Potassium Phosphate Buffer pH Research Articles

The development and validation of a stability indicating Rp-UPLC method for the simultaneous estimation of clarithromycin, amoxicillin, and vonoprazan in a physical mixture

A novel technique was developed for simultaneous quantification of clarithromycin (CLA) amoxicillin (AMO), and vonoprazan (VON) in a mixture using the reverse phase ultra-performance liquid chromatography (RP-UPLC) technique and validated as per International Council for Harmonization (ICH) guidelines as there was no literature published for its estimation by UPLC. The method was developed using an acquity UPLC system from waters corporation with Hibar Bis phosphonate C18 column (100 × 2.1 mm, 2 μm) at 35°C and tunable ultra-violet detector (TUV) with detection wavelength at 210 nm, has a run time of below 3 minutes. The mobile phase proportion of 60:40 of 0.1 N monobasic potassium phosphate buffer (pH 3.8) and acetonitrile at a flow velocity of 0.2 ml/ minute was utilized. Linearity was observed for CLA, AMO, and VON between the concentration ranges of 25–150, 25–150, and 1–6 μg/ml, respectively, and R2 was 0.999 for CLA, AMO, and VON. Accuracy and precision were within 2% of the coefficient of variation (RSD) for all drugs. The observed mean percentage recoveries for the CLA, AMO, and VON were determined to be 99.74%, 99.07%, and 99.8%, respectively. The stability of the approach was assessed using degradation studies by exposing it to acid, alkali, oxidizing agent, heat, Ultra Violet (UV) light, and water as per ICH guidelines.

Simultaneous determination of six nitroaromatic compounds and three anions in environmental matrices using a liquid chromatography-ion chromatography coupled system

Nitroaromatic compounds are used extensively in various fields such as dyes, pesticides, spices, pharmaceuticals, and explosives. However, the residual raw materials of these compounds accumulate in the environment and pose serious risks to human health. Chronic exposure to low concentrations of nitroaromatic compounds can cause anemia, cancer, and organ damage. Currently, Fenton oxidation and natural bioremediation are the processes most often used to eliminate nitroaromatic compounds from environmental water and soil. According to previous research, the presence of inorganic anions such as chloride, nitrite, and nitrate ions in the environmental matrix exerts an inhibitory effect on the biodegradation of nitroaromatic compounds. Furthermore, high nitrate levels in drinking water can lead to the production of nitrosamine carcinogens, which affect ecological safety and human health, in water bodies. Thus, the simultaneous determination of nitroaromatic compounds and chloride, nitrite, and nitrate ions in environmental soil and water matrices is critical for selecting appropriate nitroaromatic compound degradation methods and monitoring surface water quality. Traditional detection methods require two sample pretreatment steps and two instrumental analytical techniques to determine nitroaromatic compounds and inorganic anions in environmental matrices; moreover, these methods are time consuming, labor intensive, and error prone. Therefore, in this study, a method that combines high performance liquid chromatography (HPLC) and ion chromatography (IC) was developed to simultaneously detect nitroaromatic compounds and anions in environmental matrices. In this method, sample enrichment was achieved through bulk injection and enrichment column collection, which greatly simplified the pretreatment process. The HPLC instrument was connected to the IC instrument using two six-way valves and an enrichment column. The system operation can be divided into four stages: (A) sample loading to the quantitative ring, (B) separation of nitroaromatic compounds and anions, (C) enrichment of anions in an AG20 column, and (D) simultaneous determination of nitroaromatic compounds and anions by HPLC and IC, respectively. The time of the anions flowing out of the C18 column was determined by directly connecting the C18 column to a conductivity detector. Based on the retention times of the anions, the switching time of the six-way valve was optimized to ensure that the anions completely entered the IC column, thereby ensuring the accuracy of the method. During the chromatographic analysis stage, nitroaromatic compounds were separated and analyzed by HPLC system with a mobile phase composed of potassium phosphate buffer (pH 7.0) and acetonitrile (60∶40, v/v) at a flow rate of 1.0 mL/min; in the IC system, the anions were separated and analyzed using a 20 mmol/L sodium hydroxide aqueous solution as the mobile phase under a suppression current of 50 mA. Both anions and nitroaromatic compounds exhibited strong linear correlations within certain concentration ranges, with correlation coefficients greater than 0.993. The recoveries of the nitroaromatic compounds and anions ranged from 88.20% to 105.38% at three spiked levels, with relative standard deviations ranging from 2.0% to 11.5%. The contents of six nitroaromatic compounds and three anions in five surface water and five soil samples were determined using the developed method. Although no nitroaromatic compounds were detected in these samples, the three anions were detected at contents ranging from 0.41 to 55.3 mg/L in surface water samples, and 0.56 to 30.2 mg/kg in soil samples. Methodological validation and actual sample detection demonstrated that the proposed method has a high degree of automation, simple operation, good repeatability, high accuracy, wide applicability, and high sensitivity. Thus, this method is suitable for the rapid determination of chloride, nitrite, nitrate ions and nitroaromatic compounds in soil and water and can be extended to the simultaneous determination of inorganic ions and organic matters in other samples.

Stability-indicating HPLC method optimization using quality by design with design of experiments approach for quantitative estimation of organic related impurities of Doripenem in pharmaceutical formulations

Doripenem (DM), an antibiotic used for kidney, lung, and urinary infections, underwent the development of a robust high-performance liquid chromatography (HPLC) technique using the quality-by-design approach. This method, integrating design-of-experiments, detected organic impurities in both drug substances and formulated products. Operating with a C18 analytical column in a binary gradient mode with potassium phosphate buffer (pH 6.1) and acetonitrile as mobile phases, the method ran at a flow rate of 1.20 ml/minutes, with an injection volume of 20-μl and a column temperature of 45°C. Stability testing under various conditions, including hydrolysis, oxidation, heat, humidity, and light exposure, confirmed the method’s reliability without interference. Validation studies, compliant with ICH guidelines, revealed quantitation limits of 0.006%, linearity between 0.060 and 1.800 μg/ml (R2 > 0.999), and recoveries ranging from 96.8% to 99.1%. This HPLC method was effectively used for stability assessment in quality control testing, assessing doripenemic acid, doripenem assay, and organic impurities.

Selective enzymes separation from the fermentation broth of Yarrowia lipolytica using aqueous two-phase system based on quaternary ammonium compounds

Aqueous two-phase systems (ATPS) are considered efficient and sustainable downstream processing techniques in extracting and separating enzymes. In this work, we evaluated the use of ATPS based on quaternary ammonium (tetrabutylammonium bromide ([N4444]Br), tetrabutylammonium chloride ([N4444]Cl), choline chloride (ChCl), and betaine) + potassium phosphate buffer (pH 7) to separate selective lipase and protease from the fermentation broth with Y. lipolytica. Cultivation in YPD medium supplemented with 200 mM potassium phosphate buffer (pH 7) in the preparation of the culture medium positively influenced the production of lipase and protease, whereby led to lesser pH variations and better values of biomass production (15.29 g p.s. L-1), lipolytic (455.96 U L-1), and proteolytic activity (23.70 U L-1). The presence of the culture medium enhanced the two-phase region, and the phase separation followed the order of ammonium compounds hydrophobicity: [N4444]Br > [N4444]Cl > betaine > ChCl. Lipase was partitioned mainly to the ammonium-rich phase and protease migrates preferentially to the salt-rich phase in all systems studied. Remarkable extraction efficiencies of 100% for lipase and 96.87% for protease were achieved in a single step for [N4444]Cl-based ATPS. Furthermore, a high level of purification was achieved with values of 10.55 and 1.69 for lipase and protease, respectively. According to the remarkable results, quaternary ammonium-based ATPS can be considered an alternative and efficient platform to separate lipase from protease, obtaining two high-value-added compounds selectively.

OPTIMUM CONDITIONS FOR PHENYLALANINE AMMONIA LYASE EXTRACTION AND PURIFICATION FROM LOCALLY CULTIVATED GRAPE SEEDS

In the present study, Phenylalanine ammonia lyase (PAL) activity was detected in different parts of grape plant (leaves, pulp, whole seeds and defatted seeds) extracts. The results revealed that the whole seed possess highest PAL activity (69.44 U), followed by defatted seed (44.22 U), whereas extracts of leaves and pulp gave the lowest activity values (22.77 and 5.11 U, respectively). The optimum conditions for crude PAL extraction achieved using 50mM potassium phosphate buffer (pH 7) at mixing ratios of (1:7) for 1 h. extraction time. PAL was shown to precipitate out at the saturation range between 20-90%. For further purification, gel filtration chromatography using Sephadex G-200 column (1.5×70) and ion exchange chromatography using DEAE- Cellulose (DE 52) column (3.2× 10) were applied. The specific activity, number of folds and yield of purified PAL was 1157 U/mg, 19.94 and 15.32 % respectively.

EXTRACTION, PURIFICATION AND CHARACTERIZATION OF LIPASE FROM THE DIGESTIVE DUCT OF COMMON CARP CYPRINUS CARPIO L.

This study was aimed to use digestive duct of common carp Cyprinus Carpio L. as economic source for fish lipase extraction . The enzyme was extracted using potassium phosphate buffer (pH 7 , 0.2 M ). The crude extract then precipitated at the saturation range between 40 – 80 % and dialyzed . Further purified carried out through ion exchange chromatography using DEAE-Cellulose column (2.5 × 30 cm) and gel filtration chromatography by Sephadex G-100 column (1.5 x 60 cm). The purified lipase had a molecular weight of 37.150KDa ,and optimum pH and temperature for enzyme activity about 45◦C and 6.5 using olive oil as substrate .The specific activity, number of purification folds and yield of purified Lipase were 146.48U/mg, 9.76 and 40.50% respectively.

(Digital Presentation) Electrochemical Single Impact Method for Electroactive Bacterial Detection Onto Carbon Ultramicroelectrode

This work aims to design a high sensitivity and selectivity biosensor based on the electrochemistry of single impacts onto ultramicroelectrode (UME) to detect and identify various bacterial strains. The main objective is to establish a unique electrochemical signature for each bacterial cell through the individual impact event signal on the surface of the UME [1, 2]. First, we focus on the detection of well-known electroactive Gram-negative bacteria such as Shewanella oneidensis in order to be able to selectively detect these different single cells. In this case, the strategy currently used is to record a chronoamperometric curve in an aqueous potassium phosphate buffer (pH = 7.4) solution containing a redox probe at an UME polarized at the oxidation or reduction potential of the electrochemical active aqueous species and to observe a “current step” when one bacterium impacts the UME, corresponding to a “blocking effect” [3] (Figure A). The response signal expected from single bacterium collision may also be a “current spike” corresponding to either the own electrochemical activity of the bacterium toward the redox probe and the UME applied potential or a “bouncing effect” of the bacterium which does not stick onto UME surface (Figure B).In order to be able to identify the type of bacterial cell striking the UME surface, an adapted functionalization (covalent grafting via reduction of diazonium aryl salts) with appropriate affinity (bio)chemical species such as antibodies or aptamers could be performed. This strategy could confer to the UME surface a selectivity of the signal generated during single impacts. For example, the electro-grafting of diazo-pyridinium cations for microbial fuel cell electrodes showed to promote and improve the development of bacterial electroactive films [4].[1] E. Lebègue, N. L. Costa, R. O. Louro, F. Barrière, J. Electrochem. Soc. 16 (2020) 105501[2] A. T. Ronspees, S. N.Thorgaard, Electrochimica Acta. 278 (2018) 412-420[3] G. Guanyue, W. Dengchao, B. Ricardo, Z. Jinfang, M. Michael V. Anal. Chem. 90 (2018) 12123−12130[4] H. Smida, E. Lebègue, J-F. Bergamini, F. Barrière, C. Lagrost, Bioelectrochemistry. 120 (2018) 157-165 Figure 1

Optimization of Sprout Seed Abrasion Method for Efficient Inoculation of Leaf Crinkle Disease in Urdbean [Vigna mungo (L.) Hepper

Background: Urdbean leaf crinkle is one of the serious diseases of urdbean (Vigna mungo (L.) Hepper) which is caused by an ungrouped virus called Urdbean leaf crinkle virus (ULCV). The disease is highly seed transmissible under natural conditions and in experimental conditions it is mechanically transmitted through sap inoculation. Methods: The aim of this study was to standardize the protocol for sap inoculation by sprout seed abrasive method by comparing the effects of different inoculation methods, such as pin prick and without pin prick, with 0.05M potassium phosphate buffer (pH 7.0) supplemented with additives and abrasives and to further validate the standardized protocol upon different urdbean genotypes. Result: This is the first effort on the optimization of protocol in which, pin pricking as inoculation method with potassium phosphate buffer (0.05M, pH 7.0) supplemented with 0.15% sodium sulfite+0.01M 2-Mercaptoethanol and 1% celite+2% carborundum showed 100% transmissibility foremost at 2nd trifoliate stage and further validation with various urdbean genotype resulted in percent transmission ranging from 92 to 100% which evinces the efficacy and repeatability of the methodology.

Влияние продолжительности сухого созревания и состава посолочных веществ на белки высококачественной говядины

Meat quality largely depends on the maturation conditions and natural biochemical processes that affect its taste, aroma, tenderness, and technological properties. Dry maturation and technological processing attract a lot of scientific attention. The research objective was to study the effect of dry maturation time on the physicochemical parameters of high-quality beef, as well as the effect of a new curing mix on the properties of raw materials.
 The research featured dorsal-lumbar cuts of high-quality beef on maturation days 21 and 40. Matured meat was tested for the main components, the solubility of sarcoplasmic and myofibrillar proteins, the hydrophobicity of myofibrillar proteins, protein oxidation products, and the activity of catalase and peroxidase. The chemical composition was evaluated depending on the dry maturation time using the arbitration method. The solubility of proteins was determined by the calorimetric method with a biuret reagent. The hydrophobicity of myofibrillar proteins was determined by bromophenol blue reaction, and the activity of catalase and peroxidase was determined by standard methods. Proteins were extracted with potassium phosphate buffer (pH 7.2), while myofibrillar proteins were extracted with Tris-HCl and KCl buffers at pH 7.5 and 7.0. 
 The meat samples were deboned and salted using sodium chloride and a combined mix of 70% magnesium chloride and 30% sodium chloride. The solubility of myofibrillar proteins on day 21 increased by 23.95% but decreased by 14.1% by day 40. The solubility of sarcoplasmic proteins decreased continuously (22.10 and 31.12%, respectively). The obtained data matched the hydrophobicity of proteins. Dry maturation initiated protein oxidation, as demonstrated by carbonyl and sulfhydryl groups of myofibrillar proteins on maturation day 40 (27.85 nmol/L and 27.3 µmol/g of protein, respectively). Sodium chloride and its mix increased the extractability of proteins by 5.2 and 6.9% on day 21 and by 6.8 and 10.6% on day 40 but triggered protein oxidation.
 Muscle proteins of high-quality beef proved functional after 21 days of dry maturation. The new mix with reduced sodium content can be recommended for high-quality dry-aged beef production.

Bridging Pharmaceutical Analysis with Computational Chemistry to Ascertain the Pharmacokinetic and Toxicological Profiling of Febuxostat and its Diacid Impurity

Drug degradation may change the pharmacological action and results in altered therapeutic and toxicological profile of drug. Identification of degradation product is good opportunity in the drug discovery, as non-toxic impurities can be considered for the new pharmacological potentials. Additionally, development of computer based in-silico model and methods further assist in prediction of pharmacokinetic, dynamic as well as biological activities which make drug discovery process more unfailing. In present work, Febuxotat (FEB) was subjected to forced degradation experiments and the formed degradation products were separated and resolved using optimized HPLC method. Further the study was extended to enrich its alkali degradation product and was ascertained using 1H NMR spectroscopy and mass spectrometry. The ADME/Toxicological properties of alkali degradation product were determined by using Admet SAR 2.0. The method was validated as per ICH guidelines Q2 (R1). In forced degradation experiments, substantial degradation of FEB was observed under acid, alkali, wet heat, dry heat and oxidative conditions, however, FEB was found stable to photolytic conditions. The drug and its formed degradation products separated and resolved from each other on Waters SunFire C8 (250 × 4.6 mm, 5 µm) column using blend of methanol: 20 mM potassium phosphate buffer (pH 3.0) (80: 20 % v/v). From the 1H NMR spectroscopy and mass spectrometry the enriched alkali degradation product was confirmed as 2-[3-carboxy-4-(2-methylpropoxy) phenyl]-4-methyl-1,3-thiazole-5-carboxylic acid (diacid FEB). Febuxostat and diacid Febuxostat was found non-toxic for carcinogenicity (binary), eye corrosion, eye irritation and Ames mutagenesis during ADME/Tox studies. However, both were found hepatotoxic. A simple, accurate, precise, and robust stability indicating HPLC method was developed and validated for the estimation of FEB in bulk and tablet dosage form. Based on ADME/Tox both were found to hepatotoxic. Henceforth, based on our evidences, one should use FEB with precaution in patient suffering from liver disease.

Co-assembly of foxtail millet prolamin-lecithin/alginate sodium in citric acid–potassium phosphate buffer for delivery of quercetin

Foxtail millet nanoparticles with smaller mean size at ∼130 nm and narrower polydispersity index at ∼0.05 were prepared in citric acid–potassium phosphate buffer (pH 8.0). Through lecithin (Lec)/sodium alginate (Alg) coating, a hydrophobic FP core, a Lec monolayer, and a hydrophilic Alg shell were formed spontaneously. Dissociation experiment revealed that electrostatic interaction and hydrogen bonding were main driving forces for the formation and maintenance of stable FP–Lec/Alg NPs. In addition, Lec/Alg coated NPs exerted an important role in sustaining the controlled release of the encapsulated quercetin under simulated gastrointestinal tract conditions. Cellular uptake test exhibited that FP-Lec-Alg NPs cold enter epithelial cells in a time-dependent manner, showing the maximum uptake efficiency were 22% and 24%, respectively, after 2 h of incubation. About 220 nm NPs can be recovered by adding 10% (w/v) sucrose. FP-Lec-Alg NPs were found to be promising delivery materials to deliver quercetin and improve its bioavailability.

A self-powered glucose sensor based on BioCapacitor principle with micro-sized enzyme anode employing direct electron transfer type FADGDH

Diabetes mellitus is a disorder in which the body does not produce enough or respond normally to insulin; consequently, blood glucose levels increase to become abnormally high. Accordingly, the primary treatment of diabetes is to control glycemic levels continuously. To continuously control glycemic levels, several medical devices have been developed to monitor blood glucose levels, represented by sensors and monitors for the self-monitoring of blood glucose. The ultimate goal for those engaged in research to develop medical devices is to develop implantable biodevices, namely self-powered autonomously operated artificial pancreas systems. One of the most challenging issues in realizing an implantable artificial pancreas is the long-term continuous supply of electricity, which is currently dependent on rechargeable batteries, requiring periodical replacement. In this work, we report the development of a direct electron transfer type enzyme-based miniaturized self-powered glucose sensor based on the BioCapacitor principle with a micro-sized enzyme anode area (0.15 mm × 0.75 mm), which has only 0.1 mm2 of electrode surface. As a result, a BioCapacitor utilizing a biofuel cell with a micro-sized enzyme anode was operated by self-power. In addition, the glucose concentration was detected within the range from 13 mM to 100 mM based on the frequency of charge/discharge cycles of the BioCapacitor. Although further improvement of the current density of the micro-sized anode is necessary to monitor a glucose concentration range lower than 13 mM, this self-powered glucose sensor with a micro-sized electrode based on the BioCapacitor principle was operated continuously for 6.6 h at 37 °C in 100 mM potassium phosphate buffer (pH 7.0). Our success indicates the potential to realize self-powered, autonomous, and implantable sensing modules for bio devices such as glucose-sensing systems for an artificial pancreas.

A Comparative Estimation of Alprazolam in Pharmaceutical Formulations by Validated HPLC and HPTLC Techniques

The aim of present work is to develop and validate two simple and specific methods for the estimation of alprazolam hydrochloride (a psychoactive drug) in pharmaceutical formulations. The first method involves HPLC separation on the Cosmosil C-8 column using acetonitrile and potassium phosphate buffer (pH 6.0±0.1) (40:60 v/v) as mobile phase. The peak was obtained at 3.01±0.02 min. The effluent was monitored at 254 nm. The second method is based on HPTLC using Acetic acid: water: methanol: ethyl acetate (2:15:20:80 v/v/v/ v) as mobile phase and detection of the spot was carried out at 254 nm. The Rf value was found to be 0.76±0.02. The methods were validated as per ICH guidelines. The linearity curves were found to be linear over the concentration range 50-150 μg/ ml for HPLC and 50-400 ng/spot for HPTLC with a high correlation coefficient (R2 = 0.991 and 0.9987 respectively). The limit of detection and limit of quantification were found to be 1.66 and 5.04 μg/ml for HPLC and14.84 and 44.097 μg/ ml for HPTLC, respectively. The proposed methods are successfully used for the quality control of alprazolam in commercial form. Statistical analysis results confirmed that the methods are selective and precise.

RP-HPLC method for simultaneous determination of escitalopram oxalate and flupentixol HCl in tablet dosage form

RP-HPLC method was developed for simultaneous determination of escitalopram oxalate (ESC) and flupentixol HCl (FLU) in tablet dosage form. Mobile phase consisting of mixture of acetonitrile and potassium phosphate buffer (pH 7.0 with 0.1% triethylamine) in the ratio 60: 40 at flow rate of 1ml/min using C18 Grace (250mmX 4.6mm) column at 231 nm. The retention time of ESC with FLU was found to be 2.96 min and 6.98 min, respectively. The linearity range for ESC with FLU observed was 5-25 µg/ml and 10-50 µg/ml, respectively. Method was validated as per ICH guidelines. Validation parameters studied were linearity and range, recovery study, precision, LOD, LOQ and robustness. Statistical data obtained was found to satisfactorily.

Thin layer chromatography identification of flavoprotein compounds in crude local spinach extract

Spinach serves as a source of a nutritious plant rich in many compounds. Components in the leaf extract of spinach are beneficial for humans. In addition to that, spinach leaves contain flavoproteins which are a class of enzymes mediating electron transport in biological system. They possess a protein chromophore with flavin adenine dinucleotide (FAD) as prosthetic group. Spinach redox proteins are used in vitro enzymatic reconstitution studies to replace native redox proteins found in bacteria. However, datathat supports the role of spinach redox proteins as control redox partners for Mycobacterium tuberculosis (Mtb) cytochrome P450s are sparse. Therefore, this study focuses on local spinach leaves containing FAD which can transport electrons. Spinach leaves extracted using gentle mechanical process with potassium phosphate buffer (pH 7.4). Paper chromatography (PC) shows positive result for flavin, thin layer chromatography (TLC) shows compound RF value similar to standard riboflavin (0.16), spectrophotometer analysis and urea denaturation test did not shows spectra of flavin compound in sample. In chemical oxidation and reduction, spinach extracts shows positive reaction towards potassium ferricyanide (oxidant) indicating electron displacement in spinach samples. Preparative chromatography methods need to be developed in order to purify crude spinach extracts to obtain semi-pure or pure flavoproteins.

CHRONIC NEUROGENIC PAIN PROMOTES DEVELOPMENT AND GROWTH OF M1 SARCOMA CHANGING LOCAL LEVELS OF GROWTH FACTORS

Introduction. The influence of chronic neurogenic pain on local levels of growth factors in the lung and the risk of lung cancer development has been little studied.The purpose of the study was to analyze the levels of VEGF, TGF-β, IGF-I, IGF-II, FGF-21 and receptors of VEGFR2, TGF-βR2 in the lungs of white outbred rats with chronic neurogenic pain after intravenous injection of M1 sarcoma.Material and Methods. A total of 28 white outbred male rats weighing 200–250 g were divided into 4 groups: 1 – sham-operated animals (control group) (n=7); 2 – animals with chronic neurogenic pain (n=7); 3 – sham-operated animals with intravenous injection of M1 sarcoma (n=7); 4 – rats with intravenous injection of M1 sarcoma in presence of chronic neurogenic pain (n=7). Animals were decapitated, the lungs were harvested on ice; 10 % cytosolic fractions were prepared in 0.1 M potassium phosphate buffer pH 7.4 containing 0.1 % Tween-20 and 1 % BSA. Levels of VEGFR2, TGF-β and receptors of TGF-βR2, IGF-I, IGF-II (CUSABIO BIOTECH Co., Ltd., China) and FGF21 (BioVender, Czech Republic) were determined by ELISA. The data were statistically processed using Statistica 10.0 software.Results. Multifocal tumors of sarcoma in the lungs were determined only in the group of rats with chronic neurogenic pain after intravenous transplantation of M1 sarcoma. In the lungs of sham-operated animals, tumor foci were not detected after M1 sarcoma transplantation. Lung tissues with M1 growth and presence of chronic neurogenic pain demonstrated decreased VEGF-А levels with increased concentrations of TGFβ, IGF-I, IGF-II and FGF-21. Chronic neurogenic pain directly or indirectly influenced levels of some growth factors in the lung, and altered cell homeostasis making possible transplantation of M1 sarcoma into the lung.

Production of 7-methylxanthine from Theobromine by Metabolically Engineered E. coli

In this work, a novel biocatalytic process for the production of 7-methylxanthines from theobromine, an economic feedstock has been developed. Bench scale production of 7-methlxanthine has been demonstrated. The biocatalytic process used in this work operates at 30 OC and atmospheric pressure, and is environmentally friendly. The biocatalyst was E. coli BL21(DE3) engineered with ndmB/D genes combinations. These modifications enabled specific N7- demethylation of theobromine to 7-methylxanthine. This production process consists of uniform fermentation conditions with a specific metabolically engineered strain, uniform induction of specific enzymes for 7-methylxanthine production, uniform recovery and preparation of biocatalyst for reaction and uniform recovery of pure 7-methylxanthine.
 Many E. coli BL21(DE3) strains metabolically engineered with single and/or multiple ndmB/D genes were tested for catalytic activity, and the best strains which had the higher activity were chosen to carry out the N-demethylation reaction of theobromine. Strain pBD2dDB had the highest activity for the production of 7-methylxanthine from theobromine. That strain was used to find the optimum amount of cells required to achieve complete conversion of theobromine to 7-methylxanthine within two hours. It was found that the optimum concentration of pBD2dDB strain to achieve 100% conversion of 0.5 mM theobromine to 7-methylxanthine was 5 mg/mL. The cell growth of pBD2dDB strain was studied using two different growth media, (Luria-Bertani Broth and Super Broth). Super broth was found to be the best medium to produce the highest amount of cell paste (1.5 g). Subsequently, the process was scaled up in which 2 L reaction volume was used to produce 7-methylxanthine (100% conversion) from 0.5 mM theobromine catalyzed by pBD2dDB strain. The reactions was carried out at 30 oC and 250 rpm shaker speed, and the reaction medium was 50 mM potassium phosphate buffer (pH=7). 7-methylxanthines was separated by preparative chromatography with high recovery, and the product solution was collected, purified by drying at 120-140 oC for 4 hours and, recovered (127 mg). Purity of the isolated 7-methylxanthine was comparable to authentic standards with no contaminant peaks, as observed by HPLC, LC-MS, and NMR.

Quantitative Analysis of 5-Hydroxymethylfurfural in Linezolid Injection by High Performance Liquid Chromatography

Background: Linezolid is a synthetic broad-spectrum antibacterial belonging to the class of oxazolidinones. Linezolid for intravenous infusion is isotonized with dextrose. In acidic environment, the dehydration of dextrose produces furan derivatives, 5-hydroxymethylfurfural (5-HMF) being the main one. The determination of this degradation product is of fundamental importance, since there is evidence it is cytotoxic, genotoxic, mutagenic and carcinogenic. However, there is no official method for the determination of 5-HMF in drug products. Objective: The aim of this study was to develop and validate a high performance liquid chromatographic method to quantify 5-HMF in injection of linezolid. Methods: The chromatographic separation, after optimization, was performed on C18 (150 x 4.6 mm, 5 μm) column. Mobile phase was composed of 14 mM potassium phosphate buffer pH 3.0 ([H+] = 1.0 x 10-3) and methanol in gradient elution at 1.0 mL min-1. The injection volume was 10 μL and detection was performed at 285 nm. Results: The method was optimized and validated, showing selectivity, linearity in the range from 0.075 to 9.0 μg mL-1, precision (RSD ≤ 2.0%), accuracy (mean recovery of 100.07%) and robustness for temperature and pH variation. Conclusion: The method was shown to be adequate to determine 5-HMF in injection containing linezolid in routine analysis.

An eco-friendly HPLC-UV method for the determination of risedronate in its bulk and tablet dosage form with application to content uniformity, dissolution and stability testing

Risedronate is a nitrogen-containing bisphosphonate for the treatment and prevention of postmenopausal osteoporosis. The current work aims to develop a novel green HPLC-UV method for the rapid analysis of risedronate sodium in bulk and tablet formulation. The analyzed samples were separated on Waters Atlantis dC18 (150 mm × 3.9 mm; 5 μm) column using a green mobile phase consisting of potassium phosphate buffer pH 2.9 and potassium edetate buffer pH 9.5 in a ratio of 1:2, the final pH was adjusted to 6.8 with phosphoric acid, the mobile phase was pumped at a rate of 1.0 mL/min, with column temperature set at 30 °C, eluted samples were detected at 263 nm and the chromatographic run time was 3.0 min. The method was found to be linear over the concentration range of 14–140 μg/mL with a correlation coefficient (r2) of 0.9994. Accuracy and precision were evaluated from three QC samples (LQC, MQC and HQC) together with the five calibrators where the percentage accuracy was found to be 101.84%. Processed quality control samples of risedronate sodium were tested for stability at different conditions, short term, long term and freeze- thaw stability. The current method was further extended to study the content uniformity of Actonel® tablets following United States Pharmacopoeia (USP) guidelines. The proposed method was fully validated as per ICH guidelines.

Trichosanthes dioica Roxb. prevents hepatic inflammation and fibrosis in CCl4-induced ovariectomized rats

Summary Plant-derived natural products have shown beneficial effects in many diseases associated with liver. The present study aimed to appraise the effects of Trichosanthes dioica (TD) peel extract on oxidative stress, inflammation as well as fibrosis exhibited by the ovariectomized rats in which hepatic damage had been induced by the administration of carbon tetrachloride (CCl4). The CCl4 administration in the ovariectomized rats elevated the plasma ALT, AST, and ALP level in comparison with the control while TD peel extract showed significant reduction of those activities. Besides, oxidative stress and lipid peroxidation also increased in CCl4-treated rats. TD peel extract significantly reduced the oxidative stress marker's concentration and improved the catalase and SOD activities in CCl4-administered ovariectomized rats. These results suggest that TD peel extract is capable of protecting the liver from CCl4induced damage through preventing oxidative stress, lipid peroxidation, inflammation, fibrosis and boosting the diminished cellular antioxidant defense.