Statistical Optimization of Media Components for Xylanase Production by Aspergillus spp. Using Solid State Fermentation and its Application in Fruit Juice Clarification

Xylanases are enzymes that convert xylan into xylose, xylobiose, and xylotriose. The present study deals with the production and optimization of xylanase through Solid-State Fermentation (SSF) using different agricultural wastes by Aspergillus spp. The Plackett Burman (PB) design was used to screen significant media components affecting the xylanase production. The carbon sources screened were wheat bran, rice bran, sugarcane bagasse, corn cob, and orange peel. The nitrogen sources screened were yeast extract, peptone, (NH4)2SO4, Na2NO3, and urea. Also, nine different salts such as KCl, MgSO4, Na2HPO4, CaCl2, FeSO4, ZnSO4, Na2CO3, KH2PO4, and NaH2PO4 which act as trace elements were screened. The results showed that wheat bran, yeast extract, Na2NO3 and KCl are the significant factors that affect xylanase production. A 33 Full Factorial Design (FFD) was performed to optimize the significant media components (wheat bran, KCl, yeast extract) obtained from PB design using Response Surface Methodology (RSM). Statistical analysis of results showed that wheat bran, KCl, yeast extract, and interaction between wheat bran and yeast extract were found to be significant. The optimum concentration of wheat bran, KCl, yeast extract was 8 g/L, 0.1 g/L and 3 g/L. The Partial purification of xylanase was carried out using ammonium salt precipitation and dialysis. Gel filtration chromatography was performed to optimize the elution time, which was found to be 6 minutes. Application of xylanase in orange juice clarification was studied at 40 °C, 50 °C, and 60 °C. The optimum temperature obtained was 60 ºC.

Combined release of antiseptic and antibiotic drugs from visible light polymerized biodegradable nanocomposite hydrogels for periodontitis treatment

The in-situ used combination of different types of drugs revolutionized the area of periodontal therapy. The purpose of this study was to develop biodegradable nanocomposite hydrogel (NCHG) as a pH-sensitive drug delivery system. To reach the local applicability of the NCHG in dental practice routinely used blue-light photopolymerization was chosen for the preparation. The reaction time was 60 seconds, which resulted in stable hydrogel structures. Universal Britton-Robinson buffer solutions were used to investigate only the effect of pH for the release of the periodontal pocket useable drugs in the 4-12 range. The release of metronidazole was faster, it came out from the NCHGs within 12 hours, but the chlorhexidine had shown a longer elution time, which was more than 7 days as far as the antiseptic effect was maintained. The release of chlorhexidine showed strong pH depending. The biocompatibility of the NCHGs was proved by Alamar-blue test, and the effectiveness of drug release in the acidic medium was demonstrated. This fast photopolymerizable NCHG can help to approach an ideal locally useable combined drug delivery system which can be used in the amount required of the medicines and can reduce the side effects with the increased effectiveness by following pH changes.

Molecular Analytical Assessment of Thermally Precipitated α-Lactalbumin after Resolubilization

Selective thermal precipitation followed by a mechanical separation step is a well described method for fractionation of the main whey proteins, α-lactalbumin (α-la) and β-lactoglobulin (β-lg). By choosing appropriate environmental conditions the thermal precipitation of either α-la or β-lg can be induced. Whereas β-lg irreversibly aggregates, the precipitated α-la can be resolubilized by a subsequent adjustment of the solution’s pH and the ionic composition. This study reports on the analytical characterization of resolubilized α-la compared to its native counterpart as a reference in order to assess whether the resolubilized α-la can be considered close to ‘native’. Turbidity and quantification by RP-HPLC of the resolubilized α-la solutions were used as a measure of solubility in aqueous environment. RP-HPLC was also applied to determine the elution time as a measure for protein’s hydrophobicity. DSC measurement was performed to determine the denaturation peak temperature of resolubilized α-la. FTIR spectroscopy provided insights in the secondary structure. The refolding of α-la achieved best results using pH 8.0 and a 3-fold stoichiometric amount of Ca2+ per α-la molecule. The results showed that the mechanism of aggregation induced by gentle thermal treatment under acidic conditions with subsequent mechanical separation is reversible to a certain extent, however, the exact native conformation was not restored.

Quantitative determination of cyanoacetic acid content in teriflunomide drug substance by ion chromatography using conductivity detector

Background The current study focuses on the development and validation of an analytical method for quantifying cyanoacetic acid (CAA) in teriflunomide drug substance using a high-performance ion chromatography (IC) with cation suppressed conductivity detection (TFM). Water was used as the diluent for preparing the sample solution, which was injected into a standard chromatographic device with 250 mm, 4.0 mm ID, and 5.0 μm particle size Metrosep A Supp 5 Ion exchange column and a suppressed conductivity detector. At a flow rate of 0.6 mL min−1 and a temperature of 40 °C, the mobile phase was delivered in an isocratic mode. Results CAA and TFM had retention times of 12.78 and 15.82 min, respectively. CAA has a limit of detection (LOD) of 33 μg/g and a limit of quantification (LOQ) of 101 μg/g, respectively. For LOD and LOQ accuracy, the percentage RSD of CAA is 1.7 and 1.2, respectively. The average CAA recovery percentage was found to be between 98.6 and 100.1%. With a value of 0.9998, the calibration curve yielded an excellent linear correlation coefficient for CAA. According to the ICH guidelines, all verification parameters are within the range, indicating that the system is stable. Conclusion The elution time and run time in the currently developed ion chromatography analytical method have been reduced, demonstrating that the method is cost-effective and generally accepted, as well as simple and functional, and can be used in routine quality control tests in the industry.

Analytical Method Development and Validation for the Analysis of Nepafenac and its related substances using Ultra Performance Liquid Chromatography

A novel, economic and time-efficient reverse-phase ultra-performance liquid chromatographic (RP-UPLC) method has been developed for the analysis of Nepafenac in the presence of both impurities and degradation products generated by forced degradation. When Nepafenac was subjected to acid hydrolysis, oxidative, base hydrolysis, photolytic, and thermal stress, observed degradation only in oxidative and base hydrolysis. The drug was found to be stable to other stress conditions. Various method development trails were performed for the separation of drug from impurities. However, best chromatographic separation was achieved on a Waters Acquity CSHC18, 100mm x 2.1mm, 1.7µ particle size column, UV detection at 245nm, a gradient elution of Ammonium formate (pH 4.0), mixture of organic solvents (Acetonitrile, Methanol) as mobile phase for drug, its impurities and it was captured. The method was validated for specificity, precision, linearity, accuracy, robustness and can be used in quality control during manufacture and for assessment of the stability samples of Nepafenac. Total elution time was about 6.5 min and equilibration time of about 1.5 min which allowed analysis of more than 100 samples per day. The analytical method discussed in United states Pharmacopoeia was pH sensitive and compatible to LC-MS analysis.

Isocratic Reverse-Phase HPLC Method for Determination of Aspirin, Paracetamol, and Naproxen

Nonsteroidal anti-inflammatory drugs (NSAIDs), which block the activity of cyclooxygenase (COX) isoenzymes and inhibit the synthesis of prostaglandin, have been used for pain relief. We have developed a method to separate a mixture of three NSAIDs, such as aspirin, paracetamol, and naproxen, using reverse-phase high-performance liquid chromatography (RP-HPLC). An isocratic mobile phase consisting of acidic water and acetonitrile was selected to run at a low flow rate, such as 0.8 mL/min. The mixture of three NSAIDs was injected at a low volume into a C18 column that was 150 mm in length and characterized using a UV detector at 230 nm. We identified three peaks in the chromatogram indicating the three compounds. The elution time of the peaks was less than 10 minutes. To identify multiple peaks on the isocratic flow using a short column, further studies are required regarding the proposed method to generate microfluidic devices for nanoLC.

Measurement of Low Concentration of Micro-Plastics by Detection of Bioaffinity-Induced Particle Retention Using Surface Plasmon Resonance Biosensors

The issue of micro-plastics is becoming more and more important due to their ubiquity and the harm they cause to the human body. Therefore, evaluating the biological–physical interaction of micro-plastics with health cells has become the focus of many research efforts. This study focuses on the movement mode and low concentration detection development for micro-plastics in surface plasmon resonance (SPR). Firstly, 20-micrometer micro-plastics were prepared by grinding and filtering, and the movement mode was explored; then, the characteristics were investigated by SPR. Chromatographic analysis showed that the surface charge of micro-plastics dominated the elution time, and estrogen receptors (ERs) played a supporting role. A difference of micro-plastics in SPR sensorgram was observed, inferring the micro-plastics’ movement in rolling mode on the ERs. Characteristics analysis indicated that the low particle number of micro-plastics on SPR showed a linear relationship with the response unit (RU). When ERs were immobilized on the biosensor, the force of the binding of micro-plastics to ERs under an ultra-low background was equivalent to the dissociation rate constant shown as follows: PS (0.05 nM) > PVC (0.09 nM) > PE (0.14) nM). The ELISA-like magnetic beads experiment verified the specificity between ERs and micro-plastics. Therefore, by using the SPR technique, a biological-derived over-occupation of PS was found via higher binding force with ERs and longer retention time. In the future, there will be considerable potential for micro-plastics issues, such as identification in natural samples, biomarking, real-time detection in specific environments/regions and human health subject.

Analytical Method Development and Validation of Stability Indicating assay method of analysis for Dolutegravir/Lamivudine/Tenofovir Disoproxil Fumarate tablets using High Performance Liquid Chromatography

A novel, economic, simple, precise and time-efficient reverse-phase high performance liquid chromatographic (RPHPLC) method has been established for the simultaneous assay determination of Dolutegravir, Lamivudine and Tenofovir Disoproxil fumarate in tablet formulation. This research paper presents the detailed method development strategy and the outcome of validation challenges. The RPHPLC method was developed using a 150 x 4.6mm, 5µm C18 column, with a gradient mode using 0.1% (v/v) trifluoroacetic acid buffer and methanol, the detection was performed at 260nm. The method was validated for specificity, precision, linearity, accuracy, robustness and can be used in quality control during manufacture and for assessment of the stability samples of Dolutegravir/Lamivudine/Tenofovir Disoproxil fumarate tablets. Total elution time was about 5 min and equilibration time of about 2 min which allows analysis of more than 100 samples per day. The method reported in this study is compatible to mass spectrometry and is thus extremely useful for stability studies.

Multilinear Mathematical Separation in Chromatography

Chromatography is a powerful and generally applicable method for the analytical separation and quantification of the chemical constituents in complex mixtures because chromatographic separation can provide high selectivity by isolating all analytes from interferences. Multiway analysis based on the multilinear model is an increasingly widely used method for interference-free and fast determination of the chemical constituents also in complex mixtures because multilinear mathematical separation can provide high selectivity by extracting the pure signal of the analyte from the mixed signal of a real sample. By combining chromatographic separation with mathematical separation, multiway calibration method, multiway standard additions method, and multiway internal standard method can be established. Chromatography assisted by multiway analysis can reduce the requirements for complete chromatographic separation, save elution time, and decrease the consumption of the mobile phase, particularly when the peak coelution problem is difficult to solve. This review presents the fundamentals and analytical applications of multilinear mathematical separation in chromatography.

Robust optimization of gradient RP HPLC method for simultaneous determination of ivabradine and its eleven related substances by AQbD approach

This paper is aimed at developing a gradient elution reversed-phase high-performance liquid chromatography (RP-HPLC) method for the separation of a complex mixture composed of ivabradine and its eleven impurities, in a reasonable timeframe. In order to obtain a robust and reliable HPLC method for separation of this mixture, Analytical Quality by Design (AQbD) was applied. This approach demonstrated to be useful in development of a long lasting life cycle methods. Four chromatographic variables were defined as key method parameters (KMPs) and optimized towards the analytical target profile (ATP). Designated KMPs were initial and final amount of acetonitrile in the mobile phase, pH value of the aqueous phase and gradient time, while resolutions of critical peak pairs were denoted as critical method attributes (CMAs). Relationships between KMPs and CMAs were obtained with the aid of Design of Experiments (DoEs) methodology among which Box-Behnken design (BBD) was employed to gain valid mathematical models. Obtained mathematical equations were used to construct the Design Space (DS) and select reliable optimal separation conditions. They included 11% (v/v) and 34% (v/v) of initial and final amount of acetonitrile, respectively, as well as 45 min of gradient elution time and 20 mM ammonium acetate as aqueous mobile phase with pH set to 7.35. The possibility to separate the diastereoisomers of impurity X was also evaluated. It was demonstrated that this separation could not be achieved in gradient elution mode within the defined variable domains and in a reasonable time span. The developed method was validated according to ICH Q2 (R1) guideline and met all the required criteria.