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1.
J Chromatogr A ; 1627: 461430, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823119

RESUMO

There is a huge, still increasing market for synthetic and therapeutic peptides. Their quality control is commonly based on a generic reversed-phase liquid chromatography (RPLC) method with C18 stationary phase and acetonitrile gradient with 0.1% trifluoroacetic acid in the mobile phase. It performs exceptionally well for a wide variety of impurities, yet structurally closely related impurities with similar sequences, not resolved in preparative RPLC, may easily coelute in the corresponding QC run as well. To address this problem an advanced generic 2D-LC impurity profiling method was developed in this work. It employs a selective comprehensive (high resolution sampling) RP×RP 2D-LC separation using a 100×2.1 mm ID column with the common acidic generic gradient in the first dimension, while RPLC under basic pH on a short 30×3 mm ID column is used in the second dimension. Recording data with a UV detector at 215 nm after 1D separation provides the common generic 1D chromatogram. However, after the 2D separation a flow splitter enabled recording of the signals of complementary detectors comprising a diode array detector (DAD) in-line with a charged aerosol detector (CAD) and a quadrupole-time-of-flight (QTOF) mass spectrometer (MS) with an electrospray ionization (ESI) source. Generic conditions of this 2D-LC method have been established through optimization of 2D stationary and mobile phase considering different pH values and buffer concentrations. The orthogonal separation principle has been documented by a number of therapeutic peptides including Exenatide, Octreotide, Cyclosporine A and Oxytocin as well as some other proprietary synthetic peptides. The information density can be further enhanced by using the QTOF-MS detector by data independent acquisition with SWATH. Through this sequential window acquisition of all theoretical fragment ion mass spectra it became possible to collect MS/MS data comprehensively in the high-resolution sampling window, thus enabling the extraction of 2D-EICs from fragment ions and the generation of 2D-contour plots of all product ions. Using Oxytocin as an example for an important therapeutic peptide, the ability of this advanced generic sRP-UV×RP-DAD-CAD-ESI-QTOF-MS/MS method with SWATH for peptide quality control is discussed.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Peptídeos/análise , Acetonitrilos/química , Tampões (Química) , Concentração de Íons de Hidrogênio , Ocitocina/análise , Controle de Qualidade , Solventes/química , Espectrometria de Massas em Tandem
2.
Nat Commun ; 11(1): 3388, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636396

RESUMO

Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining ExM with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.


Assuntos
Corantes Fluorescentes/química , Hidrogéis/química , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Animais , Tampões (Química) , Células COS , Centríolos/metabolismo , Chlamydomonas reinhardtii/metabolismo , Chlorocebus aethiops , Simulação por Computador , Eletrólitos , Epitopos , Imageamento Tridimensional , Microtúbulos/metabolismo , Distribuição Normal , Fotoquímica
3.
Food Chem ; 329: 127175, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32516708

RESUMO

This report describes the development of a methodology based on micellar electrokinetic chromatography for the separation of alcohols on chip-based systems aiming the determination of alcoholic content in whiskey samples. The separation conditions were optimized the best results were achieved using 50 mmolL-1 phosphate buffer containing 30 mmolL-1 sodium dodecyl sulfate. The alcoholic content was determined in 16 seized whiskey samples from 4 different brands as well as in the original samples. The methodology presented herein allowed the correct classification of 75% of the seized samples as adulterated and the data obtained did not statistically differ from those recorded by a reference technique. The proposed analytical approach emerges as a promising tool to provide a rapid screening of the beverages authenticity and it may be useful to be widely explored for the quality control.


Assuntos
Bebidas Alcoólicas/análise , Cromatografia Capilar Eletrocinética Micelar/métodos , Análise de Alimentos/métodos , Tampões (Química) , Butanóis/isolamento & purificação , Cromatografia Capilar Eletrocinética Micelar/instrumentação , Etanol/isolamento & purificação , Qualidade dos Alimentos , Pentanóis/isolamento & purificação , Dodecilsulfato de Sódio
7.
J Chromatogr A ; 1622: 461128, 2020 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-32331779

RESUMO

We present high resolution fast, cost-effective and sensitive Capillary zone electrophoresis (CZE) methods for determination of enantiomeric compounds of Kynurenine pathway, i.e. D, L-Kynurenine (KYN), in human serum and urine samples by cationic-ß-CD and its synergistic dual chiral selector system (SD-CSs) with α-CD in 50 mM borax borate buffer (pH 9.0) as BGE. Acid-mediated stacking enrichment by HCl delivered 15 nM limit of detection (LOD) and 50 nM limit of quantification (LOQ). The methods gave advantages of linearity in the concentration range of 50 nM-1000 nM, reproducibility (RSD ≤ 3.35), selectivity against interfering amino acids, and remarkable recoveries. SD-CSs delivered resolution of D, L-KYN twice that of individual chiral selectors (CSs) under similar conditions. The binding constants (Kb) and electrophoretic mobilities (µeff) of D, L-KYN with different concentrations of CSs were calculated to find the migration order of enantiomers. The chiral recognition mechanism was investigated by molecular docking and molecular mechanics, which revealed strong hydrogen bonding between Kynurenine enantiomers and the SD-CSs as compared to individual CS as the key player in binding, formation of stable complexes which led to the ultimate separation.


Assuntos
Eletroforese Capilar/métodos , Cinurenina/química , alfa-Ciclodextrinas/química , beta-Ciclodextrinas/química , Aminoácidos/química , Tampões (Química) , Cátions , Humanos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Cinética , Cinurenina/sangue , Cinurenina/isolamento & purificação , Cinurenina/urina , Limite de Detecção , Simulação de Acoplamento Molecular , Estereoisomerismo
8.
Bull Environ Contam Toxicol ; 104(5): 689-700, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32303813

RESUMO

In this study, the hydrolysis of amisulbrom in buffer solutions and natural water samples were investigated. Effects of pH and temperature were tested in buffer solutions. Amisulbrom was stable in acidic and neutral aqueous solutions at 25°C, while quickly hydrolyzed with a half-life of 4.5 days (25°C) at pH 9.0. The kinetics rate equation was determined as k = 1.0234 × 1010 exp (-61.3760/R·T) (R2 = 0.9642) for hydrolysis of amisulbrom at pH 9.0. The pH, ionic strength, and solubility were important factors influencing the hydrolysis of amisulbrom in natural water samples. Furthermore, three hydrolysis products were separated and identified in buffer solution (pH 9.0) and natural water samples. A tentative transformation mechanism of amisulbrom was proposed to rationalize the formation of HPs (hydrolysis products) based on their structural identification, DFT (density functional theory), and hydrolysis profiles. Toxicity prediction using the quantitative structure-activity relationship model revealed that the HP-I, and HP-II were more toxic than the parent amisulbrom. This investigation was the first to evaluate the behavior of amisulbrom hydrolysis in aquatic systems.


Assuntos
Água Doce/química , Indóis/química , Modelos Químicos , Praguicidas/química , Triazóis/química , Poluentes Químicos da Água/química , Tampões (Química) , Água Doce/análise , Concentração de Íons de Hidrogênio , Hidrólise , Indóis/análise , Cinética , Modelos Moleculares , Estrutura Molecular , Concentração Osmolar , Praguicidas/análise , Solubilidade , Soluções , Temperatura , Triazóis/análise , Poluentes Químicos da Água/análise
12.
J Food Sci ; 85(4): 910-917, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32198767

RESUMO

The pH of most acid food products depends on undefined and complex buffering of ingredients but is critically important for regulatory purposes and food safety. Our objective was to define the buffer capacity (BC) of ingredients in salad dressing products. Ingredients of salad dressings were titrated individually and in combination using concentrations typical of dressing products. Titration curves from pH 2 to 12 were generated with sodium hydroxide and hydrochloric acid, which were then used to generate BC curves. A matrix of concentration and pK values for a series of monoprotic buffers approximated the pH of each ingredient. Some buffer series required anion or cation corrections for accurate pH prediction, possibly due to the presence of salts of acid or bases. Most buffers had BC values less than 10-fold the BC of acetic acid (0.25 ß) typically in dressing formulations and had little influence on the final product pH of the dressings tested. Unexpectedly, we found that sugars in dressing formulations, including sucrose or corn syrup, exhibited buffering at pH values greater than 11 (0.035 ß and 0.059 ß, respectively), which was likely due to weakly acidic hydroxyl groups on the sugar molecules. However, the concentration and pK for buffers above pH 11 or below pH 2 were difficult to quantify due to the BC of water. The BC data may help to quantify the effects of salad dressing ingredients on the final product pH and benefit regulatory agencies and manufacturers in assessing product pH and safety. PRACTICAL APPLICATION: Buffer capacity data for salad dressing ingredients may help determine the influence ingredient addition will have on the final pH of a salad dressing product. The addition of low acid ingredients with little or no buffering may not significantly alter pH. The modeling method may be useful for regulatory purposes to estimate the effects of low acid ingredients on pH changes for food safety and may also be useful for product development of acid and acidified foods.


Assuntos
Condimentos/análise , Ingredientes de Alimentos/análise , Ácido Acético/análise , Tampões (Química) , Concentração de Íons de Hidrogênio , Sais/análise
13.
J Food Sci ; 85(4): 918-925, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32199038

RESUMO

Standard ionic equilibria equations may be used for calculating pH of weak acid and base solutions. These calculations are difficult or impossible to solve analytically for foods that include many unknown buffering components, making pH prediction in these systems impractical. We combined buffer capacity (BC) models with a pH prediction algorithm to allow pH prediction in complex food matrices from BC data. Numerical models were developed using Matlab software to estimate the pH and buffering components for mixtures of weak acid and base solutions. The pH model was validated with laboratory solutions of acetic or citric acids with ammonia, in combinations with varying salts using Latin hypercube designs. Linear regressions of observed versus predicted pH values based on the concentration and pK values of the solution components resulted in estimated slopes between 0.96 and 1.01 with and without added salts. BC models were generated from titration curves for 0.6 M acetic acid or 12.4 mM citric acid resulting in acid concentration and pK estimates. Predicted pH values from these estimates were within 0.11 pH units of the measured pH. Acetic acid concentration measurements based on the model were within 6% accuracy compared to high-performance liquid chromatography measurements for concentrations less than 400 mM, although they were underestimated above that. The models may have application for use in determining the BC of food ingredients with unknown buffering components. Predicting pH changes for food ingredients using these models may be useful for regulatory purposes with acid or acidified foods and for product development. PRACTICAL APPLICATION: Buffer capacity models may benefit regulatory agencies and manufacturers of acid and acidified foods to determine pH stability (below pH 4.6) and how low-acid food ingredients may affect the safety of these foods. Predicting pH for solutions with known or unknown buffering components was based on titration data and models that use only monoprotic weak acids and bases. These models may be useful for product development and food safety by estimating pH and buffering capacity.


Assuntos
Ácidos/análise , Análise de Alimentos , Algoritmos , Tampões (Química) , Cromatografia Líquida de Alta Pressão , Inocuidade dos Alimentos , Concentração de Íons de Hidrogênio , Modelos Biológicos , Sais/análise
14.
J Chromatogr A ; 1620: 461032, 2020 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-32199675

RESUMO

The prominent biological effects of adrenaline (A), noradrenaline (NA) and dopamine (DA) as well as the clinical importance of their metabolites (such as dihydroxyphenylacetic acid (DOPAC), methoxy­4-hydroxyphenyl glycol (MHPG), dihydroxyphenylglycol (DHPG), metanephrine (M), normetanephrine (NM), vanillylmandelic acid (VMA), homovanillic acid (HVA)) have forced researchers to evaluate new analytical methodologies for their isolation and preconcentration from biological samples. For this reason, the three most popular extraction techniques (dispersive liquid-liquid microextraction (DLLME), solid-phase extraction (SPE), solid-phase microextraction (SPME)) were tested. Micellar electrokinetic chromatography (MEKC) - a mode of capillary electrophoresis - with a diode array detector (DAD) was applied to assess the extraction efficiency. Next, the enrichment factor (EF) of each applied method was calculated in respect to standard mixtures of the analytes at the same concentration levels. The EF results of seven selected metabolites of biogenic amines (BAs) from urine after sample preparation procedures based on twenty-five different protocols (one DLLME, thirteen SPE and eleven SPME) were calculated and compared using hierarchical cluster analysis (HCA). The SPE as well as SPME procedures were proved to be the most effective approaches for the simultaneous extraction of the chosen compounds. Moreover, an ionic liquid (IL) - 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide - added to methanol in SPME additionally could successfully improve the extraction efficiency. It was also confirmed that the HCA approach could be considered a supportive tool in the selection of a suitable sample preparation procedure for that group of endogenous substances.


Assuntos
Cromatografia Capilar Eletrocinética Micelar/métodos , Redes e Vias Metabólicas , Tirosina/análise , Aminas Biogênicas , Tampões (Química) , Análise por Conglomerados , Eletrólitos/química , Humanos , Reprodutibilidade dos Testes , Extração em Fase Sólida , Tirosina/química , Tirosina/urina
15.
Biochemistry ; 59(12): 1289-1297, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32167292

RESUMO

Cobalt-mimochrome VI*a (CoMC6*a) is a synthetic mini-protein that catalyzes aqueous proton reduction to hydrogen (H2). In buffered water, there are multiple possible proton donors, complicating the elucidation of the mechanism. We have found that the buffer pKa and sterics have significant effects on activity, evaluated via cyclic voltammetry (CV). Protonated buffer is proposed to act as the primary proton donor to the catalyst, specifically through the protonated amine of the buffers that were tested. At a constant pH of 6.5, catalytic H2 evolution in the presence of buffer acids with pKa values ranging from 5.8 to 11.6 was investigated, giving rise to a potential-pKa relationship that can be divided into two regions. For acids with pKa values of ≤8.7, the half-wave catalytic potential (Eh) changes as a function of pKa with a slope of -128 mV/pKa unit, and for acids with pKa of ≥8.7, Eh changes as a function of pKa with a slope of -39 mV/pKa unit. In addition, a series of buffer acids were synthesized to explore the influence of steric bulk around the acidic proton on catalysis. The catalytic current in CV shows a significant decrease in the presence of the sterically hindered buffer acids compared to those of their parent compounds, also consistent with the added buffer acid acting as the primary proton donor to the catalyst and showing that acid structure in addition to pKa impacts activity. These results demonstrate that buffer acidity and structure are important considerations when optimizing and evaluating systems for proton-dependent catalysis in water.


Assuntos
Cobalto/química , Deuteroporfirinas/química , Hidrogênio/química , Metaloproteínas/química , Prótons , Tampões (Química) , Catálise , Concentração de Íons de Hidrogênio , Água/química
16.
J Vis Exp ; (157)2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32202528

RESUMO

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (> 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 °C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.


Assuntos
Química Farmacêutica/métodos , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos , Verde de Indocianina/administração & dosagem , Lipídeos/química , Lipossomos/química , Microfluídica , Sulfato de Amônio , Tampões (Química) , Cromatografia , Concentração de Íons de Hidrogênio , Dispositivos Lab-On-A-Chip , Temperatura
17.
J Chem Phys ; 152(4): 045102, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32007034

RESUMO

The physical chemistry of liquid-liquid phase separation (LLPS) of polymer solutions bears directly on the assembly of biologically functional dropletlike bodies from proteins and nucleic acids. These biomolecular condensates include certain extracellular materials and intracellular compartments that are characterized as "membraneless organelles." Analytical theories are a valuable, computationally efficient tool for addressing general principles. LLPS of neutral homopolymers is quite well described by theory, but it has been a challenge to develop general theories for the LLPS of heteropolymers involving charge-charge interactions. Here, we present a theory that combines a random-phase-approximation treatment of polymer density fluctuations and an account of intrachain conformational heterogeneity based on renormalized Kuhn lengths to provide predictions of LLPS properties as a function of pH, salt, and charge patterning along the chain sequence. Advancing beyond more limited analytical approaches, our LLPS theory is applicable to a wide variety of charged sequences ranging from highly charged polyelectrolytes to neutral or nearly neutral polyampholytes. This theory should be useful in high-throughput screening of protein and other sequences for their LLPS propensities and can serve as a basis for more comprehensive theories that incorporate nonelectrostatic interactions. Experimental ramifications of our theory are discussed.


Assuntos
Biopolímeros/química , Modelos Químicos , Polieletrólitos/química , Polímeros/química , Tampões (Química) , Ensaios de Triagem em Larga Escala , Extração Líquido-Líquido/métodos
18.
Nat Protoc ; 15(3): 1132-1157, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32005983

RESUMO

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is time-consuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes or clarified cell lysates. In the liquid chromatography coupled to mass spectrometry (LC-MS) approach described in this protocol, samples in MS-incompatible conditions are injected onto a short size-exclusion chromatography column. Proteins and protein complexes are separated from small molecule non-volatile buffer components using an aqueous, non-denaturing mobile phase. Eluted proteins and protein complexes are detected by the mass spectrometer after electrospray ionization. Mass spectra can inform regarding protein sample purity and oligomerization, and additional tandem mass spectra can help to further obtain information on protein complex subunits. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.


Assuntos
Técnicas de Química Analítica/métodos , Espectrometria de Massas/métodos , Proteínas/química , Tampões (Química) , Cromatografia em Gel
19.
Science ; 367(6476): 364-365, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31974233
20.
Pharm Res ; 37(3): 42, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31989335

RESUMO

PURPOSE: The design of biorelevant conditions for in vitro evaluation of orally administered drug products is contingent on obtaining accurate values for physiologically relevant parameters such as pH, buffer capacity and bile salt concentrations in upper gastrointestinal fluids. METHODS: The impact of sample handling on the measurement of pH and buffer capacity of aspirates from the upper gastrointestinal tract was evaluated, with a focus on centrifugation and freeze-thaw cycling as factors that can influence results. Since bicarbonate is a key buffer system in the fasted state and is used to represent conditions in the upper intestine in vitro, variations on sample handling were also investigated for bicarbonate-based buffers prepared in the laboratory. RESULTS: Centrifugation and freezing significantly increase pH and decrease buffer capacity in samples obtained by aspiration from the upper gastrointestinal tract in the fasted state and in bicarbonate buffers prepared in vitro. Comparison of data suggested that the buffer system in the small intestine does not derive exclusively from bicarbonates. CONCLUSIONS: Measurement of both pH and buffer capacity immediately after aspiration are strongly recommended as "best practice" and should be adopted as the standard procedure for measuring pH and buffer capacity in aspirates from the gastrointestinal tract. Only data obtained in this way provide a valid basis for setting the physiological parameters in physiologically based pharmacokinetic models.


Assuntos
Bicarbonatos/química , Ácidos e Sais Biliares/química , Líquidos Corporais/química , Líquidos Corporais/metabolismo , Trato Gastrointestinal Superior/química , Trato Gastrointestinal Superior/metabolismo , Tampões (Química) , Famotidina/administração & dosagem , Famotidina/metabolismo , Absorção Gastrointestinal , Humanos , Concentração de Íons de Hidrogênio , Ibuprofeno/administração & dosagem , Ibuprofeno/metabolismo , Intestino Delgado , Sais/química , Estômago
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