Effect of ethoxylated sorbitan ester surfactants on the chromatographic efficiency of poly(ethylene glycol)-based monoliths.

The effect of adding ethoxylated sorbitan ester surfactants (Tweens®) to poly(ethylene glycol) diacrylate-based monolithic recipes was investigated. Five different Tweens® have been evaluated to investigate the exact role of non-ionic surfactants in poly(ethylene glycol) diacrylate-based monolith preparations. These monoliths were characterized by scanning electron microscopy, infrared spectroscopy, and nitrogen physisorption analysis. Different morphological features, and surface areas were observed when different types of Tween® were included in the recipe; Tween® 20 and 85 showed small globules, while Tween® 40, 60 and 80 exhibited larger globular structures with different sizes and degrees of coalescence. The different Tween®-based monoliths were investigated for the chromatographic separation of mixtures consisting of hydroxybenzoic acids and alkylbenzenes. These columns were mechanically stable, except for Tween® 80. The highest methylene selectivity and the best overall performance were achieved by Tween® 60. The efficiency was increased by increasing the concentration of the Tween® 60 and the amount of poly(ethylene glycol) diacrylate Mn 700 in the recipes up to 30 wt%, each. Further increases in either Tween® 60 or poly(ethylene glycol) diacrylate Mn 700 led to formation of non-permeable columns. The optimized column was successfully used for separation of mixtures of nonsteroidal anti-inflammatory and sulfa drugs, with a maximum efficiency of 60,000 plates/m.

Evaluation of Residual Monomer Release After Polymerization of Colored Compomer Materials

ABSTRACT Objective: To evaluate the amount of residual monomers released after polymerization by the compomers in different colors and viscosities over time. Material and Methods: The compomer samples of different colors and viscosities (flowable compomers; blue-pink and packable compomers; A2-blue-pink-gold) were prepared in molds with an inner diameter of 5 mm and a height of 2 mm. In polymerization of samples, a LED unit was used. The amount of monomers released from the samples kept in 75% ethanol/water solution was measured by a high-performance liquid chromatography (HPLC) instrument in the 10th minute, in the 1st hour, and in the 1st, 7th, and 14th days. For statistical analyses, the paired sample t-test, independent sample t-test, and one-way ANOVA with Tukey's post hoc test were used. Results: The amount of residual monomers released from all materials increased over time. At the end of the 14th day, the most released monomer from all compomer samples was BisGMA. The total amounts of released monomers from the packable compomers were Gold>A2>blue>pink. The amount of residual monomers released from flowable compomers was higher in blue than in pink. Conclusion: The color and the viscosity are the factors affecting the residual monomer release in compomers.

Analytical lifecycle management oriented development and validation of a new stability-indicating ultra-fast liquid chromatographic method: case study of Glycopyrrolate

A new stability-indicating liquid chromatographic method was developed and validated for the estimation of glycopyrrolate in pharmaceutical formulations. A contemporary approach to analytical life-cycle management was followed to develop a robust and reliable chromatographic method. Scouted method variables such as % methanol, the strength of tetra butyl ammonium hydrogen sulfate and mobile phase flow rate were optimized using the design of experiment approach and their effect on critical quality attributes was studied. The critical quality attributes viz. retention time, theoretical plate count and symmetry factor were highly influenced by the three critical method variables. Optimum chromatography was attained on a C-18 column with a mobile phase methanol: 10 mM tetra butyl ammonium hydrogen sulfate (80:20, v/v) flowing at 1.0 mL.min-1. Chromatographic method specificity was ensured by degrading the drug forcefully. Validation studies postulated method acceptability and suitability for estimating glycopyrrolate in both bulk as well as injection formulation. Results for parameters viz. linearity (5-250 µg.mL-1), accuracy (>99%) and precision (<2%) advocated method reliability. Overall the method was reliable and of optimum quality and, possess the potential of application in routine and bio-analytical purposes

Two novel extraction chromatographic resins containing benzene-centered tripodal diglycolamide ligands: Actinide uptake, kinetic modeling and isotherm studies.

Two novel extraction chromatographic resins (EC), termed as RL-1 and RL-2, were prepared by impregnating two benzene-centered tripodal iglycolamide ligands (Bz-T-DGA) containing different spacer groups where the ligands are termed as L-1 and L-2, respectively. They were employed for the uptake of actinide and fission product ions, viz. Am3+, Eu3+, UO22+, Np4+, Pu4+, Sr2+, and Cs+, from acidic feeds. Weight distribution coefficient (Kd) values were measured by the batch method and the loaded metal ions were back extracted using a 0.01 M EDTA solution at pH 4. Kinetic modeling of the sorption data of Am(III) on both resins suggested pseudo-second order rate kinetics with rate constants of 1.68 × 10-6 and 2.47 × 10-6 g/cpm.min for the resins containing L-1 and L-2, respectively. Sorption isotherm studies indicated the Langmuir monolayer chemisorption phenomenon with Eu(III) experimentally determined saturation uptake capacities of 6.02 ± 0.11 and 5.49 ± 0.14 mg per g of RL-1 and RL-2 resins, respectively. As the batch uptake study results appeared encouraging, column studies were also carried out using both resins. The resin reusability data indicated a marginal change in the Kd values for the RL-1 resin up to three repeat runs beyond which a steady decrease of the Kd value was seen. On the other hand, in the case of RL-2 a steady decrease in the Kd values was observed for three repeat runs beyond which there was marginal change.

Online porous graphic carbon chromatography coupled with tandem mass spectrometry for post-translational modification analysis.

RATIONALE: Porous graphic carbon chromatography (PGC) has a different mechanism in the retention of tryptic peptides compared with reversed-phase chromatography and in this study we show that coupling PGC with tandem mass spectrometry offer advantages for the quantitation of phosphorylation stoichiometry and characterization of site-specific glycosylation. METHODS: Digests of protein standards (horse myoglobin, bovine fetuin and ß-casein) were analyzed with a capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) system by coupling an Agilent 1100 HPLC system to a Synapt G2-Si HDMS (Waters). Peptides were separated using a HyperCarb PGC column (300 µm i.d. × 100 mm) packed with 3 µm particles. MS/MS data were collected in data-dependent mode and three MS/MS scans were acquired after the full MS scan. RAW data were transformed to .mgf by PLGS (Waters) and searched against the Swissprot database by Mascot. Chromatograms and MS/MS spectra of identified compounds were extracted with Masslynx (Waters) and imported to Origin for analysis. Glycan composition and peptide sequence were manually annotated. RESULTS: PGC/MS/MS enabled accurate quantitation of the stoichiometry of specific phosphorylation sites from ß-casein by efficient separation of the phosphopeptide and its non-phosphorylated counterpart, which cannot be achieved by reversed-phase chromatography. PGC/MS/MS also enabled comprehensive characterization of protein sialoglycosylation as isomeric glycopeptides with different combinations of α2-3- and α2-6-linked sialic acids can be separated and the ratios of each combination were verified by exoglycosidase digestion. CONCLUSIONS: PGC has demonstrated superior separation of peptides with phosphorylation and glycosylation and can be used as an alternative in the proteomic characterization of post-translational modifications (PTMs) by polar groups.

Analyzing Mechanisms of Metastatic Cancer Cell Adhesive Phenotype Leveraging Preparative Adhesion Chromatography Microfluidic.

An integrated, parallel-plate microfluidic device is engineered to interrogate and fractionate cells based on their adhesivity to a substrate surface functionalized with adhesive ligand in a tightly controlled flow environment to elucidate associated cell-intrinsic pathways. Wall shear stress levels and endothelial presentation of E-selectin are modeled after the inflamed vasculature microenvironment in order to simulate in vitro conditions under which in vivo hematogenous metastasis occurs. Based on elution time from the flow channel, the collection of separate fractions of cells-noninteracting and interacting-at high yields and viabilities enables multiple postperfusion analyses, including flow cytometry, in vivo metastasis modeling, and transcriptomic analysis. This platform enables the interrogation of flow-regulated cell molecular profiles, such as (co)expression levels of natively expressed selectin ligands sLex , CD44, and carcinoembryonic antigen, and cancer stem cell marker CD24. This additionally reveals E-selectin adhesivity exhibited by metastatic human colon carcinoma cells to be a transient phenotype. Facile and rapid, this methodology for unbiased, label free sorting of large populations of cells based on their adhesion in flow represents a method of studying flow-regulated adhesion in vitro for the identification of molecular drug targets for development as antimetastatic cancer therapeutics.

Analytical methodologies used for the determination of colistin in biological fluids. Is it still a challenge?

Colistin is a multicomponent polypeptide antibiotic consisting mainly of colistin A and colistin B, produced by selected strains of Bacillus polymyxa var. Colistinus. Only recently, the prodrug of colistin, colistimethate sodium, is widely used as last resort antibiotic for infections caused by resistant gram-negative bacteria. Colistin having been discovered several years ago, has not subjected to the drug development and regulatory approval processes that are applied today. However, pharmacological and pharmacokinetic information are necessary for its optimal use thus, during the last decades several studies are carried out in order to shed light on this issue. In the current review, the analytical methodologies of colistin assessment in biological material are summarized and the analytical challenges are critically discussed and critical aspects of the determinations such as the method of detection, the sample pretreatment methodology etc. are compared. Furthermore, critical quality aspects of the assessment methodologies such as the sensitivity of the currently developed methodologies are presented. Lastly, some future trends that should be incorporated in the determination pipeline of modern drugs are suggested.

Characterization of Seed Oils of Pterodon emarginatus Vogel (Sucupira) Obtained by Different Extraction Methods

Abstract The present study aims to evaluate the influence of extraction methods on the quality of bioactive compounds and antioxidant capacity in Pterodon emarginatusVogel (sucupira) oils. The oils were extracted from the sucupira seeds by Soxhlet and by the extraction system of Bligh & Dyer. The oils were analyzed as to fatty acid profile, tocopherols, phytosterols, carotenoids, total phenolics, and total antioxidant capacity. The extraction by Soxhlet showed better yield of total lipids and was more efficient to extract tocopherols, phytosterols, and carotenoids, besides presenting better antioxidant activity by DPPH•. However, this method showed insufficient capacity to extract the polar lipid components of the sample, as evidenced by the low results of total phenolic compounds. On the other hand, the Bligh & Dyer method preserved the fatty acid profile and was also effective to extract higher phenolic compounds content and presented superior antioxidant activity when assessed by FRAP, ABTS•+, and oxidative stability index.

Integrating Next-Generation Genomic Sequencing and Mass Spectrometry To Estimate Allele-Specific Protein Abundance in Human Brain.

Gene expression contributes to phenotypic traits and human disease. To date, comparatively less is known about regulators of protein abundance, which is also under genetic control and likely influences clinical phenotypes. However, identifying and quantifying allele-specific protein abundance by bottom-up proteomics is challenging since single nucleotide variants (SNVs) that alter protein sequence are not considered in standard human protein databases. To address this, we developed the GenPro software and used it to create personalized protein databases (PPDs) to identify single amino acid variants (SAAVs) at the protein level from whole exome sequencing. In silico assessment of PPDs generated by GenPro revealed only a 1% increase in tryptic search space compared to a direct translation of all human transcripts and an equivalent search space compared to the UniProtKB reference database. To identify a large unbiased number of SAAV peptides, we performed high-resolution mass spectrometry-based proteomics for two human post-mortem brain samples and searched the collected MS/MS spectra against their respective PPD. We found an average of ∼117 000 unique peptides mapping to ∼9300 protein groups for each sample, and of these, 977 were unique variant peptides. We found that over 400 reference and SAAV peptide pairs were, on average, equally abundant in human brain by label-free ion intensity measurements and confirmed the absolute levels of three reference and SAAV peptide pairs using heavy labeled peptides standards coupled with parallel reaction monitoring (PRM). Our results highlight the utility of integrating genomic and proteomic sequencing data to identify sample-specific SAAV peptides and support the hypothesis that most alleles are equally expressed in human brain.

Glycosaminoglycanomics: where we are.

Glycosaminoglycans regulate numerous physiopathological processes such as development, angiogenesis, innate immunity, cancer and neurodegenerative diseases. Cell surface GAGs are involved in cell-cell and cell-matrix interactions, cell adhesion and signaling, and host-pathogen interactions. GAGs contribute to the assembly of the extracellular matrix and heparan sulfate chains are able to sequester growth factors in the ECM. Their biological activities are regulated by their interactions with proteins. The structural heterogeneity of GAGs, mostly due to chemical modifications occurring during and after their synthesis, makes the development of analytical techniques for their profiling in cells, tissues, and biological fluids, and of computational tools for mining GAG-protein interaction data very challenging. We give here an overview of the experimental approaches used in glycosaminoglycomics, of the major GAG-protein interactomes characterized so far, and of the computational tools and databases available to analyze and store GAG structures and interactions.

Cellufine sulfate column chromatography as a simple, rapid, and effective method to purify dengue virus.

Conventional method to purify/concentrate dengue virus (DENV) is time-consuming with low virus recovery yield. Herein, we applied cellufine sulfate column chromatography to purify/concentrate DENV based on the mimicry between heparan sulfate and DENV envelope protein. Comparative analysis demonstrated that this new method offered higher purity (as determined by less contamination of bovine serum albumin) and recovery yield (as determined by greater infectivity). Moreover, overall duration used for cellufine sulfate column chromatography to purify/concentrate DENV was approximately 1/20 of that of conventional method. Therefore, cellufine sulfate column chromatography serves as a simple, rapid, and effective alternative method for DENV purification/concentration.

Highly efficient and selective enrichment of glycopeptides using easily synthesized magG/PDA/Au/l-Cys composites.

Highly selective and efficient enrichment of glycopeptides from complex biological samples is necessary. In this study, novel zwitterionic hydrophilic polydopamine-coated magnetic graphene composites (magG/PDA/Au/l-Cys) were synthesized and applied to the enrichment of glycopeptides. The size, morphology, and composition of magG/PDA/Au/l-Cys composites were investigated by transmission electron microscopy, scanning electron microscopy, FT-infrared spectroscopy, and X-ray diffraction. The composites possessed a number of desirable characteristics, including good biocompatibility easy separation property and excellent hydrophilicity. By virtue of the features contributed by different ingredients, the prepared composites demonstrated superior performance for glycopeptide enrichment with high sensitivity (0.1 fmol), efficiency, selectivity (1:100), and repeatability (at least ten times). In addition, the composites were successfully applied to the enrichment of glycopeptides from human serum and 40 unique N-glycosylation peptides from 31 different N-linked glycoproteins were identified. The superior hydrophilic material is of great potential for the analysis of glycoproteins.

Sequential enrichment of singly- and multiply-phosphorylated peptides with zwitterionic hydrophilic interaction chromatography material.

An interesting and novel method for the selective and sequential enrichment of singly- and multiply-phosphorylated peptides with a zwitterionic material "Click TE-Cys" is presented. Retention mechanisms between phosphopeptides and Click TE-Cys are systematically investigated by checking the influence of acetonitrile content, pH value, and buffer concentration on the retention of phosphopeptides. Both hydrophilic interaction and electrostatic interaction are involved in retention between phosphopeptides and Click TE-Cys. Based on these results, an optimized method is established for selective enrichment of phosphopeptides using Click TE-Cys. This method not only exhibits high selectivity for phosphopeptides, but also fractionates singly- and multiply-phosphorylated peptides into two fractions. This method was evaluated using relatively complex samples, including peptide mixtures of α-casein and bovine serum albumin (BSA) at a molar ratio of 1:10 and skim milk. This efficient and optimized protocol has great potential for enriching multiply-phosphorylated peptides and could be a valuable tool for specific enrichment of phosphopeptides in phosphoproteome analysis.

Preparation of a biomimetic polyphosphorylcholine monolithic column for immobilized artificial membrane chromatography.

The present work aims to prepare a novel phosphatidylcholine functionalized monolithic stationary phase by in situ co-polymerization of 12-methacryloyl dodecylphosphocholine (MDPC) and ethylene dimethacrylate (EDMA) for immobilized artificial membrane chromatography. Scanning electron microscopy, energy dispersive X-ray spectroscopy, FT-IR spectroscopy, pore size distribution analysis, ζ-potential analysis and micro-HPLC were used to evaluate the monolithic structure and physicochemical properties. Satisfactory morphology, high mechanical stability, good permeability and chromatographic performance were obtained on the optimized monolithic columns. A typical reverse-phase retention mechanism was observed over a wide range of organic solvent content (acetonitrile< 80%). The optimized poly(MDPC-co-EDMA) monolith exhibited good selectivity for proteins and basic drugs. Good correlation was observed between the retention on commercial IAM column (IAM.PC.DD2) and poly(MDPC-co-EDMA) monolith. This novel poly(MDPC-co-EDMA) monolith exhibited good potential for studying the drug-membrane interaction.

Separation of salvianic acid A from the fermentation broth of engineered Escherichia coli using macroporous resins.

Salvianic acid A (also known as danshensu) is a plant-derived polyphenolic acid, and has a variety of physiological and pharmacological activities. Our laboratory previously constructed an unprecedented artificial biosynthetic pathway in Escherichia coli and established the fermentation process to produce salvianic acid A. Here, we developed an efficient method for separating salvianic acid A from the fermentation broth of engineered Escherichia coli by macroporous resins. Among ten tested macroporous resins, the static and dynamic adsorption/desorption experiments demonstrated that X5 resin was the best to separate salvianic acid A from fermentation broth. Other parameters during static and dynamic procedures were also investigated. Under the optimum separation conditions, the average adsorption capacity of SAA were 10.66±0.54 mg/g dry resin and the desorption ratio was 85.6±4.1%. The purity and recovery yield of salvianic acid A in the final dry product were 90.2±1.5 and 81.5±2.3%, respectively. The results show that adsorption separation with macroporous resin X5 was an efficient method to prepare salvianic acid A from fermentation broth. This work will benefit the development and application of plant-derived salvianic acid A and its derivatives.

Optimization of reactive simulated moving bed systems with modulation of feed concentration for production of glycol ether ester.

In this article, we extend the simulated moving bed reactor (SMBR) mode of operation to the production of propylene glycol methyl ether acetate (DOWANOL™ PMA glycol ether) through the esterification of 1-methoxy-2-propanol (DOWANOL™ PM glycol ether) and acetic acid using AMBERLYST™ 15 as a catalyst and adsorbent. In addition, for the first time, we integrate the concept of modulation of the feed concentration (ModiCon) to SMBR operation. The performance of the conventional (constant feed) and ModiCon operation modes of SMBR are analyzed and compared. The SMBR processes are designed using a model based on a multi-objective optimization approach, where a transport dispersive model with a linear driving force for the adsorption rate has been used for modeling the SMBR system. The adsorption equilibrium and kinetics parameters are estimated from the batch and single column injection experiments by the inverse method. The multiple objectives are to maximize the production rate of DOWANOL™ PMA glycol ether, maximize the conversion of the esterification reaction and minimize the consumption of DOWANOL™ PM glycol ether which also acts as the desorbent in the chromatographic separation. It is shown that ModiCon achieves a higher productivity by 12-36% over the conventional operation with higher product purity and recovery.

5-O-caffeoylshikimic acid from Solanum somalense leaves: advantage of centrifugal partition chromatography over conventional column chromatography.

Solanum somalense leaves, used in Djibouti for their medicinal properties, were extracted by MeOH. Because of the high polyphenol and flavonoid contents of the extract, respectively, determined at 80.80 ± 2.13 mg gallic acid equivalent/g dry weight and 24.4 ± 1.01 mg quercetin equivalent/g dry weight, the isolation and purification of the main polyphenols were carried out by silica gel column chromatography and centrifugal partition chromatography. Column chromatography led to 11 enriched fractions requiring further purification, while centrifugal partition chromatography allowed the easy recovery of the main compound of the extract. In a solvent system composed of CHCl3/MeOH/H2O (9.5:10:5), 21.8 mg of this compound at 97% purity was obtained leading to a yield of 2.63%. Its structure was established as 5-O-caffeoylshikimic acid by mass spectrometry and NMR spectroscopy. This work shows that S. somalense leaves contain very high level of 5-O-caffeoylshikimic acid (0.74% dry weight), making it a potential source of production of this secondary metabolite that is not commonly found in nature but could be partly responsible of the medicinal properties of S. somalense leaves.

Monolithic silica rods grafted with thermoresponsive anionic polymer brushes for high-speed separation of basic biomolecules and peptides.

Thermoresponsive anionic copolymer brushes, poly(N-isopropylacrylamide-co-acrylic acid-co-tert-butylacrylamide) [P(IPAAm-co-AAc-co-tBAAm)], were grafted onto a monolithic silica rod column through surface-initiated atom-transfer radical polymerization (ATRP) to prepare an effective thermoresponsive anionic chromatography matrix. An ATRP initiator was attached to the rod surface. N-Isopropylacrylamide (IPAAm), tert-butyl acrylate (tBA), tert-butylacrylamide (tBAAm), and the ATRP catalyst CuCl/CuCl2/tris[2-(N,N-dimethylamino)ethyl]amine were dissolved in 2-propanol, and the reaction mixture was pumped into the initiator-modified column. After grafting P(IPAAm-co-tBA-co-tBAAm) on the monolithic silica surfaces, deprotection of the tert-butyl group of tBA was performed. Chromatographic analysis showed that the prepared column was able to separate catecholamine derivatives and angiotensin subtypes within a shorter analysis time (5 min) than a silica-bead-packed column modified with the same copolymer brush could. These results indicated that the prepared copolymer-modified monolithic silica rod column may be a promising bioanalytical and bioseparation tool for rapid analysis of basic bioactive compounds and peptides.

Enrichment and antioxidant properties of flavone C-glycosides from trollflowers using macroporous resin.

Orientin, vitexin and other flavone C-glycosides are functional ingredients abundant in trollflowers. In this study, an efficient method for enrichment of these ingredients from the flowers of Trollius chinensis Bunge was developed using macroporous resin. Separation characteristics of six typical macroporous resins were investigated by static adsorption/desorption and dynamic separation experiments, and HPD450 was selected as optimal one. Dynamic adsorption/desorption experiments on HPD450 columns were conducted to obtain the optimal parameters, followed by a scale-up experiment. Six fractions, TC-1-TC-3 together with their acid hydrolysates were further prepared to evaluate their antioxidant capacities by 2,2-diphenyl-1-picrylhydrazyl radical and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonate) radical cation scavenging assays. They all have notable concentration-dependent antioxidant activities, with TC-1 showing strong antioxidant capacity higher than orientin. The separation process was high-efficient, low-cost and environmental-friendly, which could afford a potential approach for industrial applications.

Nanoparticle-modified monolithic pipette tips for phosphopeptide enrichment.

We have developed nanoparticle-modified monoliths in pipette tips for selective and efficient enrichment of phosphopeptides. The 5 µL monolithic beds were prepared by UV-initiated polymerization in 200 µL polypropylene pipette tips and either iron oxide or hydroxyapatite nanoparticles were used for monolith modification. Iron oxide nanoparticles were prepared by a co-precipitation method and stabilized by citrate ions. A stable coating of iron oxide nanoparticles on the pore surface of the monolith was obtained via multivalent electrostatic interactions of citrate ions on the surface of nanoparticles with a quaternary amine functionalized poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith. Hydroxyapatite nanoparticles were incorporated into the poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith by simply admixing them in the polymerization mixture followed by in situ polymerization. The nanoparticle-modified monoliths were compared with commercially available titanium dioxide pipette tips. Performance of the developed and commercially available sorbents was demonstrated with the efficient and selective enrichment of phosphopeptides from peptide mixtures of α-casein and ß-casein digests followed by off-line MALDI/MS analysis.