High-performance Liquid Chromatography Fractions Research Articles

Marinobufagenin extraction from Rhinella marina toad glands: Alternative approaches for a systematized strategy.

Marinobufagenin is a bufadienolide compound detected mainly in skin and parotoid gland secretions of Rhinella marina (L.) toad. Bufadienolides regulate the Na+ /K+ -ATPase pump by inhibiting the cardiotonic steroid dependent-site and act as cardiac inotropes with vasoconstrictive properties. Marinobufagenin and other bufadienolides, such as telocinobufagin and bufalin, are thought to be found endogenously in mammals in salt-sensitive hypertensive states such as essential hypertension, congestive heart-failure, and preeclampsia. The role of marinobufagenin as antimicrobial agent and its cytotoxic potential have also been recognized. The particular interest around marinobufagenin prompts us to consider the Rhinella marina toad venom as a possible source for molecules with pharmacological and/or diagnostic potential. In this article, two different approaches of extraction and purification of marinobufagenin from Rhinella marina (L.) venom are studied: (i) Preparative thin-layer chromatography combined to mass spectrometry and/or ultraviolet detection and (ii) solid-phase extraction coupled with fractionation on high-performance liquid chromatography. Different chromatographic conditions are tested for each approach. The solid-phase extraction combined with high-performance liquid chromatography fractionation approach was preferred as it offered a greater yield, was less time-consuming and allowed us to selectively isolate marinobufagenin. Both protocols aim to provide efficient and convenient methods for toad venom extraction, based on an easily automatable and systematized strategy.

Acute toxicity evolution during ozonation of mono-chlorophenols and initial identification of highly toxic intermediates.

Acute toxicity changes during ozonation of 2-chlorophenol (2-CP), 3-chlorophenol (3-CP) and 4-chlorophenol (4-CP) under various conditions were studied using the luminescence inhibition test. Though the concentrations of mono-chlorophenols (CPs) and TOC decreased constantly, the acute toxicity evidently increased with reaction time until a maximum value was reached during ozonation of 2-CP at pH 3 to 7, 3-CP at pH 3 to 10, and 4-CP at pH 3 and 5. Principal component analysis (PCA) was applied to understand the relationships between acute toxicity and other physicochemical parameters. The intense interrelations between acute toxicity and SUVA280 were evident under all the pH conditions for 2-CP and 3-CP, and except at pH 12 for 4-CP, which implied that high-weight intermediates were formed and contributed to the increased toxicity. SUVA280 was a possible surrogate measure of acute toxicity during the ozonation of all three CPs. Profiling of acute toxicity in ozonated CPs was conducted using solid phase extraction with a series of different cartridges, high performance liquid chromatography (HPLC) fractionation and the luminescence inhibition bioassay. Considering the acute toxicity evolution under different conditions and profiles of acute toxicity in HPLC fractions, the highly toxic intermediates probably included same non-chlorine carboxylic acids though the three CPs had different substituent positions with higher polarity than their parent compounds. In this study, the acute toxicity evolution during ozonation of CPs and the forcing agents were thoroughly discussed. Therefore, ozonation is expected to be carefully operated and monitored, and toxicity tests should be used to evaluate whether effluent detoxification takes place.

Biochemical and biological validations of a faecal glucocorticoid metabolite assay in mandrills (Mandrillus sphinx).

Stress is a major factor in determining success when releasing endangered species into the wild but is often overlooked. Mandrills (Mandrills sphinx) are vulnerable to extinction due to habitat loss and demand for bush meat and the pet trade. To help bolster in situ populations, rehabilitated rescued mandrills recently were released into a protected area in the Republic of Congo. The goal of this study was to validate the use of faecal glucocorticoid metabolite enzyme immunoassays (EIAs) in mandrills and test field-friendly faecal hormone extraction techniques that can subsequently be used to monitor the stress physiology and welfare of mandrills throughout the release process. Using faecal samples collected from ex situ mandrills, we tested cortisol, corticosterone, 11β-hydroxyetiocholanolone (69a), and 11-oxoetiocholanolone EIAs. Absolute concentrations, hormone profiles following medical procedures or translocation, and high-performance liquid chromatography fraction immunoreactivity showed that the 69a assay was the best choice to monitor the stress response in this species. Samples with delayed extraction or drying times had 40-80% lower 69a concentrations than samples extracted immediately post-collection and frozen. The 69a EIA is an appropriate assay for monitoring welfare in this species in situ or ex situ, and results indicated that consistent extraction methods are important for accurate comparisons.

High-yield production of spider short-chain insecticidal neurotoxin Tx4(6–1) in Pichia pastoris and bioactivity assays in vivo

Short-chain insecticidal neurotoxin Tx4(6–1) from the spider Phoneutria nigriventer can be prepared by reversed-phase high-performance liquid-chromatography (HPLC) fractionation of PhTx4, but this is difficult and represents an obstacle preventing analyses of its insecticidal activity against agricultural insect pests. Herein, we performed secretory expression of recombinant Tx4(6–1) using Pichia pastoris strain X33 as the host, and screened transformants using enzyme-linked immunosorbent assay (ELISA). In flasks, ∼5 mg/l rTx4(6–1) was expressed as a secreted protein following induction with methanol, and this was increased to 45 mg/l rTx4(6–1) in a fed-batch reactor. Approximately 4 mg of high-purity rTx4(6–1) was purified from a 400 ml fed-batch culture supernatant by Ni+-nitriloacetic acid affinity chromatography, followed by carboxymethyl (CM) sepharose ion-exchange chromatography. Purified rTx4(6–1) was determined by mass spectrometry (MS) analysis, revealing a molecular weight (MW) of 7660.5 Da, larger than the expected size due to O-linked glycosylation. Insect bioactivity tests of rTx4(6–1)-treated fifth-instar silkworm larvae (Bombyx mori Linnaeus) showed neurotoxin symptoms such as contraction paralysis, abdominal contraction, and mouth movement syndrome, with a half lethal dose at 12 h post-injection of ∼4.5–8.5 μg/g body weight. Dietary toxicity was not observed in silkworm larvae.

Responses of Heterodera glycines and Meloidogyne incognita Infective Juveniles to Root Tissues, Root Exudates, and Root Extracts from Three Plant Species.

The infective juvenile (J2) stage of endoparasitic plant nematodes uses plant chemical signals, released from roots, to localize and infect hosts. We examined the behaviors of soybean cyst nematode (Heterodera glycines) and root-knot nematode (Meloidogyne incognita) J2 in the presence of root signals from marigold (Tagetes patula), soybean (Glycine max), and pepper (Capsicum annuum). Signals were obtained from sources commonly used in phytoparasitic nematode chemotaxis studies: root tips, root exudates, and root extracts. Root tips from each plant species attracted M. incognita but H. glycines was attracted only to soybean. In contrast, root exudates prepared from marigold, pepper, or soybean seedlings were attractive to H. glycines but were repellent to M. incognita. Root extracts had the same effect as exudates. Fractionation of exudates by reversed-phase high-performance liquid chromatography (HPLC) (acetonitrile [CH3CN] and 0.1% trifluoroacetic acid) revealed highly polar and less polar components affecting behaviors. Fractions eluting at 12% CH3CN from all three plants attracted H. glycines and repelled M. incognita. None of the less polar HPLC fractions (>15% CH3CN) affected H. glycines but those from G. max and T. patula repelled M. incognita. Differences among exudates and effects of fractionation on behavior are discussed.

In vitro antioxidant and anti-inflammatory activities of Melipona beecheii honey protein fractions

Proteins extracted from Melipona beecheii honey were assessed for its antioxidant and anti-inflammatory activity by in vitro assays. Protein content was 3.56 mg/mL and after reverse phase high-performance liquid chromatography fractionation eight distinctive peaks were detected with molecular weight ranged from 7.04 to 95.35 kDa. Low molecular weight fraction constituted by proteins with weights of 7.04, 14.07 and 24.16 kDa, exhibited the highest antioxidant activities with the lowest IC50 values for radical scavenging activity (0.4262 mg/mL), chelating effects (0.3763 mg/mL), and reducing power (0.3060 mg/mL). Same fraction exhibited the lowest IC50 values for inhibition of protein thermal denaturation (0.4115 mg/mL) and cell membrane stabilization (0.5563 mg/mL). For its part, the complete protein extract exhibited the lowest inhibition of proteolytic activity (4.3548 mg/mL). From the results, it is concluded that proteins of honey produced by Melipona beecheii are partially related with its antioxidant and anti-inflammatory activities. These findings validate the use that many Mesoamerican cultures have made of the honey produced by M. beecheii for the healing of lesions and dermic pathologies. Once deeper studies and the pertinent clinical trials have been carried out, the honey of M. beecheii and its proteins could be incorporated into products for pharmaceutical use.

Removal of bias in C37 alkenone-based sea surface temperature measurements by high-performance liquid chromatography fractionation

Sea surface temperature determinations based on marine sedimentary C37 alkenone distributions have provided a wealth of data for paleoclimatic studies, including those performed at high resolution. The success of this approach results from several characteristics of alkenone compounds, e.g. their geochemical properties (such as unequivocal synthesis by certain widespread haptophyte algae, plus chemical stability/preservation of the original alkenone distributions during sedimentation), and their analytical properties (such as fast clean up procedures using alkaline hydrolysis of sediment extracts, followed by robust instrumental methods allowing large scale sample processing). Here we show that, in sediments under the influence of continental inputs, coelution of these compounds with cholest-5-enyl 3β-undecenyl ether and 24-methylcholesta-5,22-dienyl 3β-undecenyl ether deviate the SST measurements despite alkaline hydrolysis. Here, we report a new high performance liquid chromatrography fractionation method which eliminates these interfering compounds and gathers all the alkenones into a single fraction. These fractions can then be analysed by gas chromatography as in the initial approach, providing large amounts of data as required in high resolution studies.

Exploration of antiplasmodial activity in Acalypha wilkesiana Müller Argoviensis, 1866 (family: Euphorbiaceae) and its GC-MS fingerprint.

The plant kingdom continues to hold great promise for the eradication of Malaria infection following the challenges of insecticide resistance by the vector mosquito, drug resistance by the parasite, and the development of a vaccine still being a mirage. Acalypha wilkesiana Muller Argoviensis, 1866 (family: Euphorbiaceae) leaves have the ethnopharmacological reputation for use as a remedy against dermal microbial infections in Nigeria. Here, we have studied the antiplasmodial potential of the extract of the leaves of this ornamental plant. Aqueous methanol crude extract (70%) and Prep reversed-phase high-performance liquid chromatography (RPHPLC) fractions were tested in vitro against blood stage Plasmodium falciparum 3D7 strain parasites for antiplasmodial activity using the SYBR Green assay. Results obtained were validated through Giemsa stained microscopic blood smeared slides. An IC50 of < 0.39μg/ml for fractions of the RPHPLC together with TC50 of > 100μg/ml against mammalian HUH-7 cell lines and a HC50 of > 100μg/ml against red blood cells indicate a high selectivity of this plant against Plasmodium. This is the first report of the antiplasmodial activity of this plant and a GC-MS fingerprinting of the same, opening the possibilities of identifying novel pharmacophores against the malaria parasite.

Compound Heterozygosity of β-Thalassemia and the Sickle Cell Hemoglobin in Various Populations of Chhattisgarh State, India

Hemoglobinopathies evolved as a protective mechanism against malaria, which exhibit selective advantage in the heterozygous state. However, in a homozygous recessive condition, it poses a serious socioeconomic burden. Sickle cell anemia is an autosomal recessive hemoglobinopathy associated with erythrocytes sickling, vaso-occlusive crisis (VOC), as well as multi-organ failure and death. The coinheritance of other hemoglobinopathies is known to substantially modulate the clinical manifestation of sickle cell anemia. In the present study, we aimed to analyze the coinheritance of β-thalassemia (β-thal) in Hb S (HBB: c.20A>T) patients. The study includes 918 sickle cell anemia patients from 10 ethnic populations of Chhattisgarh State, India. Complete blood counts (CBCs) and hemoglobin (Hb) high performance liquid chromatography (HPLC) fractionation data were collected from patient record books. We observed Hb S-β-thal in all the analyzed populations. Interestingly, high frequencies of Hb S-β-thal have been observed in Satnami (53.8%), Rawat (47.1%), Gond (35.1%) and Panika (30.6%) populations. Inter-population comparison of hematological parameters [Hb F (p < 0.001), Hb A2 (p < 0.001), Hb (p = 0.03) and red blood cell distribution width (RDW) (p < 0.001)] revealed significant differences. We also observed that mean Hb F levels were significantly higher in Hb S compared to Hb S-β-thal patients in the respective populations. Our study highlights the higher prevalence of β-thal as well as the compound heterozygosity for Hb S and β-thal in various populations of Chhattisgarh State, India.

Structural Identification of 19 Purified Isomers of the OPV Acceptor Material bisPCBM by 13C NMR and UV-Vis Absorption Spectroscopy and High-Performance Liquid Chromatography.

The molecular structures of 19 purified isomers of bis-phenyl-C62-butyric acid methyl ester were identified by a combination of 13C NMR and UV-vis absorption spectroscopies and high-performance liquid chromatography (HPLC) retention time analysis. All 19 isomers are dicyclopropafullerenes (none are homofullerenes). There were seven isomers with C1 molecular point-group symmetry, four with C s, six with C2, one with C2 v, and one with C2 h symmetry. The C2 h, C2 v, and all five nonequatorial C1 isomers were unambiguously assigned to their respective HPLC fractions. For the other 12 isomers, the 13C NMR and UV-vis spectra placed them in six groups of two same-symmetry isomers. On the basis of the widely spaced HPLC retention times of the two isomers within each of these six groups, and the empirical inverse correlation between retention time and addend spacing, each isomer was assigned to its corresponding HPLC fraction. In addition, the missing trans-1 isomer was found, purified, and characterized.

Characterization and partial purification of an antibacterial agent from halophilic actinomycetes Kocuria sp. strain rsk4.

Introduction: The inevitable rise of antibiotic-resistant bacteria is a global health problem. These pathogens erode the utility of available antibiotics. Staphylococcus aureus is one of the major causes of community-acquired infections. The aim of work was to evaluate the marine actinomycetes for production of the antibacterial agent against pathogens. Methods: Halophilic actinomycetes were isolated, characterized and screened for production of antibacterial agent against pathogenic bacteria. The antibacterial compounds were extracted by solvent extraction and separated by TLC based bioautography. Antibacterial compound was further purified by flash chromatography followed by high-performance liquid chromatography (HPLC) techniques. The active fraction was characterized by spectroscopy techniques. The minimum inhibitory concentration of antibiotic was determined against pathogens. Results: A new halophilic actinomycetes strain rsk4 was isolated from marine water. It was designated as Kocuria sp. based on the physiological, biochemical and 16S rDNA sequencebased characters. It was able to produce broad-spectrum antibacterial compound and exhibited significant inhibitory activities against antibiotic-resistant S. aureus. The antibacterial compound was secreted optimally at 5% NaCl and neutral pH in the starch casein medium during stationary phase. The crude ethyl acetate extract was separated by chloroform-methanol, 24:1, v/v having Rf value 0.45. Bioassay of HPLC fractions confirms the presence of antibiotics picks at retention time: 3.24 minutes. The UV-Visible and mass spectra of the compound revealed that the active compound was different from other known antibiotics. The lowest minimum inhibitory concentration was recorded against S. aureus (30 µg/mL). Conclusion: The result suggests that a broad-spectrum antibacterial compound obtained from halophilic actinomycetes is effective against pathogenic bacteria. This compound may be a good alternative treatment against antibiotic-resistant pathogen S. aureus.

Bacterial cyclic β-(1,2)-glucans sequester iron to protect against iron-induced toxicity.

Cellular iron homeostasis is critical for survival and growth. Bacteria employ a variety of strategies to sequester iron from the environment and to store intracellular iron surplus that can be utilized in iron-restricted conditions while also limiting the potential for the production of iron-induced reactive oxygen species (ROS). Here, we report that membrane-derived oligosaccharide (mdo) glucan, an intrinsic component of Gram-negative bacteria, sequesters the ferrous form of iron. Iron-binding, uptake, and localization experiments indicated that both secreted and periplasmic β-(1,2)-glucans bind iron specifically and promote growth under iron-restricted conditions. Xanthomonas campestris and Escherichia coli mutants blocked in the production of β-(1,2)-glucan accumulate low amounts of intracellular iron under iron-restricted conditions, whereas they exhibit elevated ROS production and sensitivity under iron-replete conditions. Our results reveal a critical role of glucan in intracellular iron homeostasis conserved in Gram-negative bacteria.

Measurement of Alkaloids Achyranthes Aspera Linn Level Using Thin Layer Chromatography Method and High-Performance Liquid Chromatography

One of the plants with the potential to be developed into a raw material for anti-breast cancer drugs is jarong (Achyranthes aspera linn). Allegedly alkaloid of Achryranthes aspera linn leaves showed anticancer activity in vitro in mice myeloma cells is extremely potent. In vivo study showed that the alkaloid Achyranthes aspera linn causes apoptosis and healing in breast cancer cells in mice induced by benzopyrene. Alkaloids of Achyranthes aspera linn leaves as anticancer been listed on Incestual Property Rights (IPR) since October 12, 2012, with number P00201299839. This study aims to prove the existence of alkaloids in the leaves of Achyranthes aspera linn using thin layer chromatography (TLC), determine the level of alkaloids in the leaves alkaloid fraction of Achyranthes aspera using High-Performance Liquid Chromatography (HPLC) and alkaloid fraction dose as an anticancer and dosing for the study. Alkaloid qualitative measurement results with thin layer chromatography showed alkaloid in orange. Alkaloid levels using HPLC has a retention time of 3.482 minutes, a wavelength of 254 nm, there are 13 kinds of chemical substances with the major components amounted to 52.36% alkaloids, the other component is the green substance with a peak area of 13.0624%, while other chemical substances in a very small peak area of between 0.428% until 8.3598%. Alkaloid fraction dose of Achyranthes aspera used in the study as anticancer is controlled the dose of 0 mg/kg, 1 treatment dose of 60 mg/kg, 2 treatment dose of 75 mg/kg and 3 treatment dose of 90 mg/kg. Keywords: Alkaloids, Achyranthes aspera linn, TLC, HPLC.

Identification of CALM as the Potential Serum Biomarker for Predicting Recurrence of Nasopharyngeal Carcinoma Using Mass Spectrometry-Based Comparative Proteomic Approach

To date, there are no serum biomarkers for prediction of recurrent nasopharyngeal carcinoma (rNPC). Diagnosis of rNPC depends on imaging and biopsy of diseased tissue mostly, both two ways work mostly if target tumor in an advanced stage. Therefore, predicting recurrent biomarkers are urgently required. In this study, we used tandem mass tags(TMT) labeling and high performance liquid chromatography(HPLC)fractionation followed by liquid chromatograph-tandem mass spectrometry (LC-MS/MS) analysis to identify differentially expressed proteins, and serum was collected from 40 nasopharyngeal carcinoma (NPC)patients (recurrence (n = 20) and no-recurrence (n = 20). Compared to nrNPC, we found 59 proteins significantly dysregulated in rNPC, most were previously reported to play a role in carcinogenesis. Dysregulation of Calmodulinis (CALM) was confirmed in 74 new patients (recurrence (n = 32) and no-recurrence (n = 42) by ELISA. Moreover, we performed a preliminary pathway analysis showed Oxidative phosphorylation was altered in rNPC compared to nrNPC patients. These data together identify potential diagnostic biomarker for rNPC and elucidate the potential molecular mechanisms that are dysregulated and contribute to the pathogenesis of rNPC.

Optimization of total vaporization solid-phase microextraction (TV-SPME) for the determination of lipid profiles of Phormia regina, a forensically important blow fly species.

A new method has been developed for the determination of fatty acids, sterols, and other lipids which naturally occur within pupae of the blow fly Phormia regina. The method relies upon liquid extraction in non-polar solvent, followed by derivatization using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) w/ 1% trimethylchlorsilane (TMCS) carried out inside the sample vial. The analysis is facilitated by total vaporization solid-phase microextraction (TV-SPME), with gas chromatography-mass spectrometry (GC-MS) serving as the instrumentation for analysis. The TV-SPME delivery technique is approximately a factor of five more sensitive than traditional liquid injection, which may alleviate the need for rotary evaporation, reconstitution, collection of high performance liquid chromatography fractions, and many of the other pre-concentration steps that are commonplace in the current literature. Furthermore, the ability to derivatize the liquid extract in a single easy step while increasing sensitivity represents an improvement over current derivatization methods. The most common lipids identified in fly pupae were various saturated and unsaturated fatty acids ranging from lauric acid (12:0) to arachinoic acid (20:4), as well as cholesterol. The concentrations of myristic acid (14:0), palmitelaidic acid (16:2), and palmitoleic acid (16:1) were the most reliable indicators of the age of the pupae. Graphical abstract Blow fly pupae were extracted prior to emerging as adults. The extracts were analyzed via total vaporization solid-phase microextraction (TV-SPME), revealing a complex mixture of lipids that could be associated with the age of the insect. This information may assist in determining a post-mortum interval (PMI) in a death investigation.

Excited-State Relaxation and Förster Resonance Energy Transfer in an Organic Fluorophore/Silver Nanocluster Dyad.

A single-stranded DNA-based (ssDNA) dyad was constructed comprising 15 silver atoms stabilized by a ssDNA scaffold (DNA-AgNC) and an Alexa 546 fluorophore bound to the 5′ end. The Alexa 546 was chosen to function as a Förster resonance energy transfer (FRET) donor for the AgNC. Time-correlated single photon counting (TCSPC) experiments allowed unraveling the excited-state relaxation processes of the purified DNA-AgNC-only system. The TCSPC results revealed slow relaxation dynamics and a red shift of the emission spectrum during the excited-state lifetime. The results from the model systems were needed to understand the more complicated decay pathways present in the collected high-performance liquid chromatography fraction, which contained the dyad (37% of the emissive population). In the dyad system, the FRET efficiency between donor and acceptor was determined to be 94% using TCSPC, yielding a center-to-center distance of 4.6 nm. To date, only limited structural information on DNA-AgNCs is available and the use of TCSPC and FRET can provide information on the center-to-center distance between chromophores and provide positional information in nanostructures composed of AgNCs.

Antibacterial activity of 3,3',4'-Trihydroxyflavone from Justicia wynaadensis against diabetic wound and urinary tract infection.

The present investigation was designed to study the effect of an active compound isolated from Justicia wynaadensis against multi drug resistant organisms (MDRO's) associated with diabetic patients. The drug resistant pathogens implicated in wound and urinary tract infection of diabetic patients were isolated and identified by molecular sequencing. Solvent–solvent fractionation of crude methanol extract produced hexane, chloroform, ethyl acetate and methanol–water fraction, among which chloroform fraction was found to be potent when compared with other three fractions. Further, chloroform fraction was subjected to preparatory HPLC (High-Performance Liquid Chromatography), that produced four sub-fractions; chloroform HPLC fraction 1 (CHF1) through CHF4. Among the sub-fractions, CHF1 inhibited the pathogens effectively in comparison to other three sub-fractions. The purity of CHF1 was found to be >95%. Therefore, CHF1 was further characterized by NMR and FTIR analysis and based on the structure elucidated, the compound was found to be 3,3′,4′-Trihydroxyflavone. The effective dose of this bioactive compound ranged from 32 μg/mL to 1.2 mg/mL. Thus, the present study shows that 3,3′,4′-Trihydroxyflavone isolated from J. wynaadensis is an interesting biopharmaceutical agent and could be considered as a source of antimicrobial agent for the treatment of various infections and used as a template molecule for future drug development.

Evaluation of the antihypertensive angiotensin-converting enzyme inhibitory (ACE-I) activity and other probiotic properties of lactic acid bacteria isolated from traditional Greek dairy products

The technological and probiotic potential of lactic acid bacteria isolated from artisanal Greek yoghurt and fermented milks were evaluated. Fifty-three strains were identified by rep-PCR and 16S rDNA sequencing to belong to different Lactobacillus or Enterococcus spp., as well as to Streptococcus thermophilus and Lactococcus lactis. Several strains exhibited promising technological and probiotic properties. Among them, we focused on the production of bioactive peptides with angiotensin-converting enzyme inhibitory (ACE-I) activity during milk fermentation. The majority of strains produced ACE-I peptides when grown in skimmed milk. ACE-I peptides were sometimes sequestered in the original fermented milk sample, but were released and detected following high performance liquid chromatography (HPLC) purification. Mass spectrometry analysis of major peptide peaks in HPLC fractions with ACE-I activity revealed that they derived from the N- or C-terminal of the isracidin peptide region of αS1-casein and two internal peptide fragments, one from β-casein and one from κ-casein.

Removal of novel antiandrogens identified in biological effluents of domestic wastewater by activated carbon

Environmental antiandrogenic (AA) contaminants in effluents from wastewater treatment plants have the potential for negative impacts on wildlife and human health. The aim of our study was to identify chemical contaminants with likely AA activity in the biological effluents and evaluate the removal of these antiandrogens (AAs) during advanced treatment comprising adsorption onto granular activated carbon (GAC). In this study, profiling of AA contaminants in biological effluents and tertiary effluents was conducted using effect-directed analysis (EDA) including high performance liquid chromatography (HPLC) fractionation, a recombinant yeast screen containing androgen receptor (YAS), in combination with mass spectrometry analyses. Analysis of a wastewater secondary effluent from a membrane bioreactor revealed complex profiles of AA activity comprising 14 HPLC fractions and simpler profiles of GAC effluents with only 2 to 4 moderately polar HPLC fractions depending on GAC treatment conditions. Gas chromatography–mass spectrometry and ultra-high performance liquid chromatography-nanospray mass spectrometry analyses of AA fractions in the secondary effluent resulted in detection of over 10 chemical contaminants, which showed inhibition of YAS activity and were potential AAs. The putative AAs included biocides, food additives, flame retardants, pharmaceuticals and industrial contaminants. To our knowledge, it is the first time that the AA properties of N-ethyl-2-isopropyl-5-methylcyclohexanecarboxamide (WS3), cetirizine, and oxcarbazepine are reported. The EDA used in this study was proven to be a powerful tool to identify novel chemical structures with AA activity in the complex aquatic environment. The adsorption process to GAC of all the identified antiandrogens, except WS3 and triclosan, fit well with the pseudo-second order kinetics models. Adsorption to GAC could further remove most of the AAs identified in the biological effluents with high efficiencies.

Rapid strategies for the determination of sensory and chemical differences between a wealth of similar wines

The work introduces a fast methodology for the identification of compounds responsible for the generation of the distinctive aromas in different wines. Five Verdejo musts and five Tempranillo musts from different terroirs were fermented with five different yeasts for each variety under controlled laboratory conditions. The resulting wines were first sorted in a sorting task with a description step by a panel of wine experts into different groups depending on the odor similarities between the wines. A wine belonging to each group with positive differentiator aromas was fractionated by high-performance liquid chromatography (HPLC) and the fractions whose attributes were similar to their descriptors in the sorting task were screened by gas chromatography–olfactometry (GC–O). Different compounds were related with different aromatic profiles obtained in the sorting task. This methodology led to the identification of two compounds related with green and fresh fruit aromas in wine. Thus, the sorting task followed by HPLC fractionation and GC–O analysis has been shown to be a rapid and effective methodology for the identification of compounds responsible for the distinctive aromas in different wines.