Development and Validation of Salt Gradient CEX Chromatography Method for Charge Variants Separation and Quantitative Analysis of the IgG mAb-Cetuximab

Ion exchange chromatography is widely used for charge variant analysis of proteins, including monoclonal antibodies. In this study, a simple and robust salt gradient cation exchange chromatography was developed and validated for quantitative determination of cetuximab in biopharmaceutical formulations. For this purpose, we investigated the effect of various parameters including buffer composition, column temperature, pH, gradient volume and flow rate on chromatographic separation of charge variants to achieve the acceptable peak separation, and the optimum condition was selected. Validation of the method was done in accordance with the International Conference on Harmonization (ICH) guidelines. The developed method was found to provide a linear regression over the concentration range of 0.06–2.00 mg mL^?1 yielding a correlation coefficient of 0.9972. The limits of detection and quantification for the developed method were 0.02 and 0.06 mg mL^?1, respectively. The intra-day and inter-day precision had relative standard deviation values?≤?2.7%. The robustness of the method was assessed by changes in the applied pH range of buffer, temperature, mobile phase composition, and flow rate. Specificity of the method was confirmed by evaluation of baseline resolution of the mAb variants from product excipients, which showed no interference between excipients and cetuximab. The stability indicating capability of this method was determined using photodegraded, and mechanically and thermally stressed samples. The proposed method could be applied as a simple, precise, and robust quantitative technique which can be reproduced in any labs for the high-throughput quality control and stability assessment of in-process and final product samples.     

Experimental and quantum chemical study on the IR, UV and electrode potential of 6-(2,3-dihydro-1,3-dioxo-2-phenyl-1H-inden-2-yl)-2,3-dihydroxybenzaldehyde

Electrode potential of 6-(2,3-dihydro-1,3-dioxo-2-phenyl-1H-inden-2-yl)-2,3-dihydroxybenzaldehyde (DPDB) in methanol have been calculated theoretically. For the achievement of this task, the density functional theory (B3LYP/6-31G(d)) was employed with the inclusion of the entropic and thermochemical corrections to yield the free energies of the redox reactions. The electrode potential was also obtained experimentally by means of an electrochemical technique (cyclic voltammetry). The geometric parameters, the vibrational frequency values and the UV spectrum of DPDB and 2-(2,3-dihydro-1,3-dioxo-2-phenyl-1H-inden-2-yl)-5,6-dioxocyclohexa-1,3-dienecarbaldehyde (DPDD is the oxidized form of DPDB), were computed using the same methods. The calculated IR spectrum of DPDB, used for the assignment of the IR frequencies, was observed in the experimental FT-IR spectrum. The correlation between the theoretical and experimental DPDB vibrational frequencies was 0.996. This agreement mutually verified the accuracy of the experimental method and the validity of the applied mathematical model.     

Molecular parameterization and determination of the electrode potentials of anticoagulant derivatives: Electrochemical and quantum chemical study

This research presents calculations and computation of two anticoagulant derivatives electrode potentials in methanol. For this purpose, the ab initio molecular orbital calculations (HF) and density functional theory (DFT) together with the 6-31G(d) basis set were utilized. The calculated values were compared with the experimental values obtained by linear sweep voltammetry. The observed and the calculated changes in the reduction potential of the anticoagulant derivatives differed from those of the reference compound (catechol), being less than 20 mV. In this way, a method was provided, by which the reduction potentials of the related molecules could be predicted very accurately. Actually, the resulting data illustrated that the method was likely to be useful for the prediction of biomolecules electrode potentials in different aprotic solvents. The bond lengths, bond angles and dipole moment of the studied compounds were calculated in two different solvents and the solvent effects were discussed.     

Effects of substituents in the beta-position of 1,3-dicarbonyl compounds in bromodimethylsulfonium bromide-catalyzed multicomponent reactions: a facile access to functionalized piperidines

1,3-Dicarbonyl compounds can be converted to Mannich-type products A or highly functionalized piperidines B in the presence of a catalytic amount of bromodimethylsulfonium bromide (BDMS). The combination of aromatic aldehyde, amine, and 1,3-dicarbonyl compounds in the presence of a catalytic amount of BDMS leads to the formation of Mannich-type product A when R is a non-enolizable carbon or an alkoxy group, whereas in cases when R = CH3, the same combination yielded highly functionalized piperidines B. A synthetic study and mechanistic proposal are presented.     

Detection of acetone in the human breath as diabetes biomarker based on PPy/WO3 nanocomposite sensor by FFT continuous cyclic voltammetry method

This research represents a novel detection method of acetone level in the exhaled breath samples (RH=88 %) based on polypyrrole/tungsten oxide (PPy/WO₃) nanocomposite sensor. The PPy/WO₃ sensor was fabricated by the deposition of nanocomposite on/between interdigitated electrodes (IDEs) through electrospray coating and was then characterized by FE-SEM imaging. In this detection method, the coulometric signal of the sensor was calculated using Fast Fourier Continuous Cyclic Voltammetry (FFTCCV), where cyclic voltammetry (CV) was applied to the sensor in the defined potential rang and then charge changes of the sensor was obtained by integration of the current in all scanned potential ranges. FFTCCV method enhances the sensitivity of the sensor when exposed to the gas mixtures containing acetone. In addition to its fast coulometric response time (≤5 s) in the two linear ranges of 0.7–2.8 ppm and 2.8–28.2 ppm (R²=0.99), FFTCCV method provides the low detection limit of 70 ppb, and high sensitivity toward acetone at the optimum values of the parameters. The fabricated sensor showed great selectivity toward acetone when exposed to humid air and some exhaled gas like carbon dioxide, ammonia, methanol, ethanol and alkyl amines. The results were very satisfying as the sensor was capable to detect different acetone levels in human exhaled breath as non-invasive diagnosis of diabetes with a good correlation (R²≃0.9) to the routine blood sugar test taken by different commercial glucometers results.     

Antioxidant activity of different parts of Pistacia khinjuk Stocks fruit and its correlation to phenolic composition

The fruits of Pistacia khinjuk Stocks were collected from Ilam province, Iran. The aim of this study was to analyse antioxidant capacity and phenolic composition of different parts of P. khinjuk fruit. The antioxidant capacity of extracts was measured using different assays: ferric reducing ability of plasma, 2,2-diphenyl-1-picrylhydrazyl and nitric oxide radical scavenging. The phenolic composition of P. khinjuk fruit is reported for the first time. Amongst different parts of the fruit analysed in this study, hull extract contained the highest total phenolic and flavonoid contents. We observed a high correlation between different antioxidant activity and total phenolic and flavonoid contents. Therefore, antioxidant capacity can be related to total phenolic and flavonoid contents. A correlation analysis revealed that ascorbic acid, gallic acid, rutin, caffeic acid, ferulic acid and sinapic acid were the phenolic compounds mainly responsible for antioxidant power of the fruit extracts.     

Polyvinylimidazole/sol–gel composite as a novel solid‐phase microextraction coating for the determination of halogenated benzenes from aqueous solutions

A polyvinylimidazole/sol–gel composite is proposed as a novel solid‐phase microextraction fiber to extract five halobenzenes from the headspace of aqueous solutions in combination with gas chromatography with mass spectrometry. The prepared fiber was characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. The obtained results showed that porous polyvinylimidazole/sol–gel composite was chemically deposited on fused silica fiber. The effect of important extraction parameters including extraction temperature, extraction time, and salt content were investigated. The optimum conditions were as follows: extraction temperature 25°C, extraction time 20 min, and salt concentration 30 w/v%. Detection limits and relative standard deviations of the developed method for halogenated benzenes were below 0.1 pg/mL and 15%, respectively. Repeatability of the proposed method, explained by relative standard deviation, varied between 5.48 and 9.15% (n = 5). The limits of detection (S/N = 3) ranged between 0.01 and 0.10 ng/L using gas chromatography with mass spectrometry with selected ion monitoring mode. For real sample analysis, three types of water samples with different matrices (ground, surface, and tap water) were studied. The optimized procedure was applied to extraction and method validation of halogenated benzenes in spiked water samples.     

Synthesis of Arylidene – Isoquinolinones bearing Combretastatin Skeleton by Cyclocarbopalladation/cross coupling Tandem Heck-Suzuki Miaura Reactions using nano catalyst Pd@Py-IL-SPION

Cyclocarbopalladation/cross-coupling cascade intramolecular Heck–Suzuki–Miyaura reactions is applied for the first time by palladium immobilized on pyridine-imidazolium ionic liquid supported magnetic iron oxide nanoparticle catalyst (denoted Pd@Py-IL-SPION) for the last step to synthesize trisubstituted arylidene–isoquinolinones derivatives having Combretastatin skeleton. The reaction is performed via propargylamide intermediates prepared by Ugi 4-CR reactions, which undergoes intramolecular Heck–Suzuki–Miyaura domino reaction to produce the desired trisubstituted arylidene-isoquinolinones. The method shows full regio- and stereoselectivity derives from the particular Pd-catalyzed syn-insertion of triple bond.     

ZnO/Fe3O4 nanoparticles promoted green synthesis of pyrazolo pyrimidinones: Study of antioxidant activity

The preparation of pyrazolo pyrimidinone derivatives was performed by using five component reactions of phthalaldehyde, cyanomethylamine, electron deficient acetylenic compounds, isocyanate, hydrazine, and catalytic amounts of ZnO/Fe₃O₄-magnetic nanoparticles as a high performance catalyst under ultrasonic conditions at ambient temperature in aquause media at room temperature. It should be mentioned that this catalyst was prepared using Spinacia oleracea water extract. In addition, for investigation of antioxidant ability, radical trapping by DPPH and reducing power of ferric ion experiments was performed. As a result, synthesized compounds show excellent radical trapping by DPPH and good reducing ability of ferric ion. The current procedure has the benefits for instance excellent yield of reaction, green media, and easy separation of product and catalyst.