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1.
Bioanalysis ; 16(9): 307-364, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38913185

RESUMO

The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on June 19-23, 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with this NEW Regulation" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition.As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues.This 2023 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication covers the recommendations on Mass Spectrometry Assays, Regulated Bioanalysis/BMV (Part 1A) and Regulatory Inputs (Part 1B). Part 2 (Biomarkers, IVD/CDx, LBA and Cell-Based Assays) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 16 of Bioanalysis, issues 7 and 8 (2024), respectively.


Assuntos
Proteômica , Humanos , Proteômica/métodos , Espectrometria de Massas/métodos , Biomarcadores/análise , Estados Unidos , Terapia Baseada em Transplante de Células e Tecidos , Terapia Genética , Cromatografia/métodos , Brancos
2.
Curr Protoc ; 4(6): e1068, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38837274

RESUMO

Adeno-associated virus (AAV) vectors can efficiently transduce exogenous genes into various tissues in vivo. Owing to their convenience, high efficiency, long-term stable gene expression, and minimal side effects, AAV vectors have become one of the gold standards for investigating gene functions in vivo, especially in non-clinical studies. However, challenges persist in efficiently preparing a substantial quantity of high-quality AAV vectors. Commercial AAV vectors are typically associated with high costs. Further, in-laboratory production is hindered by the lack of specific laboratory equipment, such as ultracentrifuges. Therefore, a simple, quick, and scalable preparation method for AAV vectors is needed for proof-of-concept experiments. Herein, we present an optimized method for producing and purifying high-quality AAV serotype 9 (AAV9) vectors using standard laboratory equipment and chromatography. Using ceramic hydroxyapatite as a mixed-mode chromatography medium can markedly increase the quality of purified AAV vectors. Basic Protocols and optional methods for evaluating purified AAV vectors are also described. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production of AAV9 vectors in 293EB cells Basic Protocol 2: Concentration and buffer exchange of AAV9 vectors from 293EB cell culture supernatants using tangential flow filtration Basic Protocol 3: Purification of AAV9 vectors from TFF samples using ceramic hydroxyapatite chromatography Basic Protocol 4: Analysis of the purified AAV9 vectors.


Assuntos
Cerâmica , Dependovirus , Durapatita , Vetores Genéticos , Sorogrupo , Dependovirus/genética , Dependovirus/isolamento & purificação , Vetores Genéticos/isolamento & purificação , Vetores Genéticos/genética , Humanos , Cerâmica/química , Durapatita/química , Cromatografia/métodos , Células HEK293
3.
ACS Nano ; 18(24): 15729-15743, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38839059

RESUMO

Lipid nanoparticles (LNP) have emerged as pivotal delivery vehicles for RNA therapeutics. Previous research and development usually assumed that LNPs are homogeneous in population, loading density, and composition. Such perspectives are difficult to examine due to the lack of suitable tools to characterize these physicochemical properties at the single-nanoparticle level. Here, we report an integrated spectroscopy-chromatography approach as a generalizable strategy to dissect the complexities of multicomponent LNP assembly. Our platform couples cylindrical illumination confocal spectroscopy (CICS) with single-nanoparticle free solution hydrodynamic separation (SN-FSHS) to simultaneously profile population identity, hydrodynamic size, RNA loading levels, and distributions of helper lipid and PEGylated lipid of LNPs at the single-particle level and in a high-throughput manner. Using a benchmark siRNA LNP formulation, we demonstrate the capability of this platform by distinguishing seven distinct LNP populations, quantitatively characterizing size distribution and RNA loading level in wide ranges, and more importantly, resolving composition-size correlations. This SN-FSHS-CICS analysis provides critical insights into a substantial degree of heterogeneity in the packing density of RNA in LNPs and size-dependent loading-size correlations, explained by kinetics-driven assembly mechanisms of RNA LNPs.


Assuntos
Lipídeos , Nanopartículas , Tamanho da Partícula , Nanopartículas/química , Lipídeos/química , RNA/química , Cromatografia/métodos , RNA Interferente Pequeno/química , Análise Espectral/métodos , Lipossomos
4.
J Chromatogr A ; 1728: 465034, 2024 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-38824842

RESUMO

Covalent organic frameworks (COFs) are featured with large specific surface areas, good thermal stability, and abundant pores. These properties are exactly what the sorbents used for extraction or adsorption of interest substances are desired with. While, the low density and hydrophobicity of COFs often makes them difficult to be dispersed evenly and recovered from the aqueous solution. Magnetic covalent organic frameworks (MCOFs) inherit magnetic property of the magnetic particles and porous structure of COFs. They have improved dispersity in aqueous solution and phase separation can be rapidly achieved via external magnetic fields. This review summarized the synthesis strategies for MCOFs, and their application in trace environmental organic pollutants analysis by chromatography techniques. The selection of COFs types and modification with active groups for a certain adsorption purpose is discussed, along with the exploration of adsorption mechanisms, which is beneficial for the design and synthesis of MCOFs.


Assuntos
Poluentes Ambientais , Estruturas Metalorgânicas , Adsorção , Estruturas Metalorgânicas/química , Poluentes Ambientais/análise , Poluentes Ambientais/química , Compostos Orgânicos/química , Interações Hidrofóbicas e Hidrofílicas , Porosidade , Cromatografia/métodos
5.
Se Pu ; 42(6): 533-543, 2024 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-38845514

RESUMO

Antibody drugs are becoming increasingly popular in disease diagnosis, targeted therapy, and immunoprevention owing to their characteristics of high targeting ability, strong specificity, low toxicity, and mild side effects. The demand for antibody drugs is steadily increasing, and their production scale is expanding. Upstream cell culture technology has been greatly improved by the high-capacity production of monoclonal antibodies. However, the downstream purification of antibodies presents a bottleneck in the production process. Moreover, the purification cost of antibodies is extremely high, accounting for approximately 50%-80% of the total cost of antibody production. Chromatographic technology, given its selectivity and high separation efficiency, is the main method for antibody purification. This process usually involves three stages: antibody capture, intermediate purification, and polishing. Different chromatographic techniques, such as affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, and temperature-responsive chromatography, are used in each stage. Affinity chromatography, mainly protein A affinity chromatography, is applied for the selective capture and purification of antibodies from raw biofluids or harvested cell culture supernatants. Other chromatographic techniques, such as ion-exchange chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography, are used for intermediate purification and antibody polishing. Affinity biomimetic chromatography and hydrophobic charge-induction chromatography can produce antibodies with purities comparable with those obtained through protein A chromatography, by employing artificial chemical/short peptide ligands with good selectivity, high stability, and low cost. Temperature-responsive chromatography is a promising technique for the separation and purification of antibodies. In this technique, antibody capture and elution is controlled by simply adjusting the column temperature, which greatly eliminates the risk of antibody aggregation and inactivation under acidic elution conditions. The combination of different chromatographic methods to improve separation selectivity and achieve effective elution under mild conditions is another useful strategy to enhance the yield and quality of antibodies. This review provides an overview of recent advances in the field of antibody purification using chromatography and discusses future developments in this technology.


Assuntos
Cromatografia de Afinidade , Anticorpos/isolamento & purificação , Anticorpos/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/química , Cromatografia/métodos , Cromatografia de Afinidade/métodos , Cromatografia por Troca Iônica/métodos , Interações Hidrofóbicas e Hidrofílicas
6.
J Chromatogr A ; 1727: 465008, 2024 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-38788402

RESUMO

A critical factor for automated method development in chromatography is the maximization or minimization of an objective function describing the quality (and speed) of the separation. In chromatography, this function is commonly referred to as a chromatographic response function (CRF). Many CRFs have previously been introduced, but many have unfavourable properties such as featuring multiple optima, insufficient discriminatory power, and a too strong dependence on the weight factors needed to balance resolution and time penalty components. To overcome these problems, the present study introduces a new type of CRF wherein the relative weight of the time penalty term is a self-adaptive function of the separation quality. The ability to unambiguously identify the optimal gradient settings of this newly proposed CRF is compared to that of some of the most frequently used CRFs in a study covering 100 randomly composed in silico samples. Doing so, the new CRF is found to flawlessly lead to the correct solution (=linear gradient parameters providing the highest resolution in the shortest potential time) in 100 % of the cases, while the most frequently used literature CRFs were off-target for about 50 to 60 % of the samples, even when considering the availability of spectral peak shape data. Some slight alterations to the proposed CRF are introduced and discussed as well.


Assuntos
Algoritmos , Simulação por Computador , Cromatografia/métodos , Automação
7.
Se Pu ; 42(5): 487-493, 2024 Apr 08.
Artigo em Chinês | MEDLINE | ID: mdl-38736393

RESUMO

The pharmaceutical analysis course is a three-dimensional knowledge network that connects several courses to form a new comprehensive knowledge node involving a large knowledge system and flexible knowledge structure. In this course, the subject of chromatography covers a wide range of topics. However, because accurate content is challenging to present, the teaching effect of this subject is poor. In this work, we sought to achieve the educational purpose of establishing morality and cultivating talent, as well as the goal of training highly skilled professionals, by taking the teaching of chromatography in the pharmaceutical analysis course as an example of transforming scientific research results into teaching resources. The resources obtained are integrated into the teaching process to provide innovative and scientific research ideas to students with the aim of not only helping them understand and master technical knowledge but also exercise their ability to raise and solve problems. Furthermore, we expound on how to introduce scientific development frontiers and formulate scientific problems through curriculum design. We also describe how our strategy can promote the teaching effect and achieve teaching objectives. Based on the characteristics of rapid knowledge update and equal emphasis on theory and practice in pharmaceutical analysis, the course is designed by introducing new advances in scientific development, formulating scientific problems, and adopting question- and problem-based learning methods for teaching. The teaching effect is then evaluated through diversified assessment, student feedback, and self-evaluation. The results show that the transformation of scientific research results into teaching resources plays a significant role in stimulating students' interest in learning, improving students' ability to solve problems, and achieving curriculum objectives, all of which greatly improve the teaching effect.


Assuntos
Ensino , Cromatografia , Currículo , Humanos
8.
Sci Rep ; 14(1): 8714, 2024 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-38622266

RESUMO

Green, photosynthesizing plants can be proficiently used as cost-effective, single-use, fully biodegradable bioreactors for environmentally-friendly production of a variety of valuable recombinant proteins. Being near-infinitely scalable and most energy-efficient in generating biomass, plants represent profoundly valid alternatives to conventionally used stationary fermenters. To validate this, we produced a plastome-engineered tobacco bioreactor line expressing a recombinant variant of the protein A from Staphylococcus aureus, an affinity ligand widely useful in antibody purification processes, reaching accumulation levels up to ~ 250 mg per 1 kg of fresh leaf biomass. Chromatography resin manufactured from photosynthetically-sourced recombinant protein A ligand conjugated to agarose beads demonstrated the innate pH-driven ability to bind and elute IgG-type antibodies and allowed one-step efficient purification of functional monoclonal antibodies from the supernatants of the producing hybridomas. The results of this study emphasize the versatility of plant-based recombinant protein production and illustrate its vast potential in reducing the cost of diverse biotechnological applications, particularly the downstream processing and purification of monoclonal antibodies.


Assuntos
Cromatografia , Proteína Estafilocócica A , Proteína Estafilocócica A/química , Ligantes , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos Monoclonais/metabolismo , Imunoglobulina G/metabolismo , Proteínas de Plantas/metabolismo , Cromatografia de Afinidade/métodos
9.
Molecules ; 29(8)2024 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-38675651

RESUMO

Outer membrane vesicles (OMVs) are attractive for biomedical applications based on their intrinsic properties in relation to bacteria and vesicles. However, their widespread use is hampered by low yields and purities. In this study, EVscore47 multifunctional chromatography microspheres were synthesized and used to efficiently isolate functional OMVs from Escherichia coli. Through this technology, OMV loss can be kept to a minimum, and OMVs can be harvested using EVscore47 at 11-fold higher yields and ~13-fold higher purity than those achieved by means of ultracentrifugation. Based on the results presented here, we propose a novel EVscore47-based isolation of OMVs that is fast and scalable.


Assuntos
Escherichia coli , Vesículas Extracelulares , Microesferas , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Ultracentrifugação , Cromatografia/métodos
10.
Molecules ; 29(8)2024 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-38675682

RESUMO

Drug discovery is a challenging process, with many compounds failing to progress due to unmet pharmacokinetic criteria. Lipophilicity is an important physicochemical parameter that affects various pharmacokinetic processes, including absorption, metabolism, and excretion. This study evaluated the lipophilic properties of a library of ipsapirone derivatives that were previously synthesized to affect dopamine and serotonin receptors. Lipophilicity indices were determined using computational and chromatographic approaches. In addition, the affinity to human serum albumin (HSA) and phospholipids was assessed using biomimetic chromatography protocols. Quantitative Structure-Retention Relationship (QSRR) methodologies were used to determine the impact of theoretical descriptors on experimentally determined properties. A multiple linear regression (MLR) model was calculated to identify the most important features, and genetic algorithms (GAs) were used to assist in the selection of features. The resultant models showed commendable predictive accuracy, minimal error, and good concordance correlation coefficient values of 0.876, 0.149, and 0.930 for the validation group, respectively.


Assuntos
Relação Quantitativa Estrutura-Atividade , Humanos , Albumina Sérica Humana/química , Algoritmos , Modelos Lineares , Estrutura Molecular , Fosfolipídeos/química , Interações Hidrofóbicas e Hidrofílicas , Cromatografia/métodos
12.
Methods Mol Biol ; 2744: 517-523, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38683339

RESUMO

This rapid, equipment-free DNA isolation procedure using chromatography paper is a simple method that can be performed in less than 30 min and requires no wet lab experience. With minimal expense, it offers an affordable alternative for anyone wanting to explore biodiversity. It also provides an excellent option for use in classrooms or other activities that are time limited. The method works best for plants or lichens, producing stable DNA on Whatman® chromatography paper at room temperature, which can be eluted as needed.


Assuntos
Código de Barras de DNA Taxonômico , Código de Barras de DNA Taxonômico/métodos , DNA/isolamento & purificação , DNA/genética , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Plantas/genética , Cromatografia/métodos , Líquens/genética
13.
Mar Pollut Bull ; 202: 116354, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38642479

RESUMO

In recent decades, the harmful algal blooms (HABs) caused by Prorocentrum minimum have caused serious environmental damage and economic losses. The detection of P. minimum plays an important role in warning the outbreak of P. minimum-forming HABs. By utilizing the powerful absorption of graphene oxide (GO) on short-stranded DNA, a GO-assisted nucleic acid chromatography strip (GO-NACS) was proposed here to achieve a highly sensitive, specific, intuitive, and convenient detection of P. minimum. In particular, this study used our previously reported conventional-NACS (C-NACS) as a control to evaluate the improvement of detection performance with the use of GO. The performance of GO-NACS was evaluated from the perspectives of specificity, sensitivity, stability, and practicality. The specificity test demonstrated that it had a high degree of specificity and did not display cross-reacting with non-target algal species. The sensitivity test with the genomic DNA indicated that it had a detection limit of 1.30 × 10-3 ng µL-1, representing a 10-fold higher sensitivity than C-NACS and a 100-fold higher sensitivity than agarose gel electrophoresis (AGE). The interference test with non-target algal species demonstrated that it had a good detection stability, and the interfering algal species had no obvious effect on the detection of P. minimum. The practicality test with simulated natural water samples showed that the cellular detection limit of GO-NACS was 6.8 cells mL-1, which was 10-fold and 100-fold lower than that of C-NACS and AGE, respectively. In conclusion, the established GO-NACS may offer a novel alternative technique for the detection of P. minimum while guaranteeing specificity and enhancing sensitivity without requiring extensive apparatus.


Assuntos
Grafite , Proliferação Nociva de Algas , Grafite/química , Monitoramento Ambiental/métodos , Cromatografia/métodos , Ácidos Nucleicos/análise
14.
Artigo em Inglês | MEDLINE | ID: mdl-38640794

RESUMO

Chromatography is a robust and reliable separation method that can use various stationary phases to separate complex mixtures commonly seen in metabolomics. This review examines the types of chromatography and stationary phases that have been used in targeted or untargeted metabolomics with methods such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. General considerations for sample pretreatment and separations in metabolomics are considered, along with the various supports and separation formats for chromatography that have been used in such work. The types of liquid chromatography (LC) that have been most extensively used in metabolomics will be examined, such as reversed-phase liquid chromatography and hydrophilic liquid interaction chromatography. In addition, other forms of LC that have been used in more limited applications for metabolomics (e.g., ion-exchange, size-exclusion, and affinity methods) will be discussed to illustrate how these techniques may be utilized for new and future research in this field. Multidimensional LC methods are also discussed, as well as the use of gas chromatography and supercritical fluid chromatography in metabolomics. In addition, the roles of chromatography in NMR- vs. MS-based metabolomics are considered. Applications are given within the field of metabolomics for each type of chromatography, along with potential advantages or limitations of these separation methods.


Assuntos
Cromatografia , Metabolômica , Animais , Humanos , Cromatografia Líquida/métodos , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Metabolômica/métodos , Cromatografia/métodos
15.
J Chromatogr A ; 1721: 464806, 2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38518514

RESUMO

Monoclonal antibodies (mAbs) continue to dominate the biopharmaceutical industry. Certain mAbs are prone to fragmentation and clipping and in these cases, adequate removal of these species is critical during manufacturing. Fragments can be generated during fermentation, purification, storage, formulation, and administration. Their addition to the acidic charge-variant of the purified mAb has been reported to decrease stability and potency of the final product. However, contrary to mAb aggregation, manufacturers have not given much attention to removal of fragments and clipped species and as a result most conventional mAb platforms offer at best limited capabilities for their removal. In this study, we propose a novel purification platform that uses multimodal chromatography and achieves complete removal of a range of mAb fragments and clipped products (25-120 kDa). The utility of the platform has been successfully demonstrated for 2 IgG1s and 2 IgG4s. Further, adequate removal of the various host cell impurities such as host cell proteins (<10 ppm) and host cell DNA (<5 ppb) has been achieved. Finally, the platform was able to deliver adequate removal of high molecular weight impurities (<1 %) and a 30 % clearance of the acidic charge variant. The proposed single step has been shown to deliver what the polishing chromatography and intermediate purification chromatography steps deliver in a traditional mAb platform.


Assuntos
Anticorpos Monoclonais , Cromatografia , Cricetinae , Animais , Peso Molecular , Comércio , Células CHO , Cricetulus
16.
Adv Exp Med Biol ; 3234: 163-172, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38507206

RESUMO

Small angle X-ray scattering (SAXS) is a versatile technique that can provide unique insights in the solution structure of macromolecules and their complexes, covering the size range from small peptides to complete viral assemblies. Technological and conceptual advances in the last two decades have tremendously improved the accessibility of the technique and transformed it into an indispensable tool for structural biology. In this chapter we introduce and discuss several approaches to collecting SAXS data on macromolecular complexes, including several approaches to online chromatography. We include practical advice on experimental design and point out common pitfalls of the technique.


Assuntos
Cromatografia , Espalhamento a Baixo Ângulo , Raios X , Difração de Raios X , Substâncias Macromoleculares/química
17.
Ars pharm ; 65(2): 98-106, mar. 2024. tab
Artigo em Espanhol | IBECS | ID: ibc-231946

RESUMO

Introducción: El bitartrato de epinefrina, también conocido como epinefrina, es un ingrediente farmacéutico importante en el tratamiento de diversas enfermedades, pero su medición precisa es esencial para garantizar la seguridad del medicamento. La Farmacopea de los Estados Unidos (USP) establece los estándares para su análisis, pero la elección del método afecta la precisión de las mediciones. Este estudio investiga cómo los diferentes métodos afectan la medición del bitartrato de epinefrina según las versiones USP-43 y USP-44, que tienen implicaciones significativas para la calidad y la regulación de los medicamentos en el campo. Método: Se eligieron el método volumétrico y el método cromatográfico para comparación. Se utilizaron muestras de epinefrina bitartrato de alta pureza que cumplían con los estándares de la USP-43 y USP-44.Resultados: Los resultados obtenidos por ambos métodos se comparan entre sí y se evalúan según los límites de especificación definidos por USP-43 y USP-44. Los valores obtenidos para algunos parámetros, como la concentración y la pureza del bitartrato de epinefrina, varían considerablemente entre los distintos métodos analíticos. Conclusiones: Este estudio destaca la importancia de una cuidadosa selección del método analítico al evaluar el bitartrato de epinefrina según las directrices USP-43 y USP-44. La elección de la tecnología afecta a los resultados y, por tanto, a la calidad y seguridad de los productos farmacéuticos que contienen esta sustancia. Se recomienda validar el método en cada laboratorio y comparar los resultados con los estándares USP. (AU)


Introduction: Epinephrine bitartrate, also known as epinephrine, is an important pharmaceutical ingredient in the treatment of various diseases, but its accurate measurement is essential to ensure the safety of the drug. The United States Pharmacopeia (USP) sets the standards for its analysis, but the choice of method affects the precision of the measurements. This study investigates how different methods affect the measurements of epinephrine bitartrate based on USP-43 and USP-44, which have significant implications for drug quality and regulation in the field. Method: The volumetric method and chromatographic method were chosen for comparison. High-purity epineph-rine bitartrate samples that met USP-43 and USP-44 standards were used. Results: The results obtained by both methods are compared with and evaluated according to the specification lim-its defined by USP-43 and USP-44. The values obtained for some parameters, such as the concentration and purity of epinephrine tartrate, vary considerably between the different analytical methods. Conclusions: This study highlights the importance of carefully selecting analytical methods when evaluating epi-nephrine tartrate according to USP-43 and USP-44 guidelines. The choice of technology affects the results and, therefore, the quality and safety of the pharmaceutical products containing this substance. It is recommended to validate the method in each laboratory and compare the results with USP standards. (AU)


Assuntos
Epinefrina/farmacologia , Epinefrina/análise , Titulometria , Cromatografia , Farmacopeias como Assunto
18.
J Chromatogr A ; 1720: 464825, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38507870

RESUMO

We report on a steady-state based, and hence highly accurate numerical modelling study of the effect of the top and bottom wall in the current generation of micro-pillar array columns. These have a mesoporous retention layer that not only covers the pillar walls but also the bottom wall. Our results show that the performance of these columns can in general not be improved by also covering the top wall with the same layer, despite the increased column symmetry this approach would offer. The reason for this is that the local species retardation caused by a retentive layer is much stronger than the pure flow arresting effect of an uncovered wall. At least, this has a crucial impact in high aspect-ratio systems such as micro-pillar array columns because these require a small inter-pillar distance to promote mass transfer together with a large channel depth to enable a sufficiently high flow rate. On the other hand, a notable improvement could be made if micro-pillar array would be produced without having a retentive layer at the bottom. At Péclet number Pe = 50 and aspect ratio AR = 5 for flow-channels, this gain amounts up to about 4.5 h-units at a zone retention factor k'' = 2 and 1.75 h-units at k'' = 16 (gain scales almost linearly with Pe). To verify these results, we also considered another high aspect-ratio system with a simplified geometry: the open-tubular channel with a flat-rectangular cross-section. This led to very similar observations, thus confirming the findings for the micro-pillar array. The results produced in the present study also allow us to conclude that the classic modelling paradigm adopted in chromatography, which is based on the independency and hence additivity of the hCm- and hCs-contributions, can lead to large modelling errors in chromatographic systems with a high aspect-ratio, even when their geometry is so simple as that of a straight open-tubular channel with constant cross-section. Indeed, when both zones are treated independently, the analysis misses how the vertical diffusion through the retentive layer helps suppressing the vertical gradients in the mobile zone. The diffusion through this layer occurs in a ratio of k''Ds/Dm (Dm being the diffusion coefficient in mobile phase zone and Ds being the diffusion coefficient in stationary phase zone), such that at high retention factors this diffusion contribution even becomes the dominant one.


Assuntos
Cromatografia , Difusão
19.
Anal Chem ; 96(14): 5702-5710, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38538555

RESUMO

Glass nanopipets have been demonstrated to be a powerful tool for the sensing and discrimination of biomolecules, such as DNA strands with different lengths or configurations. Despite progress made in nanopipet-based sensors, it remains challenging to develop effective strategies that separate and sense in one operation. In this study, we demonstrate an agarose gel-filled nanopipet that enables hyphenated length-dependent separation and electrochemical sensing of short DNA fragments based on the electrokinetic flow of DNA molecules in the nanoconfined channel at the tip of the nanopipet. This nanoconfined electrokinetic chromatography (NEC) method is used to distinguish the mixture of DNA strands without labels, and the ionic current signals measured in real time show that the mixed DNA strands pass through the tip hole in order according to the molecular weight. With NEC, gradient separation and electrochemical measurement of biomolecules can be achieved simultaneously at the single-molecule level, which is further applied for programmable gene delivery into single living cells. Overall, NEC provides a multipurpose platform integrating separation, sensing, single-cell delivery, and manipulation, which may bring new insights into advanced bioapplication.


Assuntos
DNA , Nanotecnologia , DNA/química , Nanotecnologia/métodos , Cromatografia
20.
Carbohydr Res ; 538: 109076, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38537364

RESUMO

Profiling of pectic arabinans and galactans by analysis of the released oligosaccharides after backbone cleavage provides information on the complexity of the polymer structure. In plants of the family Amaranthaceae, arabinan and galactan substitution with ferulates extends the polysaccharide complexity, changing its chemical properties. Knowledge of the ferulate environment is crucial to understand structure-function-relationships of feruloylated pectins. Here, we present an approach to separate enzymatically generated feruloylated and non-feruloylated arabino- and galactooligosaccharides, followed by deesterification and semiquantitative analysis by HPAEC-PAD using previously reported relative response factors. Application of this approach to sugar beet pectins and insoluble and soluble dietary fiber preparations of amaranth and quinoa suggests that ferulates are preferably incorporated into more complex structures, as nicely demonstrated for feruloylated galactans. Also, ferulate substitution appears to negatively affect enzymatic cleavage by using endo-enzymes. As a consequence, we were able to tentatively identify new feruloylated tri- and tetrasaccharides of galactans isolated from sugar beet pectins.


Assuntos
Galactanos , Pectinas , Polissacarídeos , Galactanos/química , Pectinas/química , Oligossacarídeos/química , Cromatografia , Açúcares
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