Potassium Phosphate Buffer Research Articles

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.

Chitosan-PVA-PVP/nano-clay composite: a promising tool for controlled drug delivery.

In this study, chitosan, polyvinyl alcohol (PVA), and polyvinyl pyrrolidone (PVP) were used to create ternary blends reinforced with organically modified montmorillonite nanoclay. Tramadol was used as a model drug to assess the efficacy of these ternary blends as drug delivery systems. The current work demonstrated the highly controlled release of tramadol via transdermal administration. The results of the FTIR investigation revealed the compatibility of the blending components. Among non-drug-loaded formulations, MC6 is the most stable with a 17.6% weight residue at 505 °C and MC11 is the most stable of all the drug-loaded and non-drug-loaded formulations with a weight residue of 22.0% at 505 °C. The XRD studies of the prepared formulations showed crystalline behavior. However, the SEM analysis revealed that no gaps or mixing components were uniformly dispersed in the nanocomposites. Pharmaceutical tests, such as swelling, dissolution, and permeation rates, revealed a strong influence of the PVA concentration. There was a uniform distribution of drug throughout the films with maximum encapsulation efficiency found for MC7 (96.09 ± 0.31) and minimum encapsulation efficiency for MC11 (90.56 ± 0.34)%. Compared to the sodium acetate (pH 4.5) and potassium phosphate buffers (pH 6.8) the swelling and erosion were higher in hydrochloric acid buffer (pH 1.2). An increase in PVA concentration (or decrease in PVP concentration) increases the swelling, dissolution, and permeation rates. In addition, erosion increased with increasing PVP concentration. Furthermore, the nanoclay-reinforced composite showed high permeation. Based on the obtained results, it can be concluded that the produced nanocomposite could be used as an efficient transdermal drug delivery system.

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.

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.

Studying the ability of the food additive E171 (titanium dioxide) to induce gene mutations in bacteria

Introduction. Titanium dioxide in the Russian Federation is approved for use in the food industry, in the production of medicines and hygiene products. The food additive E171 is a mixture of micro- and nanoparticles of TiO2. In 2010, IARC classified TiO2 in nanoform as a probably carcinogenic to humans (Group 2B). In vitro and in vivo studies of the genotoxicity of titanium dioxide revealed contradictory results, indicating both the presence and absence of TiO2 mutagenicity. 
 The aim of the work is to evaluate the mutagenicity of the food additive E171 in the Ames test using standard and modified protocols.
 Materials and methods. The ability of food additive E171 (China) to induce reverse gene mutations in 5 strains of Salmonella typhimurium was studied under standard and modified conditions (cultivation of bacteria in the presence of methylated b-cyclodextrin (MCD) and/or pre-incubation for 1 hour in potassium phosphate buffer, pH 5.5 containing 10 mM NaCl and/or 3M MCD).
 Results. A sample of food additive E171 based on rutile titanium dioxide does not induce gene mutations in S. typhimurium in standard experiments. Modification of the Ames test protocol (decrease of the incubation mixture pH, addition of 10 mM NaCl) revealed statistically significant dose-dependent effects in TA100, TA98, and TA97 strains under metabolic incubation conditions. However, the fold increase of the number of revertants in the experimental plates compared to the negative control was < 2.
 Limitations. The research is limited to the mutagenicity assessment of food additive E171 (titanium dioxide) in the Ames test.
 Conclusion. The evaluation of the mutagenicity of titanium dioxide in other in vitro and in vivo tests taking into account the size and shape of the particles, is necessary to resolve the issue of its genetic safety as a food dye. A full range of studies will be performed on other samples of titanium dioxide presented in the market of the Russian Federation.

Toward effect of buffer system composition in dehydrogenase activity of Escherichia coli and its electrochemical activity in mediated bioanode

The impact of composition, concentration, and pH of buffer solution on the activity and viability of Escherichia coli BB cells has been studied. Potassium/sodium phosphates with different composition and pH and TRIS buffer solutions used for microbiological and biotechnological applications as well as in microbial bioanodes were compared. The optimal buffer solution to achieve the highest dehydrogenase activity was 50 мМ potassium phosphate buffer solution, KH2PO4, isotonized by sodium chloride either with pH 7.4 for freshly obtained E. coli cells or with pH 7.8 to maintain the activity and viability of the microorganisms while storing. The cells concentration in solution was important to achieve the highest dehydrogenase activity, viz. 0.6–0.8 mg of cells (in terms of the dry weight) per mL of buffer solution with 0.2 M dextrose (substrate) was optimal. The good correlation between dehydrogenase activity and electrochemical current responses of bioanodes with E. coli and methyl orange as a redox mediator was shown.

Paired Electrosynthesis 2,5-Furandicarboxylic Acid (FDCA) and 2-Butanone in a Flow-Cell

Biobased platform chemicals and energy carriers can replace conventional, fossil-based chemicals and fuels to decrease the footprint of the chemical industry in the future. Promising chemicals for this purpose are 2,5-furandicarboxylic acid (FDCA), a monomer for biobased polymers, and 2-butanone, a solvent and energy carrier. Both molecules can be synthesized in electrochemical processes: FDCA is a product of the oxidation of Hydroxymethylfurfural (HMF) and 2-butanone is a product of the reduction of acetoin. In an electrochemical reactor, the oxidation and the reduction reaction can be coupled, so that the anode and the cathode yield a value-added product. The increased efficiency is accompanied by an increased complexity. A crucial aspect is the migration of ions between anolyte and catholyte, which impacts the pH in both electrolyte compartments thereby influences the reactions. The direction of this migration is determined by the type of ion exchange membrane, the effect of which is much less pronounced when analyzing a single reaction.This work develops a coupled process for these reactions. The individual reactions have been demonstrated prior [1,2]. The oxidation takes place on an Ni-foam electrode with a Ni(OH)2/NiOOH catalyst in 0.1 mol/L KOH in a zero-gap assembly. The reduction is carried out on a lead plate electrode in a potassium phosphate buffer at pH 9 with an electrolyte gap of 3 mm. We analyze the impact of the three different ion exchange membranes on the migration of ions and the pH in the electrolyte compartments, using anion exchange membranes (AEM), cation exchange membranes (CEM), and bipolar membranes (BPM) in a flow-cell reactor (flex-E-cell). The electrode area was 25cm². No stable operation was possible with a CEM due to the acidic pH at the interface of the CEM and the anode, which deteriorated the nickel-based catalyst. With an AEM, a stable process was made possible through pH control in the electrolytes to counteract ion migration across the membrane. With a BPM, we showed the most stable process with little to no ion permeation. However, the cell-voltage was highest for the BPM.At an increased current density to 120 mA/cm2 and a reactant concentration to 0.45 mol/L of HMF and 0.9 mol/L acetoin, respectively, we were able to maintain a high yield, for FDCA on the anode side above 90 % and for 2-butanone above 60 % on the cathode side with a BPM. The combined faraday efficiency (FE) was above 150 %. The increased product concentration of FDCA required the stabilization of the alkaline pH through the redosing of base. With an AEM however, no stable process was possible at the elevated current densities due to the extensive crossover of anions, which resulted in drastically changing pH and conductivity. To increase the economic prospect of the process, we employed a background electrolyte on the cathode side (0.5 M Na2SO4) to decrease the cell voltage by 15 % and employed NaOH instead of KOH on the anode side without significantly affecting the reaction metrices. The elevated current densities in combination with the economically interesting electrolyte system moves the process towards economic competitiveness, which we will assess in the future. With this work, we hope to establish a paired electrolysis process for the efficient and economic electrochemical valorization of biomass.

Design of an histidine-based imprinted polymer as a selective receptor of copper(II) for its detection in seawater

A protein-inspired ion-imprinted polymer (IIP) was used as an additive in a carbon paste electrode for the selective determination of copper(II) in water samples. The imprinted polymer was synthesized by precipitation polymerization of a functional monomer, methacrylamido-L-histidine (MAH), based on an amino acid, L-histidine, with ethylene glycol dimethacrylate (EGDMA) as crosslinker. The corresponding non-imprinted polymer (NIP) and a copolymer containing EGDMA and methyl methacrylate (MMA) (in the same proportions as MAH in the NIP) were also prepared in similar conditions as the IIP. The polymer particles were first chemically characterized by 13C CP-MAS NMR and FT-IR and the presence of MAH was confirmed by the colorimetric Pauly's test. It was shown by SEM and adsorption/desorption of nitrogen that the IIP and the NIP present similar morphological properties. The copper(II) binding properties of the polymers were also studied in a batch adsorption experiment, and an imprinting effect was observed for the IIP. This polymer was shown to possess binding cavities selective towards copper(II) versus common interferents (iron(III), zinc(II) and lead(II)), when compared to the NIP and to the poly(EGDMA-co-MMA). A carbon paste working electrode was prepared with the IIP particles and a limit of detection of 59 nmol/L for copper(II) was determined in a potassium phosphate buffer (0.1 M, pH = 7). The determination of copper(II) was not affected by the presence of a 50-fold excess of iron(III), zinc(II) and lead(II). Finally, the copper concentration in a real seawater was estimated to be 0.12 μmol/L, in full agreement with a measurement by polarography which was used as a reference method.

Structural effects induced by dialysis-based purification of carbon nanomaterials

Dialysis plays a crucial role in the purification of nanomaterials but its impact on the structural properties of carbon nanomaterials was never investigated. Herein, a carbon-based nanomaterial generated electrochemically in potassium phosphate buffer, was characterized before and after dialysis against pure water. It is shown that dialysis affects the size of the carbon domains, structural organization, surface functionalization, oxidation degree of carbon, and grade of amorphicity. Accordingly, dialysis drives the nanomaterial organization from discrete roundish carbon domains, with sizes ranging from 70 to 160 nm, towards linear stacking structures of small nanoparticles (<15 ​nm). In parallel, alcohol and ether (epoxide) surface groups evolve into more oxidized carbon groups (e.g., ketone and ester groups). Investigation of the as-prepared nanomaterial by electron paramagnetic resonance (EPR) revealed a resonance signal consistent with carbon-oxygen centred radicals.Additionally, this study brings to light the selective affinity of the carbon nanomaterial under study to capture Na+ ions, a property greatly enhanced by the dialysis process, and its high ability to trap oxygen, particularly before dialysis. These findings open new perspectives for the application of carbon-based nanomaterials and raise awareness of the importance of structural changes that can occur during the purification of carbon-based nanomaterials.

Eco-friendly liquid chromatography method for the quantification of ibrutinib in a pharmaceutical dosage form.

The objective of this study was to quantitatively determine Bruton's tyrosine kinase inhibitor ibrutinib in its capsule dosage form and assess the homogeneity of the dosage form using green chromatography. The chromatographic method using gradient elution mode was optimized and validated in accordance with the International Council for Harmonization guidelines. The analysis was conducted on a Zodiac C18 column (75 × 4.6 mm and 3.5 μm) using a mobile phase consisting of pH 5.5 potassium phosphate buffer (mobile phase A) and 90% ethanol in milli-Q water (mobile phase B), with a flow rate set at 0.6 mL/min. Based on the validation data, the accuracy results fell within the range of 99.1%-100.6%. The relative standard deviation (% RSD) from precision for both the assays and the uniformity of dosage by content uniformity were determined to be 0.82 and 1.16, respectively. The correlation coefficient obtained from the linearity experiment was 0.999, indicating a strong linear relationship. The greenness of the developed method was assessed using various tools, including the National Environmental Methods Index (NEMI) pictogram, Modified NEMI, Analytical Eco-score calculation, Green Analytical Procedure Index (GAPI) pictogram, Analytical GREEnness (AGREE), and AGREE preparation (AGREEprep). The obtained greenness profile suggests that the optimized LC method is an excellent greener method, supported by the analytical eco-score of 86.

Simple HPLC-UV Method for Therapeutic Drug Monitoring of 12 Antiepileptic Drugs and Their Main Metabolites in Human Plasma

The objective of the present report was to develop and validate a simple, selective, and reproducible high-performance liquid chromatography method with UV detection suitable for routine therapeutic drug monitoring of the most commonly used antiepileptic drugs and some of their metabolites. Simple precipitation of plasma proteins with acetonitrile was used for sample preparation. 10,11-dihydrocarbamazepine was used as an internal standard. Chromatographic separation of the analytes was achieved by gradient elution on a Phenyl–Hexyl column at 40 °C, using methanol and potassium phosphate buffer (25 mM; pH 5.1) as a mobile phase. The method was validated according to the FDA guidelines for bioanalytical method validation. It showed to be selective, accurate, precise, and linear over the concentration ranges of 1–50 mg/L for phenobarbital, phenytoin, levetiracetam, rufinamide, zonisamide, and lacosamide; 0.5–50 mg/L for lamotrigine, primidone, carbamazepine and 10-monohydroxycarbazepine; 0.2–10 mg/L for carbamazepine metabolites: 10,11-trans-dihydroxy-10,11-dihydrocarbamazepine and carbamazepine-10,11-epoxide; 0.1–10 mg/L for oxcarbazepine; 2–100 mg/L for felbamate and 3–150 mg/L for ethosuximide. The suitability of the validated method for routine therapeutic drug monitoring was confirmed by quantification of the analytes in plasma samples from patients with epilepsy on combination antiepileptic therapy.

The effects of vitamins C and E on the redox parameters of cytochrome P450 3A4

Abstract The research on the influence of antioxidant vitamins C and E on the catalytic activity of cytochrome P450 3A4 in electrochemical experiments is relevant, as they are widely used as additional biologically active substances in human diets and can affect biochemical processes in the body. Understanding these effects is of significant importance for pharmacology and medicine. The purpose of this study is to investigate how antioxidant vitamins C and E impact the catalytic activity of cytochrome P450 3A4 within the framework of electrochemical experiments. The voltammetry was performed in the presence of potassium phosphate buffer of neutral pH value (0.2 M) and 0.5 M erythromycin solution (Er). The ratio between the measures of current obtained in the presence of O2 and Er was 2.10 ± 0.09 (100 %, P &lt; 0.05). With vitamin C and Er, it corresponded to 2.80 ± 0.12 (133 %, P &lt; 0.05). The voltammograms of Cyt with Dic show a higher current stimulated by ascorbic acid. The effect of vitamin C on the H-dimethylation process of Er was investigated with a spectrophotometer (value 408 nm). The catalytic current of cytochrome P450 3A4 in the presence of diclofenac was stimulated by ascorbic acid, the effect of which on the N-demethylation of erythromycin was studied spectrophotometrically at 408 nm based on the formation of formaldehyde using the acetylacetone reagent. The obtained results suggest the potential impact of antioxidant vitamins not only on the stimulation of electrochemical reduction of cytochrome P450 3A4 but also on the clinical metabolism of substrates in patients. These findings provide valuable insights for further clinical investigations aimed at predicting the risk of drug interactions with antioxidants.

Simultaneous determination of praziquantel, fipronil, eprinomectin, (S)-methoprene, their key related substances and butylated hydroxytoluene (BHT) in a topical veterinary drug product by a single stability indicating high performance liquid chromatography method

A simple, robust and QC (quality control) friendly high performance liquid chromatography (HPLC) method was developed and validated for simultaneous determination of four active pharmaceutical ingredient [namely fipronil, (S)-methoprene, eprinomectin, and praziquantel] and their key degradation products in a broad-spectrum topical finished product. Typical sample of the finished product contains a total of 30-plus peaks of interest. Analytes were separated on a HALO C18 column (150 mm × 4.6 mm i.d., 2.7 µm particle size) with a gradient elution at 50 °C column temperature and 0.6 mL/min flow rate. Detection wavelength of 245 nm is used for praziquantel, eprinomectin and their degradation products, 265 nm for (S)-methoprene and its degradation products and 280 nm for fipronil and its degradation products and for the antioxidant, BHT. Mobile phase of the method is composed of 10 mM potassium phosphate buffer and 1,4- Dioxane (98/2, v/v, pH 5.0) as mobile phase-A, and EtOH/MeOH/MeCN/IPA (86/4/6/4, v/v/v/v) as mobile phase-B. All analytes of interest were adequately separated by this single HPLC method. The stability-indicating capability of the method has been demonstrated by successfully separating the degradation products in the stressed degraded samples of the finished product. Limit of quantitation (LOQ) and limit of detection (LOD) of the method is 0.3% and 0.1% of target analytical concentration for each individual API in the finished product. This method has been demonstrated to be sensitive, robust, specific, accurate and stability-indicating for analysis of the topical drug product containing praziquantel, fipronil, eprinomectin and (S)-methoprene.

Development and validation of high-performance liquid chromatography method for determination of clarithromycin in pharmaceutical tablets.

Clarithromycin is a very important macrolide antibiotic used to treat bacterial infections in human and veterinary medicine. This study reports the development and validation of cost-effective, simple, precise, accurate, and robust high-performance liquid chromatography (HPLC) for the determination of clarithromycin (CLA) in tablets. Reversed-phase chromatography was conducted using a standard column at 55°C with ultraviolet detection at 215nm. A mobile phase consisting of acetonitrile -2-methyl-2-propanol -potassium phosphate buffer was used at a flow rate of 1.0mL/min. The proposed method displayed good linearity, precision, accuracy, robustness, and specificity. The present HPLC was compared with capillary electrophoresis and bioassay methods and the results indicated that there was no significant difference between these methods. Moreover, the obtained results demonstrated the validity of the isocratic HPLC, which allows reliable quantitation of CLA in pharmaceutical samples. Thus, it can be used as a substitute alternative methodology for the routine quality control of this medicine, in situations where other methods are less accessible in the laboratory.

Dilution induced spectral and structural changes in allophycocyanin

Allophycocyanin (APC) is a key molecule of the light harvesting complex - phycobilisome of Cyanobacteria and Red Algae. Its bright color and fluorescent nature make it a suitable molecular for biomedical applications. Here, we report the dilution induced changes in the spectral and structural properties of APC, isolated from marine cyanobacterium, Phormidium sp. A09DM. We observe measurable blue shift in the light absorption and fluorescence emission properties of APC upon dilution. The blue shift in spectral properties is originated from the disruption of hydrophobic interactions and systematic dissociation of the APC into its building blocks. APC, a trimer of αβ-heterodimer, dissociates into monomer and further into individual subunits upon dilution. The observed dilution induced spectral and structural changes can be moderated in the presence of physiological osmolytes with the moderation strength in the order, Sucrose > Trehalose > Betaine = Potassium Phosphate buffer > Glycine. Our results conclude that the spectral properties of APC is a function of osmolytes concentrations, which makes it a potential candidate to be explored as the molecular probe for measuring the molecular crowding.

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.

Immobilized Phosphoenolpyruvate Carboxylase Beads for Catalytic Carboxylation in Carbon Dioxide-Containing Gas–Liquid Flow

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the Mg2+-dependent carboxylation reaction in aqueous phase with a carbon source. Performing enzymatic reactions in a bioreactor with gas–liquid contacting is challenging because proteins potentially undergo conformational change at the gas–liquid interface. In the present work, immobilized phosphoenolpyruvate carboxylase polystyrene beads (PEPC-PSBs) were prepared and applied to catalyze the carboxylation of phosphoenolpyruvate (PEP) producing oxaloacetate (OAA) in an external loop airlift bubble column (ELBC). For a typical reaction, ELBC was charged with 10 mL of a 0.2 M potassium phosphate buffer solution (pH = 7.5) initially containing 3.1 mM PEP and 9.7 mM MgSO4. When ELBC was operated at 25 °C with 20 PEPC-PSBs ([PEPC] = 2.0 μg/mL) by continuously introducing CO2 gas bubbles at the superficial gas velocity of 1.0 cm/s, the conversion of PEP of 77% was obtained at the reaction time of 300 min. After the reaction, PEPC-PSBs could be recovered from the reaction mixture followed by washing for reuse. PEPC-PSBs were storable at 4 °C and physically robust enough for handling. The catalytic activity of PEPC-PSBs was observed for the total reaction time of 900–1080 min in the gas–liquid flow. On the other hand, no detectable amount of OAA was produced with free PEPC in ELBC operated under the comparable condition to the PEPC-PSB-catalyzed reaction. Adsorption of free PEPC to CO2 gas bubbles was indicated to cause a rapid decrease in the concentration of biologically active PEPC in ELBC. The polystyrene beads with rough surface were suggested to offer an environment for the immobilized enzyme to stably catalyze the prolonged carboxylation of PEP with continuously supplied CO2-containing gas bubbles.

Purification and Characterization of Glutathione Reductase Enzyme from Arum Maculatum Leaf

Arum species grow in temperate and Mediterranean climates and have been used for hundreds of years for food and medicinal purposes, although they are highly toxic if not cooked using proper techniques. Glutathione reductase (GR) is a member of the pyridine nucleotide disulfide oxidoreductase family of flavoenzymes that catalyzes the reduction of glutathione disulfide (GSSG) to reduced GSH using NADPH or NADH. In this study, GR enzyme was characterized by partial purification processes including homogenate preparation, ammonium sulfate precipitation and dialysis from the leaf of Arum maculatum plant. The highest enzyme activity was found at 40-60% saturation range. Optimum ionic strength, pH and substrate concentration were investigated for GR enzyme from A. maculatum leaf. As a result of the study, these values were found to be 150 mM potassium phosphate buffer, pH 7.00, and 0.18 mM, respectively. The GR enzyme was partially purified from the leaf of the A. maculatum with a specific activity of 1.640 EU mg-1 in 34.9% yield, 1.108-fold. This study is the first study in terms of purification and characterization of GR enzyme from A. maculatum leaf.

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.

Development of a peptide reactivity assay for screening botanicals and natural substances: Proof of concept studies

Consumer products containing botanicals or natural substances (BNS) are often preferred because there is a perception that ‘natural’ is safe. As with any product ingredient, a thorough safety assessment must be conducted, including a determination of skin sensitization potential. A modification of the Peroxidase Peptide Reactivity Assay (PPRA) was explored for screening BNS (B-PPRA) for their reactivity to a model cysteine peptide. The PPRA incorporates a horseradish peroxidase‑hydrogen peroxide (+HRP/P) oxidation system for the activation of potential pre- and pro-haptens. BNS test materials contained <2% botanical constituent in either glycerin/water or propylene glycol/water. Stock solutions prepared in acetonitrile were diluted to 8 working concentrations. Direct reactivity was determined in reaction mixtures containing peptide and deferoxamine in potassium phosphate buffer. Enzyme-mediated reactivity determinations were performed with addition of +HRP/P. Initial studies demonstrated that results were reproducible and impact of carrier low. To determine the sensitivity of the assay, experiments were conducted with chamomile extract spiked with three sensitizers. Peptide depletion was observed in the +HRP/P reaction mixtures with isoeugenol spikes as low as 0.05%. The B-PPRA shows promise as a screening method for skin sensitization potential and could become part of a framework for the skin sensitization safety assessment of BNS.