Chromatographic Procedure Research Articles

Crystal structure of glutamate dehydrogenase 2, a positively selected novel human enzyme involved in brain biology and cancer pathophysiology.

Mammalian glutamate dehydrogenase (hGDH1 in human cells) interconverts glutamate to α-ketoglutarate and ammonia while reducing NAD(P) to NAD(P)H. During primate evolution, humans and great apes have acquired hGDH2, an isoenzyme that underwent rapid evolutionary adaptation concomitantly with brain expansion, thereby acquiring unique catalytic and regulatory properties that permitted its function under conditions inhibitory to its ancestor hGDH1. Although the 3D-structures of GDHs, including hGDH1, have been determined, attempts to determine the hGDH2 structure were until recently unsuccessful. Comparison of the hGDH1/hGDH2 structures would enable a detailed understanding of their evolutionary differences. This work aimed at the determination of the hGDH2 crystal structure and the analysis of its functional implications. Recombinant hGDH2 was produced in the Spodoptera frugiperda ovarian cell line Sf21, using the Baculovirus expression system. Purification was achieved via a two-step chromatography procedure. hGDH2 was crystallized, X-ray diffraction data were collected using synchrotron radiation and the structure was determined by molecular replacement. The hGDH2 structure is reported at a resolution of 2.9Å. The enzyme adopts a novel semi-closed conformation, which is an intermediate between known open and closed GDH1 conformations, differing from both. The structure enabled us to dissect previously reported biochemical findings and to structurally interpret the effects of evolutionary amino acid substitutions, including Arg470His, on ADP affinity. In conclusion, our data provide insights into the structural basis of hGDH2 properties, the functional evolution of hGDH isoenzymes, and open new prospects for drug design, especially for cancer therapeutics.

Electrophilic Etherification of α-Heteroaryl Carbanions with Monoperoxyacetals as a Route to Ketene O,O- and N,O-Acetals.

Alkyl ketene acetals are useful reactants in a variety of synthetic processes, and yet, there are limited routes to their formation as isolable products. We now report the successful synthesis and isolation of heteroaryl ketene acetals through intermolecular transfer of alkoxyl (δ+OR) from electrophilic peroxides to lithiated benzofurans, indoles, and pyridines. Primary and secondary peroxyacetals enable selective transfer of the nonanomeric alkoxy group in moderate to high yield; substrates bearing an electron-donating substituent show enhanced reactivity toward electrophilic oxygen. Heteroaryl ketene acetals are remarkably stable throughout traditional purification techniques; the superior stability of ketene N,O-acetals compared to ketene O,O-acetals is presumably due to increased aromaticity of the indole and pyridine structures. The presented method overcomes typical problems associated with alkyl ketene acetal synthesis as reported products withstood workup and flash column chromatography procedures.

Anti-Syncytium (MC99+1A2) and Anti-Bacterial Activities from Twigs and Stems of Ficus dubia

Aims: To study the anti-HIVs and anti-bacterial activity and investigate the chemical compositions of the crude extracts and isolated compounds from stems and twig of Ficus dubia.
 Methodology: Twigs and stems of Ficus dubia were collected from Si Sawat District, Kanchanaburi Province, Thailand. Bioactivity assay and phytochemical analysis of ficus dubia were processed under standard method including, anti-HIV1-RT, antibacterial activities, and CC chromatography procedures. Structures of the compound were elucidated by spectroscopic techniques.
 Results: Bioactivities assay exposed that, the hexane, ethyl acetate, and methanol extracts of Ficus dubia were verified for anti-HIV and anti-bacterial activities. The ethyl acetate and methanol extract showed evidence of anti-HIVs-1RT inhibition at 69.02 % and 69.24 %, respectively. Anti-syncytium (MC99+1A2) among evaluated all three extracts: it afforded the lowest EC50; hexane 9.65 (TI 4.39), ethyl acetate 11.15 (TI 3.84), and methanol 43.61 (TI 3.04). Moreover, an antibacterial study on extract was also performed. The antibacterial study was evaluated using nine strains (Staphylococcus aureus, Enterobacter aerogenes, Escherichia coli 0157: H7, Escherichia coli (ETEC), Escherichia coli (EPEC), Proteus mirabilis, Salmonella typhimurium, Shigella flexneri, and Vibrio cholera) by Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) method. The MeOH extract was the most effective antibacterial with a MIC in the range >0.1 mg/mL and MBC in the range >0.2 mg/mL. However, the isolated compounds from this plant had not been studied in all bioassay. Phytochemical examination of stems and twigs from Ficus dubia had directed to the isolation of four known compounds, epifriedelanol, β-sitosterol, stigmasterol, and bergapten.
 Conclusion: The crude extract of twigs and stems of Ficus fubia were specifically active toward MC99+1A2. With these results, it could be concluded that Ficus dubia may be an accomplished candidate in pill and future medicine.

Biosynthesis of Cylindrospermopsin in Cyanobacteria: Characterization of CyrJ the Sulfotransferase.

7-Deoxy-desulfo-cylindrospermopsin was purified at small-scale from the supernatant of a culture of the cyanobacterium Oscillatoria sp. PCC 10702. This metabolite was obtained in a pure form using a three-step chromatographic procedure, and its identity was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS quantification showed that this metabolite was excreted in the culture medium of Oscillatoria sp. PCC 10702. Isotopic incorporation studies using [2-13C,15N]glycine, a cylindrospermopsin precursor, and Oscillatoria sp. PCC 10702 cells showed that glycine was incorporated into 7-deoxy-desulfo-cylindrospermopsin, 7-deoxy-cylindrospermopsin, 7-epi-cylindrospermopsin, and cylindrospermopsin. The isotopic incorporation rate was consistent with the following metabolic flux: 7-deoxy-desulfo-cylindrospermopsin → 7-deoxy-cylindrospermopsin → 7-epi-cylindrospermopsin and cylindrospermopsin. We have cloned the cyrJ gene into an expression vector and overproduced the putative sulfotransferase CyrJ in Escherichia coli. The purified protein CyrJ catalyzed, in vitro, the transfer of a sulfonate group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to 7-deoxy-desulfo-cylindrospermopsin to give 7-deoxy-cylindrospermopsin. Kinetic analysis afforded the following apparent constants: KMapp. (PAPS) = 0.12 μM, Vmaxapp. = 20 nM/min, KMapp. (7-deoxy-desulfo-cylindrospermopsin) = 0.12 μM, and KIapp. (7-deoxy-desulfo-cylindrospermopsin) = 4.1 μM. Preliminary data suggested that CyrJ catalyzed the reaction through a ternary-complex kinetic mechanism. All these data confirmed that CyrJ catalyzed a sulfotransfer during the penultimate step of the biosynthesis of cylindrospermopsin.

Bioactivity flavonoids from roots of Euphorbia tirucalli L.

Euphorbia tirucalli (Euphorbiaceae) is a species widely utilized for popular medicine in northeast of Brazil because of its innumerable reported medicinal properties. The HPLC-Qtrap-MS/MS analysis from E. tirucalli roots ethanol extract revealed that this species presents 8 flavonoids and 5 shikimate derivative compounds. Chemical studies of these ethanolic extract using chromatographic procedures allowed the isolation of four previously unreported compounds for this species: myricetin, 3,3'-dimethoxy-4-O-α-rhamnopyranoside-ellagic acid, 4-O-methyl-gallic acid and ampelopsin. All structures were determined using 1D and 2D NMR spectroscopy analysis. The ethanol extract and four isolated compounds were evaluated for antimicrobial activity against Staphylococcus aureus, Escherichia coli, Sporothrix brasiliensis and Candida albicans. All the analyzed compounds presented activity against all evaluated bacteria and fungi, with MIC values ranging from 8 to 2048 μg/mL.

Hepatoprotective effects of Tagetes lucida root extract in carbon tetrachloride-induced hepatotoxicity in Wistar albino rats through amelioration of oxidative stress

Context The roots of Tagetes lucida Cav. (Asteraceae) have antioxidant and antimicrobial properties. Objective This study aimed to examine the hepatoprotective effects of T. lucida roots ethanol extract (TLRE) using carbon tetrachloride (CCl4)-induced hepatotoxicity in rats. Materials and methods The active ingredients of TLRE were identified by high-performance liquid chromatography, infra-red spectrum, and mass spectrometric procedures. Ninety rats were distributed into four main groups: positive, therapeutic, protective, and negative group. The therapeutic group was implemented using CCl4 (a single dose of 2 mL/kg) before TLRE or silymarin administration. Meanwhile, the protective group was implemented by administering CCl4 (a single dose of 2 mL/kg) after force-feeding TLRE or silymarin. Each therapeutic and protective group was divided into three subgroups: force-fed with saline, TLRE (500 mg/kg), and silymarin (25 mg/kg). The positive group was split into two subgroups that were force-fed TLRE and silymarin. Positive, therapeutic, and protective groups were compared to the negative group (untreated rats). CCl4, TLRE, and silymarin were orally administrated using a gastric tube. Results In the therapeutic and protective groups, TLRE significantly reduced liver enzymes, i.e., aspartate aminotransferase (12.47 and 6.29%), alanine aminotransferase (30.48 and 11.39%), alkaline phosphatase (17.28 and 15.90%), and cytochrome P450-2E1 (39.04 and 48.24%), and tumour necrosis factor-α (53.72 and 53.72%) in comparison with CCl4-induced hepatotoxicity controls. Conclusions TLRE has a potent hepatoprotective effect with a good safety margin. After a repeated study on another type of small experimental animal, their offspring, and an experiment with a large animal, this study may lead to clinical trials.

On line kinetic analysis of permeation profiles for UV filter loaded microemulsions using an automatic system with spectroscopic detection and a chemometric approach.

The analysis of the permeation kinetics of new UV filter formulations is of great importance since the kinetic parameters are related to the effectiveness of the product over time. The dynamics of this process can be evaluated by means of the calculation of the permeation kinetic constants, which can be obtained from the respective permeation profiles. This paper is aimed at improving the analytical performance of permeation assays using an on-line automatic system with spectrometric detection avoiding the chromatographic procedure and the usually manual sampling steps required using the traditional Franz diffusion cell. Then, the kinetics of permeation of octyl p-methoxycinnamate loaded in different microemulsions through a synthetic membrane (polyamide) was analysed at real time by UV-Vis and fluorescence spectroscopies. The spectral data were obtained at regular intervals of time (5 min) during 60 min, and the concentration of the permeated UV-filter was at each time calculated using univariate linear calibration. The interference caused by the presence of basil essential oil (oily phase) in some microemulsion samples was overcome using synchronous fluorescence spectroscopy (Δλ = 60 nm) and partial least squares. In all cases, the permeation profiles were obtained (first-order kinetics) and the respective permeation kinetic constants were calculated. The validation of the proposed method was assessed by gas chromatographic-mass spectroscopy and non-significant differences for the obtained permeation kinetic constants were found between methods (p = 0.05). Additionally, a commercial sample was analysed with the proposed methods and the results were validated by high performance liquid chromatography technique.

A Simple Reversed Phase High Performance Liquid Chromatography Method for the Estimation of Related Substances, Assay of Cabozantinib and Nivolumab and its Application to Dissolution Studies

The present work describes the development and subsequent validation of a novel, simple, selective and stability indicating gradient reversed phase high performance liquid chromatography method for the quantitative determination of related substances, assay of cabozantinib and nivolumab and its application to dissolution studies. The chromatographic method was optimized using the impurity-spiked solution. A good resolution between the peaks was achieved under selected chromatographic conditions. The separation was accomplished on an X-Bridge C18, 150×4.6 mm, 3.5 μ column connected to a photo diode array detector using 0.1 % orthophosphoric acid in water as mobile phase A and acetonitrile as mobile phase B, under gradient elution. The mobile phase flow rate was maintained at 1.0 ml/min. The detection of the constituents was done at 216 nm using a ultra-violet detector. Recovery studies were satisfactory and the correlation coefficient for two active pharmaceutical ingredients and their related substances, 0.999 indicates the linearity of the method within the limits. Limit of detection and limit of quantification for all impurities and cabozantinib and nivolumab were established with respect to the test concentration. Specificity, accuracy, precision, ruggedness and robustness were determined as part of the method validation. The performance of the method was validated according to the current International Council for Harmonisation requirements. Moreover, the dissolution study was performed on active pharmaceutical ingredients to estimate the recovery using the same method. Validation of the developed reversed phase high performance liquid chromatography procedure revealed that all the degradation products formed during stress conditions and related impurities were well separated from their active pharmaceutical ingredients and peaks were well resolved from each other with appropriate retention time. The method was characterized by good linearity, specificity, low values of limit of detection and quantisation, accuracy, precision, ruggedness and robustness. All statistical results were within the acceptance criteria and the proposed method is simple, fast, accurate, precise and reproducible hence, it can be applied for routine dissolution analysis and employed for quality control of drug samples during stability studies.

Identification and Isolation of Caffeic, Chlorogenic and Ferulic Acids in Aerial Parts of Capparis spinosa wildly grown in Iraq

Capparis spinosa is one of the oldest genera grown in Iraqi land with worldwide traditional medicinal uses beside the culinary uses. These uses were own to the presence of many phytochemical including flavonoids, polyphenols. Among the reported polyphenolic acids are caffeic, chlorogenic and ferulic acids with well-known powerful antioxidant properties. The present work aimed to identify the presence of these polyphenolic acids in Iraqi caper naturally gown in the rural area of middle Iraq following standard chromatographic procedures. Aerial parts of the plant (buds, berries and leaves) were extracted with hydroalcoholic solvent by maceration method. Thin layer chromatographic techniques and HPLC analysis were performed to identify the presence of polyphenolic acids in ethyl acetate fraction of the hydroalcoholic extracts. The result obtained in this work revealed the presence of these phenolic acids in investigated extracts. Chromatographic analysis confirms the presence of considerable amounts of these acids in ethyl acetate fraction when the separated spots were compared with Rf values and UV spectra of standards. Such data give a promising use of aerial parts of Iraqi caper for globally reported medicinal uses.

Polyphenolic compounds of Euphorbia umbellata (Pax) Bruyns (Euphorbiaceae) improved endothelial dysfunction through arginase inhibition.

Euphorbia umbellata is used for its anti-inflammatory properties; however, there are limited data available regarding its effects on vascular function. Its bark is rich in polyphenolic compounds, which potentially improve endothelial dysfunction (ED). This study proposes to investigate the effects of E. umbellata bark extracts and its polyphenolic compounds on arginase (ARG) activity and nitric oxide (NO)-related targets. Chromatographic procedures were used for the chemical characterisation of the extracts. Furthermore, in silico (molecular docking), in vitro (ARG inhibition), in vivo (streptozotocin-induced hyperglycemia model), and ex vivo (l-arginine metabolism, vascular reactivity, western blot, and biochemical) techniques were carried out. Quercetin, gallic acid, and ellagic acid were identified in the extracts. In silico screening predicted that gallic acid and quercetin would have the most promising interactions with ARG -identified cavities. This was confirmed in vitro as both compounds had a direct inhibitory effect on ARG, as was the case regarding the extracts. Oral treatment preserved endothelium-dependent vasodilation through ARG inhibition together with an increase in l-arginine bioavailability and endothelial NO synthase expression. Biochemical parameters determined the lack of toxicity for sub-chronic treatment. E. umbellata bark extracts and its compounds can contribute to ED treatment, at least partly, through the inhibition of vascular ARG.

Anti-esophageal Cancer Effect of Corilagin Extracted from Phmllanthi Fructus via the Mitochondrial and Endoplasmic Reticulum Stress Pathways

Headings ethnopharmacological relevanceCorilagin (β-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-d-glucose) is a tannin isolated from the traditional ethnopharmacological plant Phmllanthi Fructus, which is widely used in not only traditional Chinese medicine but also tropical and subtropical medicine to ameliorate various diseases. Aim of the studyThis study was designed to isolate the potential anti-esophageal cancer (EC) component corilagin from Phmllanthi Fructus and explain its anti-EC mechanism. Materials and methodsCorilagin was isolated from Phmllanthi Fructus by extraction and chromatographic procedures, and its anti-esophageal cancer effect was evaluated by in vitro and in vivo experiments. In vitro experiments included MTT analysis, flow cytometry, and the Transwell assay and were used to observe corilagin-mediated inhibition of EC cell growth. Western blotting was used to analyze the apoptotic pathway of EC cells. In vivo experiments used tumor-bearing nude mice to evaluate the antitumor effect of corilagin, and its potential mechanism was explored by Western blotting. ResultsCorilagin showed significant anti-EC activity in vitro and in vivo. Corilagin was significantly cytotoxic to EC cells and induced apoptosis in EC cells. Corilagin induced G0/G1 phase arrest by altering key G0/G1 cell cycle regulatory markers and significantly reducing the migration of EC cells and the number of cells in a time- and dose-dependent manner. Additionally, corilagin inhibited the growth of transplanted tumors in nude mice without significant toxicity. Regarding the anticancer mechanism of corilagin, the results showed that corilagin inhibited esophageal cancer progression by activating mitochondrial and endoplasmic reticulum stress signaling pathways. ConclusionsCorilagin shows significant anti-EC activity in vitro and in vivo. The mechanism of the anti-EC activity of corilagin may be due to activating mitochondrial and endoplasmic reticulum stress signaling pathways.

A linearized non-isothermal general rate model for studying thermal effects in liquid chromatographic columns

A linearized non-isothermal general rate model is formulated and analytically solved to quantify the effects of temperature variations in fixed-bed chromatographic columns. The model contains a set of four coupled PDE accounting for energy transfer resistances, inner and outer particle-pore diffusions, and interfacial mass and axial dispersion. The Laplace transform, the eigenvalue-decomposition technique, and a conventional technique for the solutions of ODE are jointly employed for the solution of the model equations. A few numerical test studies are considered to assess the impact of system parameters on the performance of packed-bed adsorption columns. To access the range of applicability and to get the scope of the appropriateness of calculated analytical results, the numerical results are also obtained by applying a high resolution finite volume scheme. The analytical solutions obtained can be used as an invaluable tool for analyzing, optimizing, and upgrading the non-isothermal liquid chromatographic procedures.

Разработка методики определения потенциально опасных летучих органических соединений (трихлорэтилен и тетрахлорэтилен) в атмосферном воздухе

The article dwells on results obtained via experimental research on working out a gas chromatography procedure for determining trichloroethylene and tetrachloroethylene in ambient air. Experiments were performed on substances which had low limits of detection with gas-liquid chromatography with electron capture detection (GLC/ECD) when examined substances were absorbed from ambient air on Tenax TA sorbent. Optimal gas chromatography parameters were established with a hardware-software complex based on «Crystal-5000» gas chromatographer and use of a column from IDBPX-VOL series, 60m⋅0.32mm⋅1.8µm, under the following temperatures: column, 50–230о С; evaporator, 250о С; detector, 250о С. The developed capillary gas chromatography procedure allows determining trichloroethylene in concentrations ranging from 0.000146 to 0.00146 mg/m3, and tetrachloroethylene, from 0.000081 to 0.00081 mg/m3 with inaccuracy not exceeding 25.0%. We performed metrological assessment of the procedure and it allowed determining quality of analysis results for trichloroethylene and tetrachloroethylene; they were as follows: precision, 21.97% and 14.3%: repeatability, 4.22% and 3.38%; reproducibility, 5.66% and 4.9%. Limit of detection (LOD) for trichloroethylene and tetrachloroethylene was =0.0000038 mg/dm3 and =0.00000083 mg/dm3 accordingly. Limit of quantitative determination (LOQ) was =0.000013 mg/m3 for trichloroethylene, and = 0.0000028 mg/m3 for tetrachloroethylene. The developed procedure allowed detecting contents of the examined substances in ambient air near a construction site and a dry-cleaner’s, trichloroethylene in a range from 0.00001 mg/m3 to 0.0009 mg/m3, tetrachloroethylene, from 0.000011 mg/m3 to 0.00039 mg/m3. This unified high-sensitive and selective procedure is recommended for systemic control over potentially hazardous volatile organic compounds in ambient air as it allows providing objective and reliable hygienic assessment of chemical safety and quality of the environment and health risk assessment.

Working out a procedure for determining potentially hazardous volatile organic compounds (trichloroethylene and tetrachloroethylene) in ambient air

The article dwells on results obtained via experimental research on working out a gas chromatography procedure for determining trichloroethylene and tetrachloroethylene in ambient air. Experiments were performed on substances which had low limits of detection with gas-liquid chromatography with electron capture detection (GLC/ECD) when examined substances were absorbed from ambient air on Tenax TA sorbent. Optimal gas chromatography parameters were established with a hardware-software complex based on «Crystal-5000» gas chromatographer and use of a column from IDBPX-VOL series, 60m⋅0.32mm⋅1.8µm, under the following temperatures: column, 50–230о С; evaporator, 250о С; detector, 250о С. The developed capillary gas chromatography procedure allows determining trichloroethylene in concentrations ranging from 0.000146 to 0.00146 mg/m3, and tetrachloroethylene, from 0.000081 to 0.00081 mg/m3 with inaccuracy not exceeding 25.0%. We performed metrological assessment of the procedure and it allowed determining quality of analysis results for trichloroethylene and tetrachloroethylene; they were as follows: precision, 21.97% and 14.3%: repeatability, 4.22% and 3.38%; reproducibility, 5.66% and 4.9%. Limit of detection (LOD) for trichloroethylene and tetrachloroethylene was =0.0000038 mg/dm3 and =0.00000083 mg/dm3 accordingly. Limit of quantitative determination (LOQ) was =0.000013 mg/m3 for trichloroethylene, and = 0.0000028 mg/m3 for tetrachloroethylene. The developed procedure allowed detecting contents of the examined substances in ambient air near a construction site and a dry-cleaner’s, trichloroethylene in a range from 0.00001 mg/m3 to 0.0009 mg/m3, tetrachloroethylene, from 0.000011 mg/m3 to 0.00039 mg/m3. This unified high-sensitive and selective procedure is recommended for systemic control over potentially hazardous volatile organic compounds in ambient air as it allows providing objective and reliable hygienic assessment of chemical safety and quality of the environment and health risk assessment.

Chiral Analysis of Amphetamine and Methamphetamine in Urine by Liquid Chromatography-Tandem Mass Spectrometry Applying Mosher Derivatization

Methamphetamine (MA) and amphetamine (AM) are central nervous system doping agents, listed in the WADA Prohibited List, which in the past years showed the highest prevalence among all stimulants. Specifically, concerning these stimulants, it is essential to distinguish between d and l-isomers of AM and MA due to their distinction in illicit or legal nonprescription use. The main separation methods used for the urinary determination of MA and AM enantiomers are based on chromatographic procedures, such as gas chromatography after chiral derivatization or liquid chromatography (LC) applying chiral columns, followed by the mass spectrometer (MS) detection. The use of the optically pure reagent R-(-)-α-methoxy-α-(trifluoromethyl) phenylacetic acid chloride (R-MTPA-Cl – Mosher’s Reagent) allows the isomeric separation by both chromatographic procedures while preserving the isomeric composition of the substances. Therefore, in the present work, an LC-MS2 approach has been developed for AM and MA detection in urine after Mosher derivatization. The method was validated and can be used for the enantioselective confirmation of AM and MA for doping control purposes and forensic analysis in general, being useful for the distinction between the medicinal and illicit use of both stimulants.

Cerium stable isotope analysis of synthetic and terrestrial rock reference materials by MC-ICPMS

Cerium is the most abundant rare earth element (REE) in the solar photosphere, CI chondrites, and the Earth. It has four main stable isotopes (masses: 136,138,140, and 142), with 138Ce being the most studied species, used in geochronology and petrogenesis. In addition, more abundant 140Ce and 142Ce are suggested to be potentially applicable in geochemical investigations. In this work, we developed a modified four-step ion chromatography procedure for Ce chemical separation. Using a MC-ICPMS, we designed a cup configuration to measure 142Ce/140Ce ratio of the samples with an optimized Nd correction equation. A 0.03‰ (2SD) reproducibility was obtained for Ce Ames metal standard. We analyzed ten different igneous and one sedimentary geochemical reference materials. Mean δ142Ce range from −0.07 to 0.32‰. Most of the samples show a heavier Ce isotopic composition than the Ce Ames standard. The majority of rocks have a homogenous δ142Ce. The δ142Ce does not show any correlation with rock chemical composition including their Ce content or rock types. A carbonatite (SARM 40) has a mean δ142Ce of −0.07 ± 0.13‰ (2SD), lower than the other rocks, suggesting the possibility of a pronounced isotopic fractionation. Our work demonstrates the applicability of the developed methodology and the potential of Ce stable isotopes for future geochemical studies. Production of a larger database of δ142Ce values is required to obtain a clearer view on the similarities and differences between different geological material and explaining Ce stable isotope dynamics.

Detection of clostebol in sports: Accidental doping?

The detection of clostebol misuse in sports has been growing recently, especially in Italy, due to the ample availability of pharmaceutical formulations containing clostebol acetate (Trofodermin®) and the use of more sensitive instrumentation by the antidoping laboratories. Most of these cases have been claimed to be related to a nonconscious use of the drug or through contact with relatives or teammates using it. We have investigated, through the application of the well-known and currently used gas chromatographic mass spectrometric procedures, the likelihood of these allegations and have demonstrated that after a single transdermal administration of 5 mg of clostebol acetate and a transient contact with the application area, it is possible to generate adverse analytical findings in antidoping controls. We have reviewed the Phase I and Phase II clostebol metabolism in order to generate evidences that may help the sport authorities reviewing these cases. The main clostebol metabolite (4-chloro-androst-4-en-3α-ol-17-one, M1) generally used at the screening level as well as other three metabolites (M2-M4) are mainly excreted as glucuronides, whereas M5 (4ζ-chloro-5ζ-androstan-3β-ol-17-one) is predominantly excreted as sulfate. Neither the 5α-reductases activity (impaired by the presence of the chlorine in C4) nor specific sulfotransferases present in the skin allowed a clear distinction of the administration route. Studies with a larger number of volunteers and probably investigating another physiological fluid allowed in antidoping such as blood are needed for a deeper investigation. It is not unreasonable to establish a reporting level for M1, maybe creating some false negatives but excluding nonintentional doping scenarios.

Radiation-Induced Transformations of 2-Fluoroethanol in Aqueous Solutions

Steady-state radiolysis has been used to study the radiation-induced transformations of 2-fluoroethanol in dilute aqueous solutions as a model system for examining the autoradiolytic dehalogenation of the radiopharmaceutical drug 2-[18F]fluorodeoxyglucose ([18F]FDG). A derivatization–gas chromatography procedure for determining the fluoride anion in the presence of organofluorine compounds has been developed. It has been established that •OH and H• induced the dehalogenation of 2-fluoroethanol in aqueous solutions. Unlike bromo- and chlorohydrins, 2-fluoroethanol does not interact with $${\text{e}}_{{{\text{aq}}}}^{ - }$$ according to the dissociative attachment mechanism. It has been shown that О2 halved the radiation-chemical yield of fluoride due to the oxidation of hydroxyl-containing carbon-centered radicals of 2-fluoroethanol. The constant of unimolecular dehalogenation of the 2-fluoroethanol radical is 3.9 × 106 s−1, as calculated using the method of competing reactions.

Enhancing the potential exploitation of Salvia miltiorrhiza Bunge: Extraction, enrichment and HPLC-DAD analysis of bioactive phenolics from its leaves

Salvia miltiorrhiza Bunge leaves, a crop waste, have potential as a source of bioactive phenolics. In this work, ultrasound-assisted extraction (UAE) was employed as a green extraction technology to extract phenolics from S. miltiorrhiza leaves. UAE parameters were optimized using response surface methodology (RSM) based on Box-Behnken design, achieving the total phenolics from S. miltiorrhiza leaves (SMLTP) yield of 71.16 ± 0.57 mg gallic acid equivalent per g of dry material (mg GAE/g DM) under optimal conditions. Afterwards, NKA-2 resin was selected to enrich SMLTP, the data for adsorption of SMLTP on NKA-2 resin fitted well with pseudo-second-order and Langmuir models. A NKA-2 resin-based chromatography procedure was optimized by studying breakthrough curves and desorption curves to enrich SMLTP from crude extract. A lab-scale enrichment experiment was performed under optimal conditions, the content of SMLTP in the product was 78.33 ± 0.61 %, which was 3.7 times higher than that in the crude extract, the recovery yield of SMLTP was 82.14 ± 1.62 %. Finally, HPLC-DAD analysis showed the contents of five major phenolic compounds including caffeic acid, rutin, isoquercitrin, rosmarinic acid, and salvianolic acid B significantly increased through enrichment. Certainly, this study will enhance S. miltiorrhiza industry to be more ecologic and economic.

Perylenequinones production induced by co-culturing Setophoma sp. and Penicillium brasilianum

Abstract Co-culture strategy can be applied to produce structural diversity of natural products. This approach also enables the understanding of the interaction mechanisms in microbial ecology. The present study aimed at the secondary metabolism diversification of the endophytic fungus Setophoma sp. by co-culturing it with Penicillium brasilianum and Talaromyces wortmannii, species also isolated as endophytes, and with Penicillium digitatum, a phytopathogenic fungus dangerous to Brazilian citriculture. The increase of perylenequinones (stemphyperylenol and derivatives) produced by Setophoma sp. strain was observed in the co-cultures in response to the endophytic P. brasilianum. The induced stemphyperylenol was isolated by combined chromatographic procedures and identified by spectroscopic techniques. Other perylenequinone derivatives were detected by UHPLC-DAD-HRMS based dereplication data analysis. Stemphyperylenol exhibited antifungal effects not only against P. brasilianum, but also against P. digitatum, the main postharvest pathogen of orange fruits, and Aspergillus fumigatus, an important human pathogenic fungus ubiquitous in soil. Preliminary insights concerning the mode of action of stemphyperylenol were determined by evaluating a collection of A. fumigatus mutants. Thus, stemphyperylenol may have the potential for agricultural applications, and it is also promising as a human antifungal compound. Moreover, the co-culture of an endophyte-endophyte system may represent a potential way to achieve the chemical diversity of bioactive molecules.