Potassium Phosphate Buffer Research Articles

A novel RP-HPLC method for the simultaneous quantification of azithromycin and dexamethasone in marketed ophthalmic drops

A straightforward, precise, accurate, specific, and sensitive RP-HPLC technique has been established for the quantification of Azithromycin Dihydrate and Dexamethasone Sodium Phosphate in their commercial formulation. The wavelength maxima for Azithromycin and Dexamethasone were considered to be 215 nm for estimation. In RP-HPLC separations were carried out on a Zorbax SB C18 column (250mm X 4.6mm, 5 μm) and mobile phase consisting of Acetonitrile: Monobasic Potassium Phosphate (KH2PO4) buffer in the proportion of 75:25 v/v and pH of the buffer was adjusted to 5.5 with OPA. The estimation was carried out at 215 nm using UV detector keeping the flow rate of 1.2 ml/min and injection volume of 50 μl. The current method demonstrates good linearity over the range of 20 - 120 μg/ml and 2 - 12 μg/ml for Azithromycin and Dexamethasone respectively. No interference of excipients was found during estimation. The recommended techniques were verified and determined to be specific, accurate, and exact. The approaches were effectively used for the concurrent quantification of both medicines in pharmaceutical formulations, making them suitable for regular quality control examination.

Antibacterial activity of Savory (Satureja hortensis) extract against pure culture strains of

The paper presents the results of a study of the antibacterial activity of supercritical carbon dioxide extract of the aboveground part of Savory (Satureja hortensis) against Escherichia coli, Salmonella spp. and Staphylococcus aureus bacteria.To study the antibacterial properties of Savory extract (Satureja hortensis) against infections caused by Staphylococcus aureus, Escherichia coli and Salmonella spp.In the course of the experiment, Savory extract obtained by supercritical carbon dioxide extraction was studied for antimicrobial activity against these bacteria. The component composition of the extract was determined by chromatography–mass spectrometry. To determine the antibacterial activity, we used the method of dilution in a liquid medium with subsequent measurement of the growth of the suspension culture biomass. The activity of the extract was compared with certified drugs included in the protocols for the treatment of bacterial infections. DMSO and potassium phosphate buffer were used as a negative control.The studies showed that the extract exhibits high antibacterial activity against all the studied bacterial strains and has a direct positive correlation with its concentration. Chromatographic mass‐spectrometry analysis showed that the extract of garden Savory contains 28 substances, the main ones being thymol (30.51 %), gamma‐terpinene (15.27 %), para‐ cymene (14.25 %) and Carvacrol (9.18 %), which both individually and in combination could exhibit antibacterial activity.Supercritical carbon dioxide extract exhibits pronounced antimicrobial activity against reference strains of S. aureus, Escherichia coli and Salmonella spp.

Evaluation of blue textile dye decolorization by immobilized polyphenol oxidase using pumice stone under optimum conditions

Industrial dyes are major pollutants in wastewater and river water with an initial visible concentration of 1 mg/L. Recent studies have shown the possibility of using polyphenol oxidase in catalytic biological treatment due to its ability to oxidize a large number of dyes and pollutants in wastewater and the flexibility to work in wide ranges of temperature, pH and salinity. It is easy availability as well as the low economic cost resulting from its use in biological treatments, this enzyme polyphenol oxidase was used. The findings in this study showed that the extraction of polyphenol oxidase (PPO) from potato peel was homogenized with potassium phosphate buffer (0.1 M, pH 7) at a ratio of 1:10 (weight: volume) for two min. The result of enzyme purification by ion exchange chromatography showed that the yield of this step was 64 % and the specific activity was 6541.67 U/mg. The polyphenol oxidase was immobilized covalently on the functionalized pumice stone (25.2 U/gm) compared with other methods. The characterization results demonstrated that the optimum pH for immobilized and free enzyme activity was 6.0 while the pH range of free and immobilized polyphenol oxidase stability was from 4.5 -7.0 and 3.5 - 8.5 respectively. The better temperature for free and immobilized polyphenol oxidase activity was 25 ºC, whereas the free and immobilized polyphenol oxidase was stable at 15-35 and 15-50 °C respectively. The outcomes showed that the decolorization efficiency of blue textile dye employing immobilized polyphenol oxidase reached 99.85 % after 24 hr. for 100 mg/L concentration while for other concentrations 200, 300, 400, 500 and 1000 mg/L the decolorization efficiencies were 85.96, 76.15, 72.54, 66.94 and 63.5 % respectively. Based on the results, the immobilized polyphenol oxidase on pumice stone is highly efficient in removing textile dyes at large concentrations and in different environmental conditions.

Graphene-Based Nano Drug Delivery of Curcumin for the Treatment of Dengue Fever.

Graphene, which is defined as a single-layered sp2 hybridized carbon atoms placed in a honeycomb-like crystal lattice when converted to graphene oxide, has proven its worth by showing its tremendous capacity in controlling the release rate of drugs by entrapping in the numerous pockets present inside it through π-π stacking interaction when used as a nanocarrier. In this experiment graphene oxide was synthesized through traditional Hummer’s method and was used as a nanocarrier to target nanosized drugs, which was prepared by loading curcumin drug into the PVP functionalized graphene oxide by using necessary procedures. After the successful confirmation of the stability, functionalization, and compatibility through various characterization procedures and effective drug loading into the systems, the release behavior of the formulations has been checked through dialysis bag diffusion technique using potassium phosphate buffer saline (pH 7.2, 4.0 and 9.2) for selection of the best medium for determination of the successful sustained-release behavior of the drugs through the dialysis membrane which concluded with satisfactory, sustained and constant drug release phenomenon in the acidic environment reflecting an idea of the fact its favorable release in acidic pH. It is a fact known that dengue fever causes systemic acidosis i.e., reduction of the pH of blood below 7. Thus, when used in-vivo there is a possibility that the formulation has easy access to its target and, performs the desired mechanism of action and can be used as a therapeutic delivery system against the Dengue virus.

Determination of solid-state acidity of lyophilized trehalose containing citrate, phosphate, and histidine buffers using UV/VIS diffuse reflectance and solid-state NMR spectroscopy

Changes in the protonation state of lyophilized proteins can impact structural integrity, chemical stability, and propensity to aggregate upon reconstitution. When a buffer is chosen, the freezing/drying process may result in dramatic changes in the protonation state of the protein due to ionization shift of the buffer. In order to determine whether protonation shifts are occurring, ionizable probes can be added to the formulation. Optical probes (dyes) have shown dramatic ionization changes in lyophilized products, but it is unclear whether the pH indicator is uniform throughout the matrix and whether the change in the pH indicator actually mirrors drug ionization changes. In solid-state NMR (SSNMR) spectroscopy, the chemical shift of the carbonyl carbon in carboxylic acids is very sensitive to the ionization state of the acid. Therefore, SSNMR can be used to measure ionization changes in a lyophilized matrix by employing a small quantity of an isotopically-labeled carboxylic acid species in the formulation. This paper compares the apparent pH of six trehalose-containing lyophilized buffer systems using SSNMR and UV-Vis diffuse reflectance spectroscopy (UVDRS). Both SSNMR and UVDRS results using two different ionization probes (butyric acid and bromocresol purple, respectively) showed little change in apparent acidity compared to the pre-lyophilized solution in a sodium citrate buffer, but a greater change was observed in potassium phosphate, sodium phosphate, and histidine buffers. While the trends between the two methods were similar, there were differences in the numerical values of equivalent pH (pHeq) observed between the two methods. The potential causes contributing to the differences are discussed.

A novel QbD HPLC approach for the concurrent analysis of amoxicillin, amprolium, and ethopabate in food samples and dosage forms

A simple, rapid, and sensitive reversed-phase HPLC method coupled with photodiode array detection (PDA) was developed for the simultaneous estimation of three co-administered antibacterial drugs, amprolium, ethopabate, and amoxicillin for the first time. An analytical quality-by-design (QbD) approach was adopted to optimize the developed approach. A two-level full factorial design (25 FFD) was implemented to optimize how variable factors affected chromatographic responses. On a Hypersil BDS C18 column, the chromatographic separation was carried out using isocratic elution at 40.1 °C. The mobile phase consisted of methanol: potassium phosphate buffer containing 5.76 mM heptane sulphonate (9.05 mM; pH 3.32, 44.8:55.2 % v/v) pumped at a flow rate of 0.8 mL/min and an injection volume of 20.0 μL. In less than 7 min, the suggested method was able to separate the ternary mixture with a challenging ratio of 1: 15.6: 1 for amoxicillin, amprolium, and ethopabate, respectively. The calibration curves showed excellent linearity over the concentration ranges of 0.50–30.0, 0.30–30.0, and 0.05–20.0 μg/mL, respectively. Recovery percentages ranged from 90.30 % to 111.13 % with %RSD less than 2 % were obtained upon spiking to chicken tissues, liver, and egg samples, indicating the suitability of the proposed method for determining of the studied drugs in complicated food matrices. Furthermore, the proposed method was successfully applied to determine the studied drugs in their commercial formulations. Full validation was carried out according to ICHQ2 (R1) guidelines. Finally, the method's greenness and ecofriendliness were evaluated using various metrics tools, including the Green Analytical Procedure Index (GAPI) and Analytical GREEnness (AGREE).

Integrated Carbon Layer and CoNiP Cocatalyst on SnWO4Film for Enhanced Photoelectrochemical Water Splitting.

α-SnWO4 is a promising semiconductor for solar water splitting, however, its performance is limited by weak water oxidation and poor charge transfer. In this study, we employ a vapor deposition method to uniformly implement a carbon layer onto the surface of SnWO4 coupled with a CoNiP cocatalyst, successfully constructing the integrated CoNiP/C/SnWO4 film photoanode and alleviating the oxidation of Sn2+ when loading electrocatalyst. Incorporating the carbon layer enhances the interface charge conduction behavior between the SnWO4 substrate and the CoNiP cocatalyst, thereby mitigating charge recombination. The synergistic interplay between the carbon layer and CoNiP leads to a remarkable achievement, as evidenced by the photocurrent of 1.72 mA cm-2 (1.23 V vs. RHE) observed for SnWO4 film measured in 0.2 M potassium phosphate buffer solution. In this work, we demonstrate the viability of tailoring SnWO4 photoanode and provide valuable insights for prospective advancements in modifying SnWO4 photoanode.

Large-Scale Artificial-Photosynthetic Production of Formate and Isolation of Pure Formic Acid

We proposed a concept of an artificial-photosynthetic system to produce pure formic acid using CO2, water, and solar energy, generating no waste, as illustrated in Fig. 1.1 The system consists of an artificial-photosynthetic reaction process and a product isolation process. We proved the concept by small-scale experiments, and constructed a practically large-sized solar-driven CO2 electrolyzer as the first step toward widespread use of artificial photosynthesis.2,3 We adopted a single-compartment configuration of the solar-driven CO2 electrolyzer without ion-exchange membranes and a near-neutral pH electrolyte of a potassium phosphate buffer aqueous solution to simplify the reactor structure. The large-sized electrolyzer was composed of eight-stacked cathode-anode-electrode pairs of 1 m×1 m in size and powered by crystalline silicon solar cells installed on the electrolyzer housing via a DC-DC converter. The raw material of 100% CO2 gas was dissolved in the electrolyte and supplied to the cathode-electrode surfaces uniformly with the help of well-designed flow channels. The electrochemically synthesized formic acid was dissolved in the electrolyte and ejected from the electrolyzer. Insertion of nanoporous separator between the facing anode/cathode electrodes prevented O2 bubbles produced on the anodes from reaching the cathodes, suppressing the crossover reaction of O2 reduction. We developed a novel Ru complex polymer (RuCP)-based cathode catalyst loaded on carbon supports and an IrO x -based anode catalyst, and achieved a Faradaic efficiency as high as 96% for the formic acid synthesis at an extremely low operating voltage of 1.65 V (overpotential of only 0.22 V) and a large current of 65 A, resulting in a high solar-to-chemical energy conversion efficiency of 10.5%. This significant performance was realized by the low-resistive Ti substrates of the cathode and anode electrodes, sufficient CO2 supply to whole of the cathode surfaces, etc., which have greater impacts on a larger electrolyzer, in addition to the high catalytic activity of the RuCP and IrO x . Further, we tackled to improve both activity and durability of the anode and cathode electrodes,4,5 and to realize direct reduction of CO2 in a flue gas to eliminate energy-consuming CO2 capture processes.6 Moreover, we designed tandem solar modules suitable for direct coupling with the electrolyzers using organic-inorganic hybrid perovskite solar cells.7 The next challenge was isolation of the synthesized formic acid dissolved in the aqueous electrolyte. The formic acid cannot be isolated by distillation because its boiling point (101 °C) is virtually the same as that of water. Thus, we developed an isolation process on the basis of reactive extraction, including three sequential steps.1 The first step was reactive extraction of the formic acid from the electrolyte using an extraction solution composed of an organic base (tri-n-octylamine, TOA, boiling point 366 °C) as an extractant and an organic solvent (dichloromethane, DCM, boiling point 40 °C) as a diluent. Although formic acid is highly miscible in water, it was extracted into DCM because the formic acid formed a complex salt with TOA that is insoluble in water. The second step was removal of DCM from the mixture of the extracted formic acid, TOA, and DCM produced in the first step, by evaporation at 40 °C at 0.03 MPa. Finally, the residue was heated at 160 °C at 0.02 MPa for isolation of the formic acid by distillation in the third step. The large differences in the boiling points among these three chemicals secured few contaminations in the second and third steps. In addition, the composition of the electrolyte was tuned for promotion of the formation of the formic acid-TOA complex salt. Thus, we achieved an over 90% isolation yield of pure formic acid. The new electrolyte was confirmed to lower the energy conversion efficiency by only 10%(relative) and to secure similarly high durability compared with the original electrolyte. The great feature of the isolation process is that all the electrolyte, TOA, and DCM are reused for the next synthesis and isolation after the formic acid is isolated. Thus, we established a highly sustainable artificial-photosynthetic system that consumes no chemicals other than the raw materials of CO2 and water, or generates no waste.References M. Shiozawa,et al., Energy & Fuels, submitted.N. Kato, et al., Joule, (2021) 5, 687.N. Kato, et al., ACS Sustain. Chem. Eng.,(2021) 9, 16031.M. Shiozawa, et al., Electrocatalysis, (2022)13, 830.N. Kato, et al., submitted to 245th ECS Meeting , Symposium I04: Electrosynthesis of Fuels 8 (2024).Y. Takeda, et al., J. CO2 Util., (2023)71, 102472.Y. Takeda, et al., J. Appl. Phys., (2022)132, 075002. Figure 1

Modern chromatographic method for estimating Loratadine and affections on healthcare

PurposeThe purpose of this study is to establish Loratadine [LRD] quantification in purified and capsule formulations using a precise and specific Reversal Phase with a very high-performance liquid Chromatographic [RP-HPLC] technique. The approach was evaluated in agreement with the principles of the International Conference on Harmonization [ICH]. Arcus EP-C18 Ion Pac column, 5 m, 4.6 mm, 250 mm, mobile phase Methanol: Acetonitrile (60:40) v/v. Dibasic potassium phosphate buffer, pH 7.2, flow rate 1.0 ml/min.Design/methodology/approachThe HPLC system used a 340 nm UV detector for testing. A 10-min run time was used for the analysis. At concentrations ranging from 2 to 10 g/ml, the technique was linear (R2 = 0.9998), exact (intra-day and inter-day relative standard deviation [RSD] values 1.0%), accurate (range recovery = 96%–102%), exclusive and strong.FindingsThe detecting and quantitation limits were 0.92 g/ml and 2.15 g/ml, respectively.Originality/valueThe findings demonstrated that the proposed method could accurately determine LRD in bulk and pill dose formats quickly and accurately.

Ionic liquid–based aqueous biphasic system as an alternative tool for enhanced bacterial DNA extraction

BackgroundDeveloping an alternative and benign method for DNA extraction is imperative due to the high cost and potential harms associated with conventional techniques. Investigation of Ionic liquid (IL) as a solvent for DNA storage and stability revealed the ability of IL to assist DNA processes. IL–based aqueous biphasic system emerges as a comprehensive extraction platform capitalizing on the task–specificity of ILs and the wide applicability of ABS for biomolecule extractions. Therefore, it is beneficial to optimize an IL–based ABS specifically for DNA extraction, taking into account the fundamental interactions between the IL and DNA. ResultsThe primary objective was to design ABS consisting of Ammonium based ILs, and Potassium phosphate buffer as the salting–out agent for the partitioning of salmon sperm DNA. The analysis focused on optimizing biocompatible anions for the extraction. Moreover, the stability of the DNA in the IL rich phases was analysed to validate the method. The proposed process was then employed for extracting plasmid DNA from bacteria, demonstrating results comparable to those obtained with a commercially available kit. Further validation using agarose gel electrophoresis and transformation of the extracted DNA into E.coli were conducted, producing promising outcomes. Although there is room for improvement in terms of recovery of DNA and reusability of ABS, the described approach is comparable with the conventional one while being cost–effective, and showcases a noticeable and convincing link to eco–friendly processes. SignificanceThere is limited literature on IL–based ABS for DNA extraction, and the existing studies predominantly concentrate on systems derived from Cholinium ILs. However, their high hydrophilicity limits the choice of the second–phase forming component to polymers for the formation of ABS. Ammonium ILs efficiently form biphasic systems with various available salting–out agents, and biocompatible anions are introduced to mitigate the toxicity of the ILs.

3D‐Printed and Recombinant Spider Silk Particle Reinforced Collagen Composite Scaffolds for Soft Tissue Engineering

AbstractCollagen is one main component of the extracellular matrix (ECM) in natural tissues and is, therefore, well suited as a biomaterial for tissue engineering. In this study, a method is presented to 3D‐bioprint collagen into a precipitation bath comprising recombinantly produced spider silk protein eADF4(C16) yielding a composite with excellent mechanical properties. The spider silk precipitation bath induced assembly of the collagen into fibrils, and subsequent addition of potassium phosphate buffer lead to the formation of silk particles and stabilization of the collagen fibrils. The produced collagen‐silk composite scaffolds show an internal structure of homogeneously distributed and interacting collagen fibrils and spider silk particles with significantly better mechanical properties compared to plain collagen scaffolds. Further, enzymatic degradation assays of the scaffolds over a 7‐day period show higher stability of the collagen‐silk scaffolds compared to plain collagen scaffolds in the presence of wound proteases. Using the spider silk variant eADF4(C16‐RGD) further increases compressive stress and elastic modulus compared to that of the unmodified variant. Finally, it is shown that the unique collagen‐spider silk composite scaffolds comprising the cell‐binding domains of collagen and the RGD sequence in the spider silk variant represent a promising material for soft tissue regeneration.

Development and validation of a HPLC method for the determination of chemical and radiochemical purity of O-(2-[18F]fluoroethyl-l-tyrosine ([18F]FET)), a PET radiotracer for the imaging of brain tumor

A novel HPLC method was developed and validated to determine radiochemical identity, radiochemical purity and chemical purity for the analysis of O-(2-[18F]fluoroethyl-l-tyrosine ([18F]FET). In this method, an analytical Phenomenex Gemini C18 column was used with an isocratic eluent of 7 % ethanol and 93 % 50 mM potassium phosphate buffer (pH = 6.9). The flow rate was 1.0 mL/min and the injection volume was 10 μL. A photo-diode array detector set at 220 nm was used for UV mass detection and a single channel, high sensitivity radiation detector was used. The method validation assays including specificity, linearity, precision, accuracy, and robustness were evaluated. Results show that the method was suitable for qualitative and quantitative determination of radiochemical and chemical purity of [18F]FET. This system has been routinely used for the analysis of more than 120 batches of [18F]FET with radiochemical yield 23.7 ± 6 % (no decay corrected) and molar activity 593 ± 284 GBq/μmole in our facility to support human use.

Screening of seeds from wild Pennycress (Thlaspi arvense L.) populations for sinigrin levels using a blood glucose meter and test strips

Field pennycress, Thlaspi arvense L., is a new oil seed cover crop being commercialized as a source of biofuel in the United States. Along the path to commercialization, pennycress seed and meal will be marketed as animal feed. As a member of the Brassicacaeae family, pennycress seed is abundant in glucosinolates which are anti-nutritional factors that make the seed and meal less palatable to animals. More specifically, sinigrin has been identified as the only glucosinolate present in pennycress seed. As work continues to develop varieties low in glucosinolates, a rapid assay is needed to determine the sinigrin level in pennycress seed.The objective of this study was to develop and utilize a high throughput, rapid assay to screen pennycress seeds from wild populations for total sinigrin content. Pennycress seeds were ground in potassium phosphate buffer with endogenous myrosinase to catalyze the enzymatic hydrolysis of sinigrin to equimolar amounts of glucose. Glucose content was determined using a blood glucose meter and test strips. From the molar concentration of glucose, the molar concentration of sinigrin was known and the sinigrin level in seeds was calculated in µmol/g. The results were confirmed by HPLC/MS analysis.A rapid and inexpensive method was developed and used to screen seeds of 429 wild pennycress lines for sinigrin. Sinigrin levels in seed ranged from 25 to 85 μmol/g in the pennycress lines screened. Environmental factors also seemed to contribute to the levels of sinigrin in seeds found within the line ‘tt8-t/ARV1’ grown at ten different locations in the Midwest, USA.This rapid and inexpensive assay will allow breeders to efficiently screen lines and select for varieties that produce seeds with low sinigrin levels for improved feed value.

Image Sensing of Gaseous Acetone Using Secondary Alcohol Dehydrogenase-Immobilized Mesh for Exhaled Air.

Human-borne acetone is a potent marker of lipid metabolism. Here, an enzyme immobilization method for secondary alcohol dehydrogenase (S-ADH), which is suitable for highly sensitive and selective biosensing of acetone, was developed, and then its applicability was demonstrated for spatiotemporal imaging of concentration distribution. After various investigations, S-ADH-immobilized meshes could be prepared with less than 5% variation by cross-linking S-ADH with glutaraldehyde on a cotton mesh at 40 °C for 15 min. Furthermore, high activity was obtained by adjusting the concentration of the coenzyme nicotinamide adenine dinucleotide (NADH) solution added to the S-ADH-immobilized mesh to 500 μM and the solvent to a potassium phosphate buffer solution at pH 6.5. The gas imaging system using the S-ADH-immobilized mesh was able to image the decrease in NADH fluorescence (ex 340 nm, fl 490 nm) caused by the catalytic reaction of S-ADH and the acetone distribution in the concentration range of 0.1-10 ppm-v, including the breath concentration of healthy people at rest. The exhaled breath of two healthy subjects at 6 h of fasting was quantified as 377 and 673 ppb-v, which were consistent with the values quantified by gas chromatography-mass spectrometry.

Influence of Critical Parameters on the Extraction of Concentrated C-PE from Thermotolerant Cyanobacteria.

This work aimed to identify the influence of pH, molarity, w/v fraction, extraction time, agitation, and either a sodium (Na2HPO4·7H2O-NaH2PO4·H2O) or potassium buffer (K2HPO4-KH2PO4) used in the extraction of C-phycoerythrin (C-PE) from a thermotolerant strain of Potamosiphon sp. An experimental design (Minimum Run Resolution V Factorial Design) and a Central Composite Design (CCD) were used. According to the statistical results of the first design, the K-PO4 buffer, pH, molarity, and w/v fraction are vital factors that enhance the extractability of C-PE. The construction of a CCD design of the experiments suggests that the potassium phosphate buffer at pH 5.8, longer extraction times (50 min), and minimal extraction speed (1000 rpm) are ideal for maximizing C-PE concentration, while purity is unaffected by the design conditions. This optimization improves extraction yields and maintains the desired bright purple color of the phycobiliprotein.

Development and Validation of a Simple High-Pressure Liquid Chromatography-Ultraviolet Detection Method for Simultaneous Quantitation of First-Line Anti-Tuberculosis Drugs in Formulations of Fixed-Dose Combination.

The current treatment protocol for drug-sensitive tuberculosis involves all four first-line anti-tuberculosis drugs: rifampicin, isoniazid, pyrazinamide and ethambutol hydrochloride in a single tablet, known as fixed-dose combination tablets. However, the analytical methods are scanty to test all these drugs simultaneously in a single run without any pre-sample process or using a simple method suitable for resource-limited settings. In this method, 50mM potassium phosphate buffer containing 0.2% triethylamine (without pH adjustment) added with acetonitrile (98:2, v/v) was served as mobile phase A, while mobile phase B was 100% acetonitrile. All four drugs were separated within 10.3min using a gradient mobile phase program in a C18 column (150mm × 4.6mm; 5μm) and detected at two ultraviolet wavelengths (238nm for rifampicin, isoniazid and pyrazinamide, and 210nm for ethambutol hydrochloride). The method was selective, sensitive and linear with a correlation coefficient >0.999 with the acceptable precision and accuracy (<2% relative standard deviation) for all four drugs. In conclusion, the method is simple and it does not require any pH adjustment of the buffer/mobile phase, and within 11min, the separation of all four drugs can be achieved. Overall, the method is suitable for quality testing of fixed-dose combination tablets in limited-resource settings.

Formulation development of a stable influenza recombinant neuraminidase vaccine candidate

ABSTRACT Current influenza vaccines could be augmented by including recombinant neuraminidase (rNA) protein antigen to broaden protective immunity and improve efficacy. Toward this goal, we investigated formulation conditions to optimize rNA physicochemical stability. When rNA in sodium phosphate saline buffer (NaPBS) was frozen and thawed (F/T), the tetrameric structure transitioned from a “closed” to an “open” conformation, negatively impacting functional activity. Hydrogen deuterium exchange experiments identified differences in anchorage binding sites at the base of the open tetramer, offering a structural mechanistic explanation for the change in conformation and decreased functional activity. Change to the open configuration was triggered by the combined stresses of acidic pH and F/T. The desired closed conformation was preserved in a potassium phosphate buffer (KP), minimizing pH drop upon freezing and including 10% sucrose to control F/T stress. Stability was further evaluated in thermal stress studies where changes in conformation were readily detected by ELISA and size exclusion chromatography (SEC). Both tests were suitable indicators of stability and antigenicity and considered potential critical quality attributes (pCQAs). To understand longer-term stability, the pCQA profiles from thermally stressed rNA at 6 months were modeled to predict stability of at least 24-months at 5°C storage. In summary, a desired rNA closed tetramer was maintained by formulation selection and monitoring of pCQAs to produce a stable rNA vaccine candidate. The study highlights the importance of understanding and controlling vaccine protein structural and functional integrity.

Agrobacterium Transformation of Tea Plants (Camellia sinensis (L.) KUNTZE): A Small Experiment with Great Prospects.

Tea has historically been one of the most popular beverages, and it is currently an economically significant crop cultivated in over 50 countries. The Northwestern Caucasus is one of the northernmost regions for industrial tea cultivation worldwide. The domestication of the tea plant in this region took approximately 150 years, during which plantations spreading from the Ozurgeti region in northern Georgia to the southern city of Maykop in Russia. Consequently, tea plantations in the Northern Caucasus can serve as a source of unique genotypes with exceptional cold tolerance. Tea plants are known to be recalcitrant to Agrobacterium-mediated transfection. Research into optimal transfection and regeneration methodologies, as well as the identification of tea varieties with enhanced transformation efficiency, is an advanced strategy for improving tea plant culture. The aim of this study was to search for the optimal Agrobacterium tumefaciens-mediated transfection protocol for the Kolkhida tea variety. As a result of optimizing the transfection medium with potassium phosphate buffer at the stages of pre-inoculation, inoculation and co-cultivation, the restoration of normal morphology and improvement in the attachment of Agrobacterium cells to the surface of tea explants were observed by scanning electron microscopy. And an effective method of high-efficiency Agrobacteria tumefaciens-mediated transfection of the best local tea cultivar, Kolkhida, was demonstrated for the first time.

Development of Direct Electron Transfer-Type Extended Gate Field Effect Transistor Enzymatic Sensors for Metabolite Detection.

In this work, direct electron transfer (DET)-type extended gate field effect transistor (EGFET) enzymatic sensors were developed by employing DET-type or quasi-DET-type enzymes to detect glucose or lactate in both 100 mM potassium phosphate buffer and artificial sweat. The system employed either a DET-type glucose dehydrogenase or a quasi-DET-type lactate oxidase, the latter of which was a mutant enzyme with suppressed oxidase activity and modified with amine-reactive phenazine ethosulfate. These enzymes were immobilized on the extended gate electrodes. Changes in the measured transistor drain current (ID) resulting from changes to the working electrode junction potential (φ) were observed as glucose and lactate concentrations were varied. Calibration curves were generated for both absolute measured ID and ΔID (normalized to a blank solution containing no substrate) to account for variations in enzyme immobilization and conjugation to the mediator and variations in reference electrode potential. This work resulted in a limit of detection of 53.9 μM (based on ID) for glucose and 2.12 mM (based on ID) for lactate, respectively. The DET-type and Quasi-DET-type EGFET enzymatic sensor was then modeled using the case of the lactate sensor as an equivalent circuit to validate the principle of sensor operation being driven through OCP changes caused by the substrate-enzyme interaction. The model showed slight deviation from collected empirical data with 7.3% error for the slope and 8.6% error for the y-intercept.

Modified method for obtaining a concentrate with a high total content of phycocyanins from A. platensis

The work proposes a modified method for obtaining phycocyanin concentrate including extraction of biomass of the food cyanobacterium Arthrospira platensis and subsequent stages of membrane processing, involving combined use of double microfiltration and single- stage ultrafiltration. Extraction of A. platensis biomass was carried out at a temperature of +40°C for 3 hours in potassium phosphate buffer, and the purity of the extract was 0.61. Double microfiltration of the extract and ultrafiltration increased the purity of the intermediate product to 2.7. The content of C-phycocyanin and allophycocyanin in the dry concentrate was (63.6±1.0)% and (4.3±0.4)%, respectively. The molecular weight distribution of protein and peptide fractions of intermediate and final products was characterized using high-pressure size exclusion liquid chromatography.