Determination of benzotriazole and benzothiazole derivatives in marketed fish by double-vortex-ultrasonic assisted matrix solid-phase dispersion and ultrahigh-performance liquid chromatography-high resolution mass spectrometry.

Benzotriazoles (BTRs) and benzothiazoles (BTs) are two groups of emerging concern and high production volume contaminants. Via the biomagnification of the food web, they could jeopardize human health. In this work, rapid determining the presence of five BTRs and two BTs in marketed fish was performed by a novel double-vortex-ultrasonic assisted matrix solid-phase dispersion (DVUA-MSPD) and UHPLC-electrospray ionization (+)-quadrupole time-of-flight mass spectrometry detection. Unlike traditional MSPD, we simplified the method without the use of mortar/pestle and SPE-column procedures. The DVUA-MSPD factors were screened by a multilevel categorical design, and then optimized by Box-Behnken Design plus with response surface methodology. The limits of quantification were 0.15-2 ng g-1 (dry weight). The satisfactory average recovery ranged from 70% to 93% with RSDs less than 9%. The developed method was successfully applied for the rapid determination of selected BTRs and BTs in fish samples at trace-level.

Isolation and Identification of Aryl Hydrocarbon Receptor Modulators in White Button Mushrooms (Agaricus bisporus).

Natural aryl hydrocarbon (AHR) ligands have been identified in food and herbal medicines, and they may exhibit beneficial activity in humans. In this study, white button (WB) feeding significantly decreased AHR target gene expression in the small intestine of both conventional and germ-free mice. High-performance liquid chromatography (HPLC) fractionation and ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) combined with an AHR-responsive cell-based luciferase gene reporter assay were used to isolate and characterize benzothiazole (BT) derivatives and 6-methylisoquinoline (6-MIQ) as AHR modulators from WB mushrooms. The study showed dose-dependent changes of AHR transformation determined by the cell-based luciferase gene reporter assay and transcription of CYP1A1 in human Caco-2 cells by BT derivatives and 6-MIQ. These findings suggested that WB mushroom contains new classes of natural AHR modulators and demonstrated HPLC fractionation and UHPLC-MS/MS combined with a cell-based luciferase gene reporter assay as a useful approach for isolation and characterization of the previously unidentifed AHR modulators from natural products.

Purification of Polyphenols from Distiller's Grains by Macroporous Resin and Analysis of the Polyphenolic Components.

We aimed to purify polyphenols from distiller's grain extract using macroporous resins and to identify its polyphenolic components. The influence of operational parameters on purification efficiency was investigated. The polyphenolic composition was analyzed by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) and then quantified by UPLC-MS using authenticated standards. The results showed that the optimal purifying conditions were D101 resin with a dosage of 3 g, four hours adsorption, three hours desorption time, and 60% ethanol as the eluent, producing the highest purification rate of 51%. The purified distiller's grain extract exhibited stronger antioxidant activity than the unpurified extracts, which was assessed using DPPH and ABTS methods (IC50 DPPH = 34.03 and 16.21 µg/mL, respectively; IC50 ABTS = 20.31 and 5.73 µg/mL, respectively). UPLC-MS results indicated that (-)-epicatechin is the major compound found in distiller's grain extract which was quantified as 562.7 µg/g extract, followed by ferulic acid (518.2 µg/g), p-hydroxybenzoic acid (417.7 µg/g), caffeic acid (217.1 µg/g), syringic acid (158.0 µg/g) and quercetin (147.8 µg/g). Two compounds, vanillic acid (66.5 µg/g) and gallic acid (41.4 µg/g), were found in lower concentrations. The findings of this study suggest that purification of polyphenolic compounds from distiller's grain by macroporous resins is feasible, providing a new and effective method for the secondary use of distiller's grain resources.

[Effect of Temperature on the Performance and Microbial Community Structure in an Integrated Anaerobic Fluidized-bed Membrane Bioreactor Treating Benzothiazole Wastewater].

An integrated anaerobic fluidized-bed membrane bioreactor (IAFMBR) was applied to treat synthetic high-strength benzothiazole wastewater. This study investigated the effect of temperature on the performance, membrane fouling and microbial community structure of IAFMBR. The results showed that decreasing temperature had an adverse effect on the performance and the cycle of membrane fouling. When temperature declined from 35℃ to 15℃, the COD efficiency dropped 7.4%, benzothiazole removal efficiency dropped 49.2%, the accumulation of total VFAs increased 225.66 mg·L-1, and methane yield (in CH4/CODremoved) dropped 0.118 m3·kg-1. The membrane fouling cycle shortened from 5.2 d to 2.5 d. For cake layer, the concentration of soluble microbial product (SMP) increased from 42.47 mg·L-1 to 70.62 mg·L-1, and the extracellular polymeric substance (EPS) content (in VSS) increased from 46.30 mg·g-1 to 82.22 mg·g-1 when the TMP was 15 kPa. For mixed liquor, the concentration of SMP increased from 36.46 mg·L-1 to 69.35 mg·L-1 and the EPS content increased from 47.47 mg·g-1 to 81.63 mg·g-1. Protein was the main component of EPS and SMP, and occurred in proportion of 80%.The microbial community structure showed that the dominant phyla were Firmicutes and Chloroflexi, which accounted for 42.6%-61.0% of the total relative abundance. The genera Clostridium (13.7%), Levilinea (15.2%), and Lactococus (17.9%) dominated with decreasing temperatures. The dominant methanogen was Methanosaeta.

Enantioseparation and determination of dufulin enantiomers in cucumber and soil by chiral liquid chromatography-tandem mass spectrometry.

A simple and rapid method for enantioselective determination of dufulin in cucumber and soil was developed by liquid chromatography with tandem mass spectrometry. The enantiomers were separated on a Superchiral S-OD chiral cellulose tris(3,5-dimethylphenylcarbamate) column at 20°C, with a mixture of acetonitrile and water (0.1% formic acid; 52:48, v/v) as mobile phase at a flow rate of 0.65 mL/min. The pretreatment conditions were optimized using an orthogonal test, and the optimized method showed good linearity and sensitivity. The limits of detection and limits of quantification of two dufulin enantiomers were 0.006 and 0.02 mg/kg, respectively. The average recoveries of S-enantiomer and R-enantiomer in cucumber and soil were 80.61-99.83% and 80.97-102.96%, respectively, with relative standard deviations of 1.30-9.72%. The method was successfully applied to determine dufulin in real cucumber and soil samples. The results demonstrate that the method could facilitate further research on the differences between individual dufulin enantiomers with respect to metabolites and environmental fate and finally help reveal the complex interactions that exist between dufulin, humans and the environment.

Determination of benzotriazoles and benzothiazoles in human urine by UHPLC-TQMS.

Benzotriazole (BTR) and benzothiazole (BTH) derivatives are extensively applied in industrial processes and consumer products, and are thus frequently detected in the environmental matrices. Due to their potential estrogenic effects reported in animal studies, the assessment of human exposure to BTRs and BTHs is important. In this study, a method was developed and validated to determine six BTRs and five BTHs in human urine by using ultra-high performance liquid chromatography coupled with a triple quadrupole mass spectrometry (UHPLC-TQMS) in positive electrospray ionization mode. After de-conjugation by ß-glucuronidase, urine samples were liquid-liquid extracted for the measurement of total concentrations of BTRs and BTHs. The linearity, detection limit, precision, recovery, accuracy and matrix effect were evaluated. The limits of detection were less than 0.5ng/mL. The validated method demonstrated good precision (RSD%<15%), linearity (R2>0.99), recovery (>80%) and accuracy (80-100%). The method was successfully applied for the analysis of 22 human urine samples. Four out of eleven targeted compounds were detected in more than half of participants at ng/mL levels.

Development and validation of a highly sensitive gradient chiral separation of pramipexole in human plasma by LC-MS/MS.

AIM: Development of a high-sensitivity chiral LC-MS/MS method was required to evaluate a combination of pramipexole (S-PPX) and its enantiomer dexpramipexole (R-PPX) in a proposed clinical trial. The previously available methods suffered from low sensitivity for the (S)-enantiomer in the presence of the more abundant (R)-enantiomer. Based on the projected dosing regimen in the clinical trial, a 5000-fold improvement in sensitivity was required for the (S)-enantiomer. METHODOLOGY: Spiked human plasma samples were extracted by liquid-liquid extraction using ethyl acetate and injected onto a CHIRALPAK ID column under pH gradient conditions. CONCLUSION: An improved analytical method was developed and validated with a final LLQ for (S)-PPX of 0.1 ng/ml in the presence of 2000 ng/ml of (R)-PPX.

A small-molecule IRF3 agonist functions as an influenza vaccine adjuvant by modulating the antiviral immune response.

Vaccine adjuvants are essential to drive a protective immune response in cases where vaccine antigens are weakly immunogenic, where vaccine antigen is limited, or where an increase in potency is needed for a specific population, such as the elderly. To discover novel vaccine adjuvants, we used a high-throughput screen (HTS) designed to identify small-molecule agonists of the RIG-I-like receptor (RLR) pathway leading to interferon regulatory factor 3 (IRF3) activation. RLRs are a group of cytosolic pattern-recognition receptors that are essential for the recognition of viral nucleic acids during infection. Upon binding of viral nucleic acid ligands, the RLRs become activated and signal to transcription factors, including IRF3, to initiate an innate immune transcriptional program to control virus infection. Among our HTS hits were a series of benzothiazole compounds from which we designed the lead analog, KIN1148. KIN1148 induced dose-dependent IRF3 nuclear translocation and specific activation of IRF3-responsive promoters. Prime-boost immunization of mice with a suboptimal dose of a monovalent pandemic influenza split virus H1N1 A/California/07/2009 vaccine plus KIN1148 protected against a lethal challenge with mouse-adapted influenza virus (A/California/04/2009) and induced an influenza virus-specific IL-10 and Th2 response by T cells derived from lung and lung-draining lymph nodes. Prime-boost immunization with vaccine plus KIN1148, but not prime immunization alone, induced antibodies capable of inhibiting influenza virus hemagglutinin and neutralizing viral infectivity. Nevertheless, a single immunization with vaccine plus KIN1148 provided increased protection over vaccine alone and reduced viral load in the lungs after challenge. These findings suggest that protection was at least partially mediated by a cellular immune component and that the induction of Th2 and immunoregulatory cytokines by a KIN1148-adjuvanted vaccine may be particularly beneficial for ameliorating the immunopathogenesis that is associated with influenza viruses.

The cooperative effect of reduced graphene oxide and Triton X-114 on the electromembrane microextraction efficiency of Pramipexole as a model analyte in urine samples.

A new design of electromembrane microextraction coupled with high-performance liquid chromatography was developed for the determination of Pramipexole as a model analyte in urine samples. The presence of reduced graphene oxide in the membrane and Triton X-114 in the donor phase augments the extraction efficiency of Pramipexole by the proposed method. Dispersed reduced graphene oxide in the organic solvent was held in the pores of the fiber wall by capillary forces and sonication. It is possible that the immobilized reduced graphene oxide acts as a sorbent, affording an additional pathway for analyte transportation. Besides, the presence of Triton X-114 in the donor phase promotes effective migration of ionic analytes across the membrane. The parameters influencing the extraction procedure, such as type and concentration of surfactant, type of organic solvent, amount of reduced graphene oxide, sonication time, applied voltage, extraction time, ionic strength, pH of the donor and acceptor solutions, and stirring rate were optimized. The linear working ranges of the method for preconcentration- determination of Pramipexole in water and urine samples were found to be 0.13-1000 and 0.47-1000ngmL-1 with corresponding detection limits of 0.04 and 0.14ngmL-1, respectively. The proposed method allows achieving enrichment factors of 301 and 265 for preconcentration of the analyte in water and urine samples, respectively. The method was successfully applied for the determination of Pramipexole in the urine samples.

Chiral separation of benzothiazole derivatives of amino acids using capillary zone electrophoresis.

A method for the separation of enantiomers of leucine and phenylalanine benzothiazole derivatives as potential antimicrobial agents was developed using capillary zone electrophoresis with a dual cyclodextrin (CD) system. The best resolution of enantiomers was achieved in 100 mmol/L phosphate background electrolyte (pH 3.5) with the dual CD system consisting of 10 mmol/L of ß-CD with 10 mmol/L of 2-hydroxypropyl-ß-cyclodextrin for leucine derivative and 10 mmol/L of 2-hydroxypropyl-γ-cyclodextrin for phenylalanine derivative, respectively. Under the optimal conditions, the highest enantioresolution of 1.25 was achieved in a noncoated-fused silica capillary at 17°C and 24 kV applied voltage.

Preparation of phosphorylcholine-based hydrophilic monolithic column and application for analysis of drug-related impurities with capillary electrochromatography.

A hydrophilic monolithic CEC column was prepared by thermal copolymerization of zwitterionic monomer 2-methacryloyloxyethyl phosphorylcholine (MPC), pentaerythritol triacrylate (PETA), either methacrylatoethyl trimethyl ammonium chloride (META) or sodium 2-methylpropene-1-sulfonate (MPS) in a polar binary porogen consisting of methanol and THF. A typical hydrophilic interaction LC retention mechanism was observed for low-molecular weight polar compounds including amides, nucleotides, and nucleosides in the separation mode of hydrophilic interaction CEC, when high content of ACN (>60%) was used as the mobile phase. The effect of the electrostatic interaction between the analytes and the stationary phase was found to be negligible. The poly(MPC-co-PETA-co-META or MPS) monolithic columns have an average column efficiency of 40 000 plates/m and displayed with a satisfactory repeatability in terms of migration time and peak areas. Finally, the column was successfully applied to determine the impurities of a positively charged drug pramipexole which are often separated by ion pair RP chromatography due to their high hydrophilicity. All four components can be baseline separated within 5 min with BGE consisting of ACN/20 mM ammonium formate buffer (pH 3.0; 80/20).

Study of the retention of benzotriazoles, benzothiazoles and benzenesulfonamides in mixed-mode solid-phase extraction in environmental samples.

In the present study, the capabilities of strong cation-exchange and strong anion-exchange sorbents for solid-phase extraction (SPE) have been evaluated for the selective retention of benzotriazoles (BTRs), benzothiazoles (BTs) and benzenesulfonamides (BSAs), which are a group of neutral analytes with interesting properties such as high polarity and the capability of delocalizing electron density. The retention of these analytes has been compared in both sorbents for the first time, using a SPE procedure specially designed to promote ionic retention of the analytes with the objective of including a washing step with an organic solvent to eliminate interferences retained by hydrophobic interactions. As a result, ionic interactions between the analytes and both sorbents were observed, which allowed the successful introduction of a washing step using methanol in the SPE procedure even when most of the analytes were in their neutral state under SPE conditions. Consequently, a method was developed and further validated for each sorbent using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). Apart from the development of an improved method, special attention was paid to the discussion of the interactions present between the sorbents and each group of analytes to explain how these analytes in their neutral state might develop ionic interactions with the sorbents. At the end, the use of these sorbents helped to simplify previous developed methods where hydrophobic/hydrophilic sorbents were used, obtaining enhanced results when evaluated in river water and effluent and influent wastewaters.

Synthesis optimization of 2-(4-N-[11C]methylaminophenyl)-6-hydroxybenzothiazole ([11C]PIB), ß-amyloid PET imaging tracer for Alzheimer's disease diagnosis.

[11C]PIB is the most used amyloid plaques-specific positron-emitting radiotracers. The radiosynthesis of this compound, carried out by methylation of its precursor with [11C]methyl triflate in 2-butanone, has been improved optimizing the initial concentration and the purification method. Two HPLC methods were compared: good radiochemical yields, specific activities, and chemical purity above 98% were achieved by using as eluant acetonitrile/citrate and formulation in 10% ethanol.

Biodegradation of benzotriazoles and hydroxy-benzothiazole in wastewater by activated sludge and moving bed biofilm reactor systems.

Two laboratory scale fully aerated continuous flow wastewater treatment systems were used to compare the removal of five benzotriazoles and one benzothiazole by suspended and attached growth biomass. The activated sludge system was operated under low organic loading conditions. The moving bed biofilm reactor (MBBR) system consisted of two serially connected reactors filled with K3-biocarriers. It was either operated under low or high organic loading conditions. Target compounds were removed partially and with different rates in tested systems. For MBBR, increased loading resulted in significantly lower biodegradation for 4 out of 6 examined compounds. Calculation of specific removal rates (normalized to biomass) revealed that attached biomass had higher biodegradation potential for target compounds comparing to suspended biomass. Clear differences in the biodegradation ability of attached biomass grown in different bioreactors of MBBR systems were also observed. Batch experiments showed that micropollutants biodegradation by both types of biomass is co-metabolic.

Development of a Validated LC Method for Separation of Process-Related Impurities Including the R-Enantiomer of S-Pramipexole on Polysaccharide Chiral Stationary Phases.

Despite the availability of a few methods for individual separation of S-pramipexole from its process-related impurities, no common liquid chromatography (LC) method is reported so far in the literature. The present article describes the development of a single-run LC method for simultaneous determination of S-pramipexole and its enantiomeric and process-related impurities on a Chiralpak AD-H (150 x 4.6 mm, 5µm) column using n-hexane/ethanol/n-butylamine (75:25:0.1 v/v/v) as a mobile phase in an isocratic mode of elution at a flow rate of 1.2 ml/min at 30°C. The chromatographic eluents were monitored at a wavelength of 260 nm using a photodiode array detector. Excellent enantioseparation with good resolutions (Rs ≥ 2.88) and peak shapes (As ≤ 1.21) for all analytes was achieved. The proposed method was validated according to International Conference Harmonization (ICH) guidelines in terms of accuracy, precision, sensitivity, and linearity. Limits of quantification of impurities (0.25-0.55 µg/ml) indicate the highest sensitivity achievable by the proposed method. The method has an advantage of selectivity and suitability for routine determination of not only chiral impurity but also all possible related substances in active pharmaceutical ingredients of S-pramipexole.

The influence of salt chaotropicity, column hydrophobicity and analytes' molecular properties on the retention of pramipexole and its impurities.

The aim of this study was to examine the interaction of the chaotropic salts of different position in Hofmeister series (CF3COONa, NaClO4, NaPF6) added to the mobile phase with the stationary phases of different hydrophobicity (C8 and C18 XTerra(®) columns), as well as their common influence on the retention behavior of pramipexole and its structurally related impurities. The extended thermodynamic approach enabled the understanding of the underlying separation mechanism. Comparing six different column-salt systems it was observed that general system hydrophobicity presented by salt chaotropicity and column hydrophobicity favors stationary phase ion-pairing over the ion-pair formation in the eluent. Further, an attempt was made to describe the influence of analytes' nature on their retention behavior in such chromatographic systems. An analysis is performed in order to select and elucidate the molecular descriptors (electrostatical, quantum-chemical, geometrical, topological, and constitutional) that best explain the experimental evidence and findings obtained by the thermodynamic approach. The results of this analysis suggest that analytes' charge distribution and its complementarity to the structure of the electric double layer formed on the surface of the stationary phase upon the addition of chaotropic additives can be useful for understanding the differences in retention of structurally related analytes. These findings provide a novel understanding of the interactions between all the components of the chromatographic system containing chaotropic additive and a good basis for further investigations suggesting the development of generally applicable predictors in structure-retention relationship studies in related chromatographic systems.

Solvent bar microextraction combined with high-performance liquid chromatography for preconcentration and determination of pramipexole in biological samples.

A simple, rapid and sensitive analytical method for preconcentration and determination of pramipexole in different biological samples has been developed using solvent bar microextraction (SBME) combined with HPLC-UV. The target drugs were extracted from 10 mL of basic aqueous sample solution into an organic extracting solvent located inside the pores of a polypropylene hollow fiber, then back-extracted into an acidified aqueous solution in the lumen of the hollow fiber. In order to obtain high extraction efficiency, the effect of different variables on the extraction efficiency was studied simultaneously using an experimental design. The experimental parameters of SBME were optimized using a Box-Behnken design after a Plackett-Burman screening design. Under the optimized conditions, an enrichment factor up to 96 was achieved and the relative standard deviation of the method was 4.64% (n = 5). The linear range was 0.05-2000 µg/L with a correlation coefficient (r) of 0.987. Finally, the applicability of the proposed method was evaluated by extraction and determination of pramipexole in plasma and urine samples. The results indicated that SBME method has excellent clean-up and high preconcentration factor and can serve as a simple and sensitive method for analysis of pramipexole in biological samples.

Isolation of a novel thioflavin S-derived compound that inhibits BAG-1-mediated protein interactions and targets BRAF inhibitor-resistant cell lines.

Protein-protein interactions mediated through the C-terminal Bcl-2-associated athanogene (BAG) domain of BAG-1 are critical for cell survival and proliferation. Thioflavin S (NSC71948)-a mixture of compounds resulting from the methylation and sulfonation of primulin base-has been shown to dose-dependently inhibit the interaction between BAG-1 and Hsc70 in vitro. In human breast cancer cell lines, with high BAG-1 expression levels, Thioflavin S reduces the binding of BAG-1 to Hsc70, Hsp70, or CRAF and decreases proliferation and viability. Here, we report the development of a protocol for the purification and isolation of biologically active constituents of Thioflavin S and the characterization of the novel compound Thio-2. Thio-2 blocked the growth of several transformed cell lines, but had much weaker effects on untransformed cells. Thio-2 also inhibited the proliferation of melanoma cell lines that had become resistant to treatment with PLX4032, an inhibitor of mutant BRAF. In transformed cells, Thio-2 interfered with intracellular signaling at the level of RAF, but had no effect on the activation of AKT. Thio-2 decreased binding of BAG-1 to Hsc70 and to a lesser extent BRAF in vitro and in vivo, suggesting a possible mechanism of action. Given that tumors frequently develop resistance to kinase inhibitors during treatment, Thio-2 and related compounds may offer promising alternative strategies to currently available therapies.

Efficient tandem solid-phase extraction and liquid chromatography-triple quadrupole mass spectrometry method to determine polar benzotriazole, benzothiazole and benzenesulfonamide contaminants in environmental water samples.

An analytical method has been developed that allows the simultaneous determination of five benzotriazole (BTRs), four benzothiazole (BTs) and five benzenesulfonamide (BSAs) derivates. The method is based on tandem solid-phase extraction (SPE) with Oasis HLB followed by a clean-up step with Florisil. The chromatographic analysis was performed in less than 15min and detection was carried out with a triple quadrupole mass analyser operating in multiple reaction monitoring (MRM) mode. A comparison was performed between Oasis HLB and Oasis MAX sorbents for the solid-phase extraction, with Oasis HLB being the sorbent that gave the highest recoveries, ranging between 75% and 106%, depending on the compound and the matrix analysed. The proposed clean-up with Florisil sorbent reduced the matrix effect to below 20%. The repeatability (%RSD, 50-3000ng/L, n=3) of the method was less than 15% for all of the compounds in all of the matrices. The limits of detection (LODs) achieved ranged from 1ng/L for BTR in river water up to 100ng/L for BT in influent sewage. All of the compounds were determined in environmental waters such as river water and sewage. The highest concentrations determined corresponded to influent sewage samples in which the sum of concentrations for all compounds were between 4.6µg/L and 8.0µg/L. These concentrations were slightly reduced in secondary effluent and tertiary effluent sewage. Moreover, samples from tertiary effluent sewage based on ultra-filtration membrane treatments were also analysed and preliminary results seem to indicate that these treatments may be most effective for removing BTR, BT and BSA derivates.