syringae) could be confirmed. Besides, a production of the phytohormone ethylene was detected for P. phaseolicola isolates from kudzu (Pueraria lobata) and for all tested P. glycinea and P. cannabina strains. Strains of P. apii, P. aptata, P. atrofaciens, and P. tomato produced antimetabolic toxins which could be detected with an agar diffusion assay with Escherichia coli as indicator strain. These antimetabolites inhibit a step in the arginine/ornithine biosynthesis. P. maculicola strains caused inhibition zones in this assay which could not be reversed by the tested amino acids. All strains with inhibitory effect against E. coli and Geotrichum candidum were also proved for their antagonistic activity against a selection of typical phyllosphere microorganisms. Most of the phytotoxins possess antimicrobial activity with different spectrum and efficiency. Only the lipodepsipeptide-producers showed antifungal activities. Our results show that the production of toxins is a widespread property among Pseudomonas syringae pathovars, and that some pathovars can produce more than one toxin. This characteristic and the antimicrobial activity of most toxins could be of advantage for the toxin-producing bacteria to adapt to different habitats.

Coronafacic acid, the polyketide component of the phytotoxin coronatine, is activated and coupled to coronamic acid via amide bond formation, a biosynthetic step presumably catalyzed by the coronafacate ligase (cfl) gene product. In the present study, cfl was fused to the carboxy terminus of malE, which encodes the maltose-binding protein (MBP), and overexpressed in Escherichia coli. Immunoblot analysis indicated that Cfl contained an ATP-binding region, a motif conserved in enzymes which activate their substrates by adenylation. MBP-Cfl was overproduced and purified from Pseudomonas syringae and the protein fusion was used to generate antisera. Anti-MBP-Cfl antibodies and a transcriptional fusion of the cfl promoter to a promoterless glucuronidase gene were used to follow the temporal expression of coronafacate ligase. The results indicated that transcription of cfl is temperature-sensitive. Furthermore, a nonpolar mutation in cfl suggested that the gene may have a role in coronafacic acid biosynthesis.

Mixtures of organic solvents are used extensively in industry. The aim of this study was to develop a method for the determination of components of commercial multi-component solvent mixtures, and to perform qualitative and quantitative analyses of the mixtures. Farbasol, Solvesso and Shellsol mixtures were analysed by gas chromatography with a mass spectrometry detector (GC/MS). The composition of mixtures was determined using the mass spectrum computer analysis. Fractions of alkyl benzene, naphthalene and diphenyl derivatives were isolated. The proportions of the individual fractions in the mixtures differed. A predominant fraction was present in each mixture. Solvents (trimethylbenzene, tetramethylbenzene, ethyltoluene, diethylbenzene, isopropylotoulene) which significantly increase health risk due to their suspected neurotoxicity, were identified in each mixture.

The labour conditions at an experimental dicyclopentadiene (DCPD) processing line was characterized by a combined action of occupational hygienic factors, mostly contamination of the working zone air with chemical substances. Both to the degree of its hazardousness and concentration levels in the working zone air, DCPD was predominant agent in the gaseous discharges. The DCPD discharge in the air was due to improper pump packing gland hermetization, manual sample taking, inadequate decontamination of the equipment prior to preventive maintenance, as well as to the desorption by the construction elements surfaces. DCPD contamination of the workers' skin and overalls was also revealed.

MK-286 is a 90:10 mixture of the (+) and (-)-enantiomers of indacrinone, a combination which induces diuresis while maintaining isouricemia. The principal (phenolic) metabolites also possess pharmacological activity and assays for the four entities were needed for clinical studies. Antisera were produced with the stereospecificity required to measure one enantiomer in the presence of the others, but significant cross-reactivity between drug and metabolite necessitated the separation of these two species by means of Sep-Pak cartridges. Recovery was assessed by concurrently fractionated reference samples. The radioligand in each case was a [I-125]-L-iodotyrosine conjugate of the analyte. Absolute assay sensitivities ranged from 2-20 pg/assay tube, corresponding to 0.2-2.0 ng/ml in serum samples. The assay was also applied to urine with analytes at concentrations greater than 16 ng/ml.

Methanol extracts of bracken frond and rhizomes prepared using a metallic extraction vessel, were proved incapable of producing bracken poisoning in calves. Nevertheless, they contained appreciable quantities of pterosins and pterosides. Thus the poisonous principle(s) in bracken responsible for the toxicological effects are not associated with these major sesquiterpenoids of the plant.

The stability of the diphenylindenonesulphonyl (disyl) derivatives of tryptophan, proline, hydroxyproline, histidine, serine, threonine and aspartic acid, as well as of glycine, alanine, alpha-amino butyric acid, phenylalanine, valine, leucine and isoleucine to acid hydrolysis is studied. The results indicate that the disyl derivatives are much more stable in comparison with the corresponding DNP- and DNS-derivatives. Hence, the dysil-chloride method possesses definite advantages over these widely used methods for determination of N-terminal groups, not only because of its higher sensitivity, but also because of the higher stability of the disyl derivatives of amino acids to acid hydrolysis.

Highly specific and sensitive GLC methods were developed for the determination of (6,7-dichloro-2-methyl-1-oxo-2-phenyl-5-indanyloxy)acetic acid, a novel saluretic-uricosuric agent, in biological fluids. The procedures involve the addition of an internal standard, (6,7-dichloro-2-cyclopentyl-2-methyl-1-oxo-5-indanyloxy)acetic acid, to the biological specimens followed by extraction of the acids into benzene at pH 1. The indanones are back-extracted into sodium hydroxide and reextracted into methylene chloride under acidic conditions. The acids are subsequently converted to the methyl esters for GLC analysis by reaction with diazomethane. The sensitivity of the method is such that 1.0 microgram of material/ml of plasma can be analyzed using a flame-ionization detector. When the derivatized samples are analyzed using a 63Ni-electron-capture detector, the sensitivity is such that 2.5 ng of compound can be detected. These levels are suitable for the analysis of samples obtained following a therapeutic dose. A recovery of 98.8 +/- 11.9% was obtained using the electron-capture method for plasma (n = 322). Recoveries using flame ionization were 99.1 +/- 4.4% (plasma, n = 207) and 99.8 +/- 4.9% (urine, n = 163). Quantitation of the major ring-hydroxylated metabolite (in chimpanzee and human) was accomplished following silylation of the methyl esters.

A high-performance liquid chromatographic (HPLC) method for the determination of a new H2 receptor antagonist, 3-amino-5-[3-[4-(piperidinoindanyloxy)]propylamino] -1-methyl-1H-1,2,4-triazole (I), in human plasma and urine was developed. The method employs liquid-liquid extraction of the analyte and an internal standard and chromatographic separation using an alkylphenyl-bonded HPLC column. The total time of chromatography was less than 10 min. Sensitivity was 10 ng/ml for the plasma analysis and 1 microgram/ml for the analysis of I from urine. The coefficients of variation, based on interpolated concentrations, were less than 10%. The method was used for more than 5000 samples during clinical pharmacokinetic studies.

A sensitive and specific high-performance liquid chromatographic method for the identification and quantitation of chlorophacinone in animal tissues has been developed. Residues were extracted with chloroform-acetone (1:1, v/v). Clean-up of extracts was accomplished with a combined gel permeation-adsorption chromatographic procedure using Bio-Beads SX-3 and incorporating an on-line Sep-Pak silica cartridge. Residues were determined by ion-pair liquid chromatography, with the tetrabutylammonium ion as counter-ion, using a fixed-wavelength UV detector at 280 nm or a diode array detector at 285 nm. Recoveries from spiked liver tissue were around 90% at levels from 0.05 to 1 mg kg-1. A detection limit of 0.001 mg kg-1 could be achieved in animal tissues. The diode array detector confirmed identification by matching spectra for residues down to 0.1 mg kg-1 and below this level by multi-wavelength monitoring.