Innovative Thin-Layer Chromatography/Fluorescence Detection Approach for Sensitive and Specific Determination of Ledipasvir in Rats' Feces and Pharmaceutical Dosage Form.

An innovative thin-layer chromatography method coupled with the fluorescence detection was developed for a specific estimation of ledipasvir. The separation was achieved on plates of silica gel 60 F254 using ethylacetate: hexane: acetonitrile: triethylamine; (6: 3.5: 1.5: 0.5, $\mathrm{v}/\mathrm{v}/\mathrm{v}/\mathrm{v}$) as a mobile phase system. The method involved the exposure of the developed thin-layer chromatography plate of ledipasvir to strong ultraviolet irradiation, resulting in a great enhancement in the fluorescence properties of ledipasvir. The irradiated plates were scanned after the excitation at 315 $\mathrm{nm}$. The method provided a sufficient separation of ledipasvir from sofosbuvir with ${R}_F$values of 0.28 and 0.36 for ledipasvir and sofosbuvir, respectively. The developed procedures were validated based on guidelines from the International Conference on Harmonization and Food and Drug Administration guidelines. The calibration curve was linear over the range of 5-50 $\mathrm{ng}/\mathrm{band}$. The excellent analytical features of the proposed method allow to the specific determination of ledipasvir in pharmaceutical tablets without interference from sofosbuvir or excipients. As the main elimination route for ledipasvir is via the fecal excretion (86%), the method was applied for the estimation of ledipasvir in fecal specimens with adequate recovery. In addition, the proposed method was applied for testing the content uniformity of ledipasvir in the pharmaceutical tablets.

A novel colorimetric and ratiometric fluorescent probe for sensing SO2 derivatives and their bio-imaging in living cells.

We report a novel fluorescent probe HBN-TCF for the detection of SO2 derivatives. This probe exhibited near-infrared fluorescence emission with an excitation wavelength of 620 nm. After reacting with SO32-, the emission channel at 664 nm decreased, while the new strong emission channel at 482 nm increased (λex = 400 nm), with a large emission distance (Δλ = 182 nm) observed. This probe exhibited the rapid and selective detection of SO2 derivatives compared with other sulfur-containing species and featured a low detection limit (82 nM). This colorimetric and ratiometric fluorescent probe showed high selectivity and sensitivity for detecting SO2 derivatives. The probe was also successfully exploited for the fluorescence imaging of intracellular and exogenous SO2 derivatives in BEL-7402 cells.

Degradation of carbendazim in water via photo-Fenton in Raceway Pond Reactor: assessment of acute toxicity and transformation products.

This study aimed at investigating the degradation of fungicide carbendazim (CBZ) via photo-Fenton reactions in artificially and solar irradiated photoreactors at laboratory scale and in a semi-pilot scale Raceway Pond Reactor (RPR), respectively. Acute toxicity was monitored by assessing the sensibility of bioluminescent bacteria (Aliivibrio fischeri) to samples taken during reactions. In addition, by-products formed during solar photo-Fenton were identified by liquid chromatography coupled to mass spectrometry (UFLC-MS). For tests performed in lab-scale, two artificial irradiation sources were compared (UVλ > 254nm and UV-Visλ > 320nm). A complete design of experiments was performed in the semi-pilot scale RPR in order to optimize reaction conditions (Fe2+ and H2O2 concentrations, and water depth). Efficient degradation of carbendazim (> 96%) and toxicity removal were achieved via artificially irradiated photo-Fenton under both irradiation sources. Control experiments (UV photolysis and UV-Vis peroxidation) were also efficient but led to increased acute toxicity. In addition, H2O2/UVλ > 254nm required longer reaction time (60 minutes) when compared to the photo-Fenton process (less than 1 min). While Fenton's reagent achieved high CBZ and acute toxicity removal, its efficiency demands higher concentration of reagents in comparison to irradiated processes. Solar photo-Fenton removed carbendazim within 15 min of reaction (96%, 0.75 kJ L-1), and monocarbomethoxyguanidine, benzimidazole isocyanate, and 2-aminobenzimidazole were identified as transformation products. Results suggest that both solar photo-Fenton and artificially irradiated systems are promising routes for carbendazim degradation.

Strong Influence of Ancillary Ligands Containing Benzothiazole or Benzimidazole Rings on Cytotoxicity and Photoactivation of Ru(II) Arene Complexes.

A new family of neutral ruthenium(II) arene complexes of the type [Ru(η6-arene)X(κ2- O, N-L)] (η6-arene = p-cym, bz; X = Cl-, SCN-; HL1 = 2-(2'-hydroxyphenyl)benzimidazole, HL2 = 2-(2'-hydroxyphenyl)benzothiazole) has been synthesized and characterized. The cytotoxic activity of the Ru(II) complexes was evaluated in several tumor cell lines (A549, HepG2 and SW480) both in the dark and after soft irradiation with UV and blue light. None of the complexes bearing benzimidazole (HL1) as a ligand displayed phototoxicity, whereas the complexes with a benzothiazole ligand (HL2) exhibited photoactivation; the sensitivity observed for UV was higher than for blue light irradiation. The interesting results displayed by HL2 and [Ru(η6- p-cym)(NCS)(κ2- O, N-L2)], [3a], in terms of photo cytotoxicity prompted us to analyze their interaction with DNA, both in the dark and under irradiation conditions, in an effort to shed some light on their mechanism of action. The results of this study revealed that HL2 interacts with DNA by groove binding, whereas [3a] interacts by a dual mode of binding, an external groove binding, and covalent binding of the metal center to the guanine moiety. Interestingly, both HL2 and [3a] display a clear preference for AT base pairs, and this causes fluorescence enhancement. Additionally, cleavage of the pUC18 plasmid DNA by the complex is observed upon irradiation. The study of the irradiated form demonstrates that the arene ligand is released to yield species such as [Ru(κ2- O, N-L2)(κ1- S-DMSO)2(µ-SCN)]2 [3c] and [Ru(κ2- O, N-L2)(κ1- S-DMSO)3(SCN)] [3d]. Such photo dissociation occurs even in the absence of oxygen and leads to cytotoxicity enhancement, an effect attributed to the presence of [3d], thus revealing the potential of [3a] as a pro-drug for photoactivated anticancer chemotherapy (PACT).

Imaging nucleus viscosity and G-quadruplex DNA in living cells using a nucleus-targeting two-photon fluorescent probe.

A two-photon fluorescent probe, TP-2Bz, was designed and synthesized and it exhibited good targeting capabilities toward cell nuclei. In particular, TP-2Bz demonstrated a high selectivity to both G-quadruplex DNA and viscosity inside the nucleus with significant ratiometric enhancement in fluorescence, providing a specific, versatile imaging tool for analyzing the viscosity and G-quadruplex DNA in living cells.

Degradation and mineralization of organic UV absorber compound 2-phenylbenzimidazole-5-sulfonic acid (PBSA) using UV-254nm/H2O2.

Various studies have revealed the non-biodegradable and endocrine disrupting properties of sulfonated organic UV absorbers, directing people's attention toward their risks on ecological and human health and hence their removal from water. In this study, UV-254nm/H2O2 advanced oxidation process (AOP) was investigated for degrading a model UV absorber compound 2-phenylbenzimidazole-5-sulfonic acid (PBSA) and a structurally similar compound 1H-benzimidazole-2-sulfonic acid (BSA), with a specific focus on their mineralization. At 4.0mM [H2O2]0, a complete removal of 40.0µM parent PBSA and 25% decrease in TOC were achieved with 190min of UV irradiation; SO4(2-) was formed and reached its maximum level while the release of nitrogen as NH4(+) was much lower (around 50%) at 190min. Sulfate removal was strongly enhanced by increasing [H2O2]0 in the range of 0-4.0mM, with slight inhibition in 4.0-12.0mM. Faster and earlier ammonia formation was observed at higher [H2O2]0. The presence of Br(-) slowed down the degradation and mineralization of both compounds while a negligible effect on the degradation was observed in the presence of Cl(-). Our study provides important technical and fundamental results on the HO based degradation and mineralization of SO3H and N-containing UV absorber compounds.

The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem.

Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4',6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. When a sample stained with DAPI only was first imaged with the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence was observed. Next, we imaged the sample with a DAPI filter set, obtaining a strong nuclear DAPI signal as expected. Upon reimaging the same samples with a FITC/GFP filter set, robust nuclear fluorescence was observed. We conclude that excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI due to excitation and emission in the FITC/GFP channel. This phenomenon can affect data interpretation and lead to false-positive results when used together with fluorochrome-labeled nuclear proteins detected with blue excitation and green emission. In order to avoid misinterpretations, extra precaution should be taken to prepare staining solutions with low DAPI concentration and DAPI (UV excitation) images should be acquired after all other higher wavelength images. Of various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion while that for DAPI and Hoechst 33258 was much stronger. Different fixation methods did not substantially affect the strength of photoconversion. We also suggest avoiding the use of mounting medium with high glycerol concentrations since glycerol showed the strongest impact on photoconversion. This photoconversion effect cannot be avoided even when using narrow bandpass filter sets.

Novel amidino-substituted thienyl- and furylvinylbenzimidazole: derivatives and their photochemical conversion into corresponding diazacyclopenta[c]fluorenes. synthesis, interactions with DNA and RNA, and antitumor evaluation. 4.

Synthesis of novel nonfused amidino-substituted thienyl- and furylvinylbenzimidazole: derivatives and their photochemical cyclization into corresponding diazacyclopenta[ c]fluorenes is described. All studied compounds showed prominent growth inhibitory effect. The fused compounds showed stronger activity than nonfused ones, whereby imidazolyl-substituted compound 11 proved to be the most active one. Besides, it induced strong G2/M arrest of the cell cycle followed by drastic apoptosis, which is in accordance with the DNA intercalative binding mode determined by the spectroscopic studies. Nonfused derivatives induced strong S phase arrest of the cell cycle followed by apoptosis that together with DNA minor groove binding mode pointed to topoisomerase I inhibition. In addition, all nonfused compounds revealed pronounced selectivity toward tumor cells in comparison with nontumor cells. On the basis of the presented results, both nonfused and fused thiophene-containing imidazolyl derivatives should be considered as promising lead compounds for further investigation.

Degradation of the fungicide carbendazim in aqueous solutions with UV/TiO(2) process: optimization, kinetics and toxicity studies.

An attempt was made to investigate the potential of UV-photocatalytic process in the presence of TiO(2) particles for the degradation of carbendazim (C(9)H(9)N(3)O(2)), a fungicide with a high worldwide consumption but considered as a "priority hazard substance" by the Water Framework Directive of the European Commission (WFDEC). A circulating upflow photo-reactor was employed and the influence of catalyst concentration, pH and temperature were investigated. The results showed that degradation of this fungicide can be conducted in the both processes of only UV-irradiation and UV/TiO(2); however, the later provides much better results. Accordingly, a degradation of more than 90% of fungicide was achieved by applying the optimal operational conditions of 70 mgL(-1) of catalyst, natural pH of 6.73 and ambient temperature of 25 degrees C after 75 min irradiation. Under these mild conditions, the initial rate of degradation can be described well by the Langmuir-Hinshelwood kinetic model. Toxicological assessments on the obtained samples were also performed by measurement of the mycelium growth inhibition of Fusarium oxysporum fungus on PDA medium. The results indicate that the kinetics of degradation and toxicity are in reasonably good agreement mainly after 45 min of irradiation; confirming the effectiveness of photocatalytic process.

Microbial toxicity of pesticide derivatives produced with UV-photodegradation.

Our study aimed at acquiring information about the biological effect of pesticides and their degradates produced by UV-treatment on microbiological activity. Five photosensitive pesticides (carbendazim, acetochlor, simazine, chlorpyrifos, EPTC) and six representative soil microbes (Bacillus subtilis, Pseudomonas fluorescens, Mycobacterium phlei, Fusarium oxysporum, Penicillium expansum, Trichoderma harzianum) were applied throughout our model experiments. The antimicrobial effects of the pesticides and their degradates were assessed with filter paper disk method. The antimicrobial effect of the degradation products exhibited marked differences in terms of pesticide types, irradiation time, and the test organisms. Acetochlor and its photolytic degradation products were found to be more toxic to bacteria than fungi. All the three bacteria proved to be sensitive to the basic compound and its degradation products as well. The end product of carbendazim was weakly antibacterial against P. fluorescens and B. subtilis but strongly antifungal against T. harzianum. Chlorpyrifos and its end product inhibited neither test organisms, but the degradates hindered the growth of four of them. The basic compound of EPTC and the degradates of simazine exhibited significant toxicity to the test bacteria. It might be claimed that the pesticide photodegradation may result in significant changes in soil microbiota, as well as formation of biologically harmful degradates.

Efficient microwave enhanced regioselective synthesis of a series of benzimidazolyl/triazolyl spiro [indole-thiazolidinones] as potent antifungal agents and crystal structure of spiro[3H-indole-3,2'-thiazolidine]-3'(1,2,4-triazol-3-yl)-2,4'(1H)-dione.

A microwave-assisted three-component, regioselective one-pot cyclocondensation method has been developed for the synthesis of a series of novel spiro[indole-thiazolidinones] (6a-l) using an environmentally benign procedure at atmospheric pressure in open vessel. This rapid method produces pure products in high yields within few minutes in comparison to a conventional two-step procedure. The crystal structure of one representative compound has been determined by X-ray diffraction. The synthesized compounds have been screened 'in vitro' for antifungal activity against Rhizoctonia solani, Fusarium oxysporum and Collectotrichum capsici. All compounds have shown good activity against these pathogens.

Complexation of the sunscreen agent, phenylbenzimidazole sulphonic acid with cyclodextrins: effect on stability and photo-induced free radical formation.

The interaction between the sunscreen agent, phenylbenzimidazole sulphonic acid (PBSA) and hydrophilic alpha-, beta-, and gamma-cyclodextrin derivatives was investigated under acidic conditions (pH 4.0) by phase-solubility analysis. Among the available cyclodextrins, hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and random methyl-beta-cyclodextrin (RM-beta-CD) had the greatest solubilizing activity. The complexation of the sunscreen agent with HP-beta-CD and RM-beta-CD was confirmed by nuclear magnetic resonance spectroscopy. Solid-phase characterization of the PBSA/cyclodextrin systems by X-ray diffractometry defined the most appropriate method (co-evaporation) and cyclodextrin concentration (10-fold molar excess) for the preparation of a stable complexed form of PBSA. Long-term stability studies demonstrated that the decrease of the sunscreen level in emulsion preparations (pH 4.0) was almost completely suppressed by HP-beta-CD, RM-beta-CD being less effective. Moreover, the irradiation-induced decomposition of PBSA in the emulsion vehicle was markedly reduced by complexation with HP-beta-CD (the extent of degradation was 3.9% for the complex compared to 9.1% for uncomplexed PBSA), whereas RM-beta-CD had no significant influence. In addition, electron paramagnetic resonance (EPR) spin-trapping studies showed that the inclusion of the sunscreen agent into the HP-beta-CD cavity completely inhibited the formation of free-radicals generated by PBSA on exposure to simulated sunlight, thereby suppressing its photosensitising potential.

Photochemical behaviour of carbendazim in aqueous solution.

To elucidate the photochemical behaviour of carbendazim (or MBC) in superficial waters, photolysis studies have been carried out in aqueous solutions at several pH using a UV light source (high pressure mercury arc lamp) or a solar light simulator (xenon arc lamp). The kinetics of photodecomposition of carbendazim was determined using HPLC-DAD and the identification of photoproducts was carried out with HPLC-MS (ESI negative and positive mode). According to the experimental results carbendazim is a rather stable molecule in the dark or in environmental conditions. The pH influence of the environmental medium on the photodegradation rate has been confirmed. The photochemical process can be considerably accelerated in alkaline solutions using HPK-quartz irradiation (quantum efficiency at pH 9 phi = 3.1 x 10(-3) degraded molecule per absorbed photon) while the photodegradation is not as efficient under a simulated sun irradiation (quantum efficiency in the suntest phi = 10(-4) at pH 7). Three photoproducts have been tentatively identified in pure water: 2-aminobenzimidazole, benzimidazole isocyanate and monocarbomethoxy-guanidine (issued from the cleavage of the benzimidazole ring). The last one seems very stable and could be accumulated in the environment.

Photophysical and photochemical studies of 2-phenylbenzimidazole and UVB sunscreen 2-phenylbenzimidazole-5-sulfonic acid.

The sunscreen agent 2-phenylbenzimidazole-5-sulfonic acid (PBSA) and its parent 2-phenylbenzimidazole (PBI) cause DNA photodamage via both Type-I and Type-II mechanisms when UVB irradiated. We have studied the photophysical and photochemical properties of these compounds and their ability to photogenerate reactive oxygen species including free radicals. PBI and PBSA exhibit both oxidizing and reducing properties in their excited state. The absorption and fluorescence properties of PBSA depend strongly upon pH, and hence the photochemistry of PBSA was studied in both neutral and alkaline solutions. PBSA showed strong oxidizing properties when UV irradiated in neutral aqueous solution (pH 7.4) in the presence of cysteine, glutathione and azide, as evidenced by the detection of the corresponding S-cysteinyl, glutathiyl and azidyl radicals with the aid of the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). However, when an aqueous anaerobic solution (pH 10) of PBSA and either nitromethane (NM) or 4-nitrobenzoic acid (4-NBA) were irradiated, the corresponding nitro anion radicals were observed. This finding suggests that both NM and 4-NBA are reduced by direct electron transfer from the excited state PBSA. During UV irradiation of an aerobic solution of PBSA, O2*- and *OH radical were generated and trapped by DMPO. Further, PBI (in ethanol) and PBSA (in ethylene glycol : water 2: 1 mixture) showed low temperature (77 K) phosphorescence (lambdamax = 443, 476 and 509 nm) and also an electron paramagnetic resonance half-field transition (deltaMs = +/-2), which is evidence for a triplet state. This triplet produced singlet oxygen (1O2) with quantum yields 0.07 and 0.04 in MeCN for PBI and PBSA, respectively. These studies demonstrate that UV irradiation of PBSA and PBI generates a variety of free radicals and active oxygen species that may be involved in the photodamage of DNA.

Single UV excitation of Hoechst 33342 and ethidium bromide for simultaneous cell cycle analysis and viability determinations on in vitro cultures of murine B lymphocytes.

Assessment of DNA content by flow cytometry has largely depended on staining techniques which do not permit exclusion of dead cells from the data set. During studies of B cell activation in vitro, the large number of nonviable cells greatly affects the cell cycle distribution and thus the accurate evaluation of proliferation flow cytometry. This report describes the development of two dual staining techniques which use Hoechst 33342 and ethidium bromide excited by a single UV source to eliminate dead cells from the DNA histogram of the viable cells in murine B cell cultures. Hoechst 33342 and 0.62 micrograms/ml of ethidium bromide permit the evaluation of cell cycle distributions on the viable cells with a ratio gate. The combination of Hoechst 33342 and 6.2 micrograms/ml ethidium bromide results in the resolution of the two populations due to fluorescence energy transfer with a single PMT. Using this technique we demonstrated the simultaneous determination of DNA and RNA content on viable cells using only two PMTs. Both these techniques can be performed on either a laser or an arc lamp flow cytometer where CVs of less than 7% and as low as 3.2% are normally achieved. Determination of the S phase using these techniques produces a high correlation with DNA synthesis determined by radiolabeled precursor determination. These techniques permit the use of flow cytometry to determine proliferation during B cell activation.

UV dose-dependent increase in the Hoechst fluorescence intensity of both normal and BrdU-DNA.

If the DNA nucleoside thymidine is replaced by bromodeoxyuridine, the fluorescence of the nuclei of Hoechst-stained cells is quenched. The decrease of fluorescence intensity determined by flow cytometry and fluorometry is neutralized independent of the degree of BrdU substitution by an UV-exposure with a dose of 5-10 kJ/m2 to the unfiltered spectrum of a 100 W mercury high-pressure lamp. This dose is equivalent to that obtained in fluorescence microscopy after exposure for about 1 s. We suppose that this approximate matching of the intensities both of normal and BrdU in the DNA resulting in no further quenching. However, the fluorescence intensity of normal Hoechst-stained DNA also is increased by a previous exposure to UV light. We explain the time pattern of the Hoechst fluorescence in the course of an exposure with constant dose rate, by the superimposition of the well-known bleaching by an additional increase of the fluorescence intensity. Our results suggest that the UV-exposure of Hoechst dye creates a brightly fluorescing photoproduct which differs spectroscopically from the original dye. This product is stable in the dark and seems to fluorochrome DNA only if it is formed when the Hoechst dye is bound to DNA, thus increasing the nuclear fluorescence. Phosphorescence was not found.