Methanol-acetic Acid Research Articles

PREPARATION OF CHITOSAN-GOLD NANOPARTICLES: PART 1 (OF 2). EFFECT OF REDUCING TECHNIQUE

Chitosan-stabilized gold nanoparticles were synthesized in aqueous formic acid, citric, or acetic acid with and without chitosan as the stabilizer. Refluxing in methanol, addition of hydrazine or sodium borohydride, photo-irradiation were employed as the reducing agents & reduction technique of gold ions. Dispersed particles of chitosan-stabilized gold were obtained in aqueous acetic acid-methanol solution. The chitosan-stabilized gold colloids (chi-Au) were dispersed and the particle size were in range of 9-30 nm, some of which were crystalline with various shapes. Chi-Au prepared with hydrazine as the reducing agent resulted in large and aggregated particles. The finest and dispersed chi-Au particles were obtained if NaBH4 was used as the reducing agent and the diameters could be controlled within 2.2-2.3 nm. Keywords: Gold nanoparticles, chitosan, size control

PREPARATION OF CHITOSAN-GOLD NANOPARTICLES: PART 2. THE ROLE OF CHITOSAN

Colloidal gold nanoparticles prepared by employing chitosan as the stabilizer in solvent of methanol-acetic acid solution were stable for months without precipitation. The mole ratio of chitosan-gold ions of 5:1 - 30:1 gave dispersed and fine gold particles in range of 9.4-10.4 nm. Gold reduction in chitosan matrix was faster at higher chitosan concentration, and molar ratio of chi : Au, from 5:1 to 40:1. Higher acidity of acetic acid (pH 2-6) led to faster reduction of gold ions. The intensity of gold metal colloid plasmon band increased at higher concentration of acetic acid. Chitosan functioned both as a stabilizer and a reducing agent for gold ions. Gold colloidal particles immobilized on chitosan coated TiO2 as the solid support gave more dispersed and smaller particles (4.6 nm) compared with gold particles supported on TiO2 without chitosan coating. Keywords: gold nanoparticles, chitosan, chitosan coated TiO2

Improved and validated HPTLC method for quantification of oenothein B and its use for analysis ofEpilobiumangustifoliumL.

Summary A selective and simplest possible high-performance TLC (HPTLC) method for quantification of oenothein B on the basis of the free gallic acid and total gallic acid content after acid hydrolysis has been developed and used for analysis of cultivated Epilobium angustifolium. HPTLC of aqueous extracts of E. angustifolium was performed on silica gel with benzene–methanol–acetic acid 90:16:8 (v/v), as mobile phase. Quantitative evaluation of the plate was performed at 570 nm after derivatization with 1% ethanolic FeCl3 solution. The method was validated for precision, repeatability, and accuracy. Average recovery of the active ingredient from the samples was in the range 95.43–104.57%. The calibration plots were linear in the range 440–2200 ng per band. The technique was used, for the first time, for estimation of the oenothein B content of cultivated E. angustifolium. The oenothein B content was highest at budding time (18.4–20.2 mg g–1) and decreased dramatically to 1.0 mg g–1 after flowering.

Studies of chemical fixation effects in human cell lines using Raman microspectroscopy

The in vitro study of cellular species using Raman spectroscopy has proven a powerful non-invasive modality for the analysis of cell constituents and processes. This work uses micro-Raman spectroscopy to study the chemical fixation mechanism in three human cell lines (normal skin, normal bronchial epithelium, and lung adenocarcinoma) employing fixatives that preferentially preserve proteins (formalin), and nucleic acids (Carnoy's fixative and methanol-acetic acid). Spectral differences between the mean live cell spectra and fixed cell spectra together with principal components analysis (PCA), and clustering techniques were used to analyse and interpret the spectral changes. The results indicate that fixation in formalin produces spectral content that is closest to that in the live cell and by extension, best preserves the cellular integrity. Nucleic acid degradation, protein denaturation, and lipid leaching were observed with all fixatives and for all cell lines, but to varying degrees. The results presented here suggest that the mechanism of fixation for short fixation times is complex and dependent on both the cell line and fixative employed. Moreover, important spectral changes occur with all fixatives that have consequences for the interpretation of biochemical processes within fixed cells. The study further demonstrates the potential of vibrational spectroscopy in the characterization of complex biochemical processes in cells at a molecular level.

Optimization and validation of a method for the direct determination of catechin and epicatechin in red wines by HPLC/fluorescence

The present paper describes a direct procedure for the determination of catechin and epicatechin concentrations in red wines employing reverse-phase high performance liquid chromatography (RP HPLC) and detection by fluorescence. The method was performed using a sample volume of 10 µL without dilution. The separation process employed a Chromolith performance RP-18e (100 mm × 4.6 mm) column, and the mobile phase was composed of solvent A: methanol–acetic acid–water (90:8:2) and solvent B: water–acetic acid–methanol (10:2:88) at a flow rate of 1.0 mL min − 1 . Linearity was observed in the range of 1 to 30 mg L − 1 , with limits of detection and quantification of 0.27 and 0.89 mg L − 1 , respectively, for catechin and 0.33 and 1.01 mg L − 1 , respectively, for epicatechin. The precisions estimated by the relative standard deviation were 3.34 and 1.09% for catechin concentrations of 0.5 and 20 mg L − 1 respectively and 2.82 and 0.49% for epicatechin concentrations of 0.5 and 20 mg L − 1 , respectively. The evaluation of the accuracy was done using an addition/recovery assay. Four wine samples were used, and the recoveries varied from 105 to 108% for catechin and from 97 to 119% for epicatechin. The method was applied to the analysis of red wine samples collected from the São Francisco region, Bahia State, Brazil. Nine samples were analyzed, and the catechin and epicatechin concentrations varied from 7.51 to 73.20 and from 5.08 to 43.32 mg L − 1 , respectively. The concentrations found agree with data reported in the literature.

Development and Validation of RP-HPLC Method for The Simultaneous Estimation of Amoxicillin Trihydrate and Bromhexine Hydrochloride from Oily Suspension

A new simple, rapid and precise reverse phase high pressure liquid chromatography (RP-HPLC) method was developed for the simultaneous estimation of amoxicillin trihydrate and bromhexine hydrochloride from oily suspension. An ODS C 18 (250 X 4.5mm ID), 5 μ particle size with mobile phase methanol and glacial acetic acid (50:50 v/v) were used. The flow rate was 1.0ml/min and responses were measured at 254 nm. The retention time for amoxicillin trihydrate and bromhexine hydrochloride were observed at 3.04 and 8.18 min. respectively. Linearity for amoxicillin trihydrate and bromhexine hydrochloride were in the range of 8-50 mcg/ml and 5-25 mcg/mL respectively. Percent recovery was 99.54% and 98.65% for amoxicillin trihydrate and bromhexine hydrochloride respectively. The proposed method can be applied for the routine analysis of amoxicillin trihydrate and bromhexine hydrochloride in combination.

Determination of free α-lipoic acid in foodstuffs by HPLC coupled with CEAD and ESI-MS

A simple and rapid method for determination of free α-lipoic acid in different food matrices has been developed. It consists of extraction of α-lipoic acid with 0.5% glacial acetic acid in methanol by sonication, quantitative analysis of the extract by isocratic RP-HPLC (acetonitrile/methanol/50 mM potassium dihydrogen phosphate buffer adjusted to pH 3 with phosphoric acid: 350/65/585, v:v:v) at a flow rate of 0.45 ml/min coupled with coulometric electrode array detection at potentials between +300 and +700 mV and qualitative analysis by LC–ESI-MS in the negative ion mode for confirmation. Egg, dried egg powder, mayonnaise, fine peas and potatoes were analysed and free α-lipoic acid contents ranged from 0.1 to 4.2 μg/g with recoveries between 70% and 94%. Limits of quantitation were between 0.1 and 0.3 μg/g. This newly developed method can be used to establish a database for the content of free α-lipoic acid in different foodstuffs.

New process for catalyst-free biodiesel production using subcritical acetic acid and supercritical methanol

Abstract The production of glycerol as a by-product is unavoidable in the current conventional biodiesel manufacturing processes. Since biodiesel production is expected to increase in the near future, effective utilization of glycerol will become an issue of interest. In this study, therefore, a process consisting of subcritical acetic acid treatment to convert rapeseed oil to fatty acids and triacetin followed by conversion of the obtained fatty acids to their fatty acid methyl esters in supercritical methanol treatment was investigated. The obtained results clearly revealed that this two-step reaction could proceed effectively at a high reaction rate, and that fatty acid methyl esters and triacetin could be obtained under milder reaction condition than the one-step process utilizing supercritical methyl acetate and supercritical methanol.

Use of Structural Descriptors to QSRR Analysis of Selected Bile Acids Separated by NP-TLC

The selected bile acids such as: cholic acid (C), glycocholic acid (GC), glycolithocholic acid (GLC), deoxycholic acid (DC), chenodeoxycholic acid (CDC), glycodeoxycholic acid (GDC), lithocholic acid (LC), were separated by using normal phase thin-layer chromatography (NP-TLC) on glass plates precoated with silica gel 60 with concentrating zone (E. Merck, #1.11845) and n-hexane-ethyl acetate–methanol-acetic acid in volume composition 20:20:5:2 as the mobile phase. The selected topological indexes based on connectivity (Mν, 0χν, 1χν, 2χν, and ), on distance matrix (W, A, 0B, 1B, and C), and selected electrotopological states (SdO(acid), SsOH(acid), SsOH(aliph), SdO(amide), and SsNH) were calculated for investigated bile acids. The most accurate prediction of the RM values of the investigated bile acids was achieved by using a monoparametric equation employing the topological index Mν.

RP-HPLC Analysis of Rhinacanthins in Rhinacanthus nasutus: Validation and Application for the Preparation of Rhinacanthin High-Yielding Extract

A reversed-phase high-performance liquid chromatographic method is described for the simultaneous determination of rhinacanthin-C, rhinacanthin-D, and rhinacanthin-N in Rhinacanthus nasutus leaves. The method involved the use of a TSK-gel ODS-80Ts column (5 microm, 4.6 x 150 mm i.d.) with the mixture of methanol and 5% aqueous acetic acid (80:20, v/v) as the mobile phase. The parameters of linearity, repeatability, accuracy, and specificity of the method were evaluated. The recovery of the method was 94.3-100.9%, and good linearity (r(2) > or = 0.9999) was obtained for all rhinacanthins. A high degree of specificity as well as repeatability and reproducibility (relative standard deviation values less than 5%) were also achieved. The limit of detection and quantification of all rhinacanthins were 0.75 and 3.0 microg/mL, respectively. The solvents for extraction of rhinacanthins from R. nasutus leaves were examined in order to obtain the leaf extract with high rhinacanthin content. It was found that ethyl acetate was an appropriate solvent for rhinacanthin extraction. Fractionation of the ethyl acetate extract using a basic anion exchange resin (Amberlite IRA-67) eluted with 10% acetic acid in methanol afforded a rhinacanthin-rich extract (HRn). The total content of rhinacanthins was increased from 37.4% w/w to 77.5% w/w. The antifungal activities of HRn against Trichophyton rubrum, T. mentagrophytes, and Microsporum gypseum were also improved.

Chromosome shattering: a mitotic catastrophe due to chromosome condensation failure

Chromosome shattering has been described as a special form of mitotic catastrophe, which occurs in cells with unrepaired DNA damage. The shattered chromosome phenotype was detected after application of a methanol/acetic acid (MAA) fixation protocol routinely used for the preparation of metaphase spreads. The corresponding phenotype in the living cell and the mechanism leading to this mitotic catastrophe have remained speculative so far. In the present study, we used V79 Chinese hamster cells, stably transfected with histone H2BmRFP for live-cell observations, and induced generalized chromosome shattering (GCS) by the synergistic effect of UV irradiation and caffeine posttreatment. We demonstrate that GCS can be derived from abnormal mitotic cells with a parachute-like chromatin configuration (PALCC) consisting of a bulky chromatin mass and extended chromatin fibers that tether centromeres at a remote, yet normally shaped spindle apparatus. This result hints at a chromosome condensation failure, yielding a "shattered" chromosome complement after MAA fixation. Live mitotic cells with PALCCs proceeded to interphase within a period similar to normal mitotic cells but did not divide. Instead they formed cells with highly abnormal nuclear configurations subject to apoptosis after several hours. We propose a factor depletion model where a limited pool of proteins is involved both in DNA repair and chromatin condensation. Chromosome condensation failure occurs when this pool becomes depleted.

Core3 O-Glycan Synthase Suppresses Tumor Formation and Metastasis of Prostate Carcinoma PC3 and LNCaP Cells through Down-regulation of α2β1 Integrin Complex

Although there are numerous reports of carbohydrates enriched in cancer cells, very few studies have addressed the functions of carbohydrates present in normal cells that decrease in cancer cells. It has been reported that core3 O-glycans are synthesized in normal gastrointestinal cells but are down-regulated in cancer cells. To determine the roles of core3 O-glycans, we transfected PC3 and LNCaP prostate cancer cells with beta3-N-acetylglucosaminyltransferase-6 (core3 synthase) required to synthesize core3 O-glycans. Both engineered cell lines exhibited reduced migration and invasion through extracellular matrix components compared with mock-transfected cells. Moreover we found that alpha2beta1 integrin acquired core3 O-glycans in cells expressing core3 synthase with decreased maturation of beta1 integrin, leading to decreased levels of the alpha2beta1 integrin complex, decreased activation of focal adhesion kinase, and reduced lamellipodia formation. Upon inoculation into the prostate of nude mice, PC3 cells expressing core3 O-glycans produced much smaller tumors without metastasis to the surrounding lymph nodes in contrast to robust tumor formation and metastasis seen in mock-transfected PC3 cells. Similarly LNCaP cells expressing core3 O-glycans barely produced subcutaneous tumors in contrast to robust tumor formation by mock-transfected LNCaP cells. These findings indicate that addition of core3 O-glycans to beta1 and alpha2 integrin subunits in prostate cancer cells suppresses tumor formation and tumor metastasis.

Pharmacokinetics of forsythoside after intravenous administration in beagle dogs

A high-performance liquid chromatography with ultraviolet detection method was established and validated for quantification of forsythoside concentrations in dog plasma. Following a single-step protein precipitation with perchloric acid, the forsythoside and internal standard were separated on a reversed-phase C18 column with water-glacial acetic acid-methanol as mobile phase at a flow rate of 1 mL/min with ultraviolet detection at 326 and 278 nm for forsythoside and IS, respectively. The calibration curve for forsythoside was linear over a range of 0.052-13.33 microg/mL with correlation coefficient of 0.999. The within- and between-batch precisions of analysis were < 8 % and accuracy was 95-107%. After intravenous administration of forsythoside at the doses of 5, 10, and 20 mg/kg, the C(max) values for forsythoside were estimated to be of 12.33, 22.90 and 54.45 microg/mL, respectively. The AUC increased with the increasing of doses, and the mean AUC(o-t) values were 5.69, 11.80, and 18.66 mg x h/L, respectively. Forsythoside was eliminated quickly and the mean T(1/2) values at doses of 5, 10, and 20 mg/kg were 1.36, 1.49, and 0.71 h, respectively. The pharmacokinetics of forsythoside in beagle dogs complied with linear kinetic course in the dose range.

ITO films on glass substrate by sol–gel technique: synthesis, characterization and optical properties

The present paper extensively demonstrates synthesis, characterization and optical properties of semiconductor indium tin oxide (ITO) thin films on glass substrate using sol–gel technique for gas sensor applications. Turbidity, pH values, wettability and rheological properties of the prepared solutions were measured to determine solution characteristics by turbidimeter, pH meter, contact angle goniometer and rheometer machines prior to coating process. Thermal, structural, microstructural, mechanical and optical properties of the coatings were characterized by differential thermal analysis–thermogravimetry (DTA/TG), fourier transform infrarared, X-ray diffraction (XRD), scanning electron microscopy, scratch tester, refractometer and spectrophotometer. Four different solutions were prepared by changing solvent concentration. Turbidity, pH, contact angle and viscosity values of the solutions were convenient for coating process. Glass substrates were coated using the solutions of InCl3, SnCl2, methanol and glacial acetic acid. The obtained gel films were dried at 300 °C for 10 min and subsequently heat-treated at 500 °C for 10 min in air. The oxide thin films were annealed at 600 °C for 60 min in air. DTA/TG results revealed that endothermic and exothermic reactions are observed at temperature between 70 and 560 °C due to solvent removal, combustion of carbon based materials and oxidation of Sn and In. The spectrum of ITO precursor film annealed at 500–600 °C shows an absence of absorption bands corresponding to organics and hydroxyls. In2Sn2O7−x phase was dominantly found as well as SnO2 with low intensity from XRD patterns. It was found that surface morphologies of the film change from coating island with homogeneous structures to regular surface and thinner film structures with increasing solvent concentration. The films prepared from the solutions with 8 mL methanol have better adhesion strength to the glass substrate among other coatings. Refractive index, thickness and band gap of ITO thin films were determined to be 1.3171, 0.625 μm and 3.67, respectively.

High‐performance liquid chromatography and LC‐ESI‐MS method for identification and quantification of two isomeric polyisoprenylated benzophenones isoxanthochymol and camboginol in different extracts of Garcinia species

A rapid, sensitive and simple reverse-phase high-performance liquid chromatography-electrospray ionization mass spectrometric method has been developed for the identification and quantification of two isomeric polyisoprenylated benzophenones, isoxanthochymol and camboginol, in the extracts of the stem bark, seeds and seed pericarps of Garcinia indica and in the fruit rinds of Garcinia cambogia. The separation of isoxanthochymol and camboginol was achieved on a Perkin Elmer RP(8) column (10 x 2.1 mm with 5.0 microm particle size) using a solvent system consisting of a mixture of acetonitrile-water (80:20, v/v) and methanol-acetic acid (99.0:1.0, v/v) as a mobile phase in a gradient elution mode. The limits of detection and quantification were 5 and 10 microg/mL for isoxanthochymol and 50 and 100 microg/mL for camboginol, respectively. The intra- and inter-day precisions were 2.34 and 3.41% for isoxanthochymol and 3.35 and 3.66% for camboginol. The identity of the two isomeric compounds in the samples was determined on a triple quadrupole mass spectrometer with ESI interface operating in the negative ion mode. The method was used to identify and quantify isoxanthochymol and camboginol in the different extracts of two Garcinia species, Garcinia indica and Garcinia cambogia.

Rapid and high-throughput purification of salvianolic acid B from Salvia miltiorrhiza Bunge by high-performance counter-current chromatography

A large-scale purification of salvianolic acid B from Salvia miltiorrhiza Bunge is presented. The method development began with selection of the solvent system, then optimization of the operating parameters and ended up with linear scale-up from an analytical to a preparative instrument. Three factors were used for method optimization and scale-up estimation: purity, process throughput and process efficiency. Preparation was achieved using a two-phase solvent system comprising hexane–ethyl acetate–methanol–acetic acid–water (1:5:1.5:0.00596:5, v/v). This preparation yielded 475 mg of salvianolic acid B with a purity of 96.1% from 1.5 g of crude extract. The process throughput of crude was 2.23 g/h while process efficiency per gram of target compound was 0.769 g/h. Two factors—process environmental risk factor and process evaluation factor were used for evaluation of the separation process.

Effect of colcemid exposure and methanol acetic acid fixation on human metaphase chromosome structure

Distribution of human lymphoid cells in the mitotic subphases, prophase, prometaphase, metaphase and anatelophase, was examined, when cells had been cultured and without colcemid exposure. A higher average length of chromosome no. I was found after overnight fixation than after short time fixation with 3:1 methanol acetic acid fixative. The probability of observing banded chromosomes on slides that had not been exposed to any postfixational band induction was higher after overnight fixation than after short time fixation. Since an increase in the extraction of histones caused by the methanol acetic acid fixation of metaphase cells led to an increase of average chromosome length as well as more frequent banding of chromosomes, it is suggested that not only non-histone proteins but also histones are involved in the formation of chromosome bands. In cells harvested without having been exposed to colcemid. most of the observed chromosomes were banded prometaphase chromosomes. It is suggested that the chromosome condensation process, which is delayed or partly inhibited when inhibitors of RNA or protein synthesis are added to the growth medium, continues as long as colcemid exposed cells remain arrested at the meta-anaphase border with free -SH groups available for oxidation.

Solid-phase extraction of tanshinones from Salvia Miltiorrhiza Bunge using ionic liquid-modified silica sorbents

New ionic liquid-modified silica sorbents were developed by the surface chemical modification of the commercial silica using synthesized ionic liquids. The obtained ionic liquid-modified particles were successfully used as a special sorbent in solid-phase extraction process to isolation of cryptotanshinone, tanshinone I and tanshinone IIA from Salvia Miltiorrhiza Bunge. Different washing and elution solvents such as water, methanol and methanol–acetic acid (90/10, v/v) were evaluated. A comparison of ionic liquid-modified silica cartridges and traditional silica cartridge show that higher recovery was observed using ionic liquid-modified silica sorbents. A quantitative analysis was conducted by high-performance liquid chromatography using a C 18 column (5 μm, 150 mm × 4.6 mm) with methanol–water (78:22, v/v, and containing 0.5% acetic acid) as a mobile phase. Good linearity was obtained from 0.5 × 10 −4 to 0.5 mg/mL ( r 2 > 0.999) with the relative standard deviations less than 4.8%.

Rapid HPLC Analysis for Quantitative Determination of the Two Isomeric Triterpenic Acids, Oleanolic acid and Ursolic acid, in Plantago Major

Plantago major has been used in traditional medicine for the treatment of various diseases. In order to evaluate the quality of P. major, a simple, rapid and accurate high-performance liquid chromatography (HPLC) method was developed for the assessment of the two bioactive triterpenic acids: oleanolic acid (OA) and ursolic acid (UA). A LiChrosorb octadecylsilyl silica (ODS) column with methanol, tetrahydrofuran and aqueous acetic acid as mobile phase and detection at 220 nm were employed for HPLC analysis. The method is precise with relative standard deviations for these two constituents that ranged between 0.5–1.1% (intraday) and 0.8–1.8% (interday). The contents of these two phytochemicals in the leaves of P. major growing in Friuli Venezia Giulia Region were determined in order to establish an effective and reproducible method.

Selective solid-phase extraction of ethynylestradiol from river water by molecularly imprinted polymer microcolumns

A selective on-column molecularly imprinted solid-phase extraction (MISPE) for ethynylestradiol (EE2) from water samples was developed. Following a non-covalent molecular imprinting approach, two molecularly imprinted polymers (MIPs) have been synthesised using methacrylic acid (MAA) as functional monomer, ethyleneglycol dimethacrylate (EGDMA) as crosslinker and EE2 as template, in two different polymerisation solvents (acetonitrile or toluene). The optimisation of the on-column MISPE conditions for the selective rebinding of the EE2 resulted in a flow rate of 2.3 mL min(-1) and volumes of 1 mL toluene and 1.5 mL methanol-acetic acid (9:1) for washing and elution steps, respectively. The selectivity of the imprinted polymer towards several related estrogens such as estriol (E3), estrone (E1) and estradiol (E2) was evaluated. The recovery of EE2 from a 50-mL spiked river-water sample was 75% when the optimised on-column MISPE was applied.