Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 18.488
Filtrar
1.
Talanta ; 236: 122833, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34635223

RESUMO

A dynamic pH junction was used in capillary electrophoresis (CE-DAD) to on-line preconcentrate, separate, and determine trace amounts of sulfonamide antibiotics (SAs) in milk and yoghurt samples in this study. A sample matrix with 0.15% acetic acid and 10% methanol (MeOH) at a pH of 4.0, and a background electrolyte (BGE) that contained 35 mM sodium citrate with 10% MeOH at a pH of 8.5, and an acidic barrage of 0.4% acetic acid with 10% MeOH at a pH of 2.5 were utilised to achieve a stacking effect for SAs through a dynamic pH junction. Under optimised conditions, the proposed preconcentration method showed good linearity (30-500 ng/mL, r2 ≥ 0.9940), low limits of detection (LODs) of 4.1-6.3 ng/mL, and acceptable analytes recovery (81.2-106.9%) with relative standard deviations (RSDs) within 5.3-13.7 (n = 9). The limits of quantification (LOQs) were below the maximum residue limit approved by the European Union (EU) in this type of matrices. Sensitivity enhancement factors of up to 129 were reached with the optimised dynamic pH junction using CE with a diode array detector (DAD). The method was used to determine SAs in fresh milk, low-fat milk, full-cream milk, and yoghurt samples.


Assuntos
Antibacterianos , Iogurte , Animais , Eletroforese Capilar , Concentração de Íons de Hidrogênio , Leite , Sulfonamidas
2.
Anal Chim Acta ; 1184: 338892, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34625256

RESUMO

Prostate cancer represents the second highest malignancy rate in men in all cancer diagnoses worldwide. The development and progression of prostate cancer is not completely understood yet at molecular level, but it has been reported that changes in the N-glycosylation of prostate-specific antigen (PSA) occur during tumor genesis. In this paper we report on the development and implementation of a high-throughput capillary electrophoresis based glycan analysis workflow for urinary PSA analysis. The technology utilizes selective, high yield single domain antibody based PSA capture, followed by preconcentration and capillary electrophoresis coupled with laser-induced fluorescence detection, resulting in high resolution N-glycan profiles. Urinary PSA glycan profiles were compared to a commercially available PSA standard revealing differences in their α2,3- and α2,6-sialylated isomers, proving the excellent selectivity of the suggested workflow. This is important as sialylation classification plays an important role in the differentiation between indolent, significant and aggressive forms of prostate cancer.


Assuntos
Antígeno Prostático Específico , Neoplasias da Próstata , Eletroforese Capilar , Glicosilação , Humanos , Masculino , Neoplasias da Próstata/diagnóstico , Fluxo de Trabalho
3.
Anal Chim Acta ; 1182: 338959, 2021 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-34602190

RESUMO

Volatile solvents are excellent extraction media for liquid-liquid extractions. However, their use in supported liquid membranes (SLMs) is limited by their evaporation from SLM and thus poor SLM stability and they have never been considered truly useful for electromembrane extraction (EME). In this contribution, volatile solvents were systematically investigated as liquid membranes for EME and their extraction characteristics were comprehensively examined for the first time. A short plug of a water immiscible volatile solvent (a free liquid membrane (FLM)) was sandwiched between two aqueous plugs (donor and acceptor solutions) in a narrow-bore polymeric tubing. Evaporation of the volatile FLM was thus completely avoided and excellent stability of the phase interface was ensured. Suitability of volatile FLMs for EMEs was justified by µ-EMEs of nortriptyline, haloperidol, loperamide and papaverine as model non-polar basic drugs. Extraction performance of µ-EME through ethyl acetate was comparable or better to that through standard non-volatile EME solvents and a high extraction selectivity was achieved for nortriptyline and haloperidol extracted through chloroform. µ-EMEs through the volatile FLMs were characterized by high extraction recoveries (62%-99% for standards and 40-89% for body fluids), low electric currents (10-1380 nA), no susceptibility to matrix ions and suitability for pretreatment of raw body fluids (human urine and serum). Resulting extracts were analysed by capillary electrophoresis with ultraviolet detection (CE/UV). Repeatability of the µ-EME-CE/UV method was excellent with intra-day and inter-day RSD values 0.8-3.2% and 1.8-4.6%, respectively. Further experiments demonstrated additional advantages of volatile FLMs by nearly exhaustive µ-EMEs of atenolol as the polar basic drug with no need for FLM modification by ionic carriers. The presented comprehensive examination of volatile solvents has broadened the range of liquid membranes suitable for EME and it is believed that this proof-of-concept study will stimulate further interest in a deeper investigation of volatile phase interfaces in EME.


Assuntos
Membranas Artificiais , Preparações Farmacêuticas , Eletricidade , Eletroforese Capilar , Humanos , Solventes
4.
Anal Chim Acta ; 1183: 338958, 2021 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-34627506

RESUMO

One of the most extensively utilized rapid characterization, release and stability testing methods of therapeutic proteins in the biopharmaceutical field today is capillary SDS gel electrophoresis using borate cross-linked high molecular weight dextran. In spite of its widespread use, however, the gel composition dependent separation characteristics of this very unique sieving matrix has not been investigated yet. Introduction of three dimensional (3D) Ferguson plots, based on simultaneous variation of the dextran (D) and borate (B) concentrations generating 16 different D/B ratio gels, allowed better understanding of the electromigration process of the SDS-protein complexes. As a result of this comprehensive study, non-linear 3D logarithmic mobility vs dextran and borate concentration surfaces were obtained. Both, the molecular weight protein standards and the new modality fusion protein etanercept resulted in concave 3D Ferguson plots. The interplay between the electroosmotic flow and the viscosity of the matrices played a key role in the resulting migration time and resolution. Selectivity values were defined and evaluated in 3D graph formats for the regular and de-N-glycosylated subunits of etanercept, as well as for the latter with the 10 kDa internal standard to understand both the dextran-borate complexation and sized based selectivities. KR plots of the retardation coefficients as the function of the logarithmic molecular weights were used to more precisely assess the Mw of the samples and to obtain information about the molecular characteristics of the electromigrating SDS-protein complexes.


Assuntos
Eletroforese Capilar , Proteínas , Géis , Peso Molecular , Dodecilsulfato de Sódio
5.
Anal Chim Acta ; 1183: 338936, 2021 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-34627519

RESUMO

While ultraviolet light (UV) absorbance detection is the most widely used detection mode in capillary electrophoresis (CE), it can yield poor concentration sensitivity and has tendencies to exhibit baseline fluctuations. In order to overcome these challenges, alternative detection strategies, including the use of dedicated wavelength lasers, have been applied, resulting in enhancements of concentration sensitivity as well as decreased baseline disturbance. In this work, using a laser driven light source for excitation, we reported a native fluorescence detection (NFD) scheme for use in a commercial CE platform, PA 800 Plus Pharmaceutical Analysis System, for protein analysis. The CE-NFD system was characterized using tryptophan and a reduced IgG. We compared NFD with UV absorbance detection as applied to sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) and capillary isoelectric focusing (cIEF). In SDS-CGE, with the reported NFD a non-reduced IgG standard sample yielded a signal-to-noise ratio which was 14.6 times higher than with UV absorbance detection at 214 nm. In cIEF analysis of NISTmAb, Humanized IgG1k, with NFD ∼170 times less sample mass was needed to obtain similar profile quality to that with UV absorbance detection at 280 nm. NFD also eliminated baseline anomalies observed with UV absorbance detection and showed less interference by other absorbing species. These results suggest that CE-NFD is a practical and powerful tool for protein characterization in the biopharmaceutical industry.


Assuntos
Eletroforese Capilar , Lasers , Focalização Isoelétrica , Luz , Espectrometria de Fluorescência
6.
Adv Exp Med Biol ; 1336: 129-137, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34628630

RESUMO

Glycomics has a growing interest in the biopharmaceutical industry and biomedical research requiring new high-performance and high-sensitivity bioanalytical tools. Analysis of N-glycosylation is very important during the development of protein therapeutics and it also plays a key role in biomarker discovery. The most frequently used glycoanalytical methods are capillary electrophoresis, liquid chromatography, and mass spectrometry. In this chapter, the capillary electrophoresis-based N-linked carbohydrate analysis methods are conferred with emphasis on its use in the biopharmaceutical and biomedical fields.


Assuntos
Produtos Biológicos , Eletroforese Capilar , Glicômica , Glicoproteínas/metabolismo , Glicosilação
7.
Adv Exp Med Biol ; 1336: 51-86, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34628627

RESUMO

This chapter aims to explore various parameters involved in achieving high-end capillary electrophoresis hyphenated to mass spectrometry (CE-MS) analysis of proteins, peptides, and their posttranslational modifications. The structure of the topics discussed in this book chapter is conveniently mapped on the scheme of the CE-MS system itself, starting from sample preconcentration and injection techniques and finishing with mass analyzer considerations. After going through the technical considerations, a variety of relevant applications for this analytical approach are presented, including posttranslational modifications analysis, clinical biomarker discovery, and its growing use in the biotechnological industry.


Assuntos
Eletroforese Capilar , Proteômica , Espectrometria de Massas , Peptídeos , Proteínas
8.
Adv Exp Med Biol ; 1336: 87-104, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34628628

RESUMO

Peptides play a crucial role in many vitally important functions of living organisms. The goal of peptidomics is the identification of the "peptidome," the whole peptide content of a cell, organ, tissue, body fluid, or organism. In peptidomic or proteomic studies, capillary electrophoresis (CE) is an alternative technique for liquid chromatography. It is a highly efficient and fast separation method requiring extremely low amounts of sample. In peptidomic approaches, CE is commonly combined with mass spectrometric (MS) detection. Most often, CE is coupled with electrospray ionization MS and less frequently with matrix-assisted laser desorption/ionization MS. CE-MS has been employed in numerous studies dealing with determination of peptide biomarkers in different body fluids for various diseases, or in food peptidomic research for the analysis and identification of peptides with special biological activities. In addition to the above topics, sample preparation techniques commonly applied in peptidomics before CE separation and possibilities for peptide identification and quantification by CE-MS or CE-MS/MS methods are discussed in this chapter.


Assuntos
Proteômica , Espectrometria de Massas em Tandem , Eletroforese Capilar , Peptídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
9.
Adv Exp Med Biol ; 1336: 159-178, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34628632

RESUMO

Capillary electrophoresis-mass spectrometry (CE-MS) is a very useful analytical technique for the selective and highly efficient profiling of polar and charged metabolites in a wide range of biological samples. Compared to other analytical techniques, the use of CE-MS in metabolomics is relatively low as the approach is still regarded as technically challenging and not reproducible. In this chapter, the possibilities of CE-MS for metabolomics are highlighted with special emphasis on the use of recently developed interfacing designs. The utility of CE-MS for targeted and untargeted metabolomics studies is demonstrated by discussing representative and recent examples in the biomedical and clinical fields. The potential of CE-MS for large-scale and quantitative metabolomics studies is also addressed. Finally, some general conclusions and perspectives are given on this strong analytical separation technique for probing the polar metabolome.


Assuntos
Eletroforese Capilar , Metabolômica , Espectrometria de Massas , Metaboloma , Software
10.
Anal Chim Acta ; 1178: 338811, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34482872

RESUMO

Capillary gel electrophoresis is widely applied for determination of sequence and size of DNA, in which the sieving gel plays an unignorable role. Herein, a pore-size controllable hydrogel was synthesized in the capillary with two symmetrical tetrahedron-like macromonomers consisting of pentaerythritoltetra (succinimidylcarboxypentyl) polyoxyethylene (PS) and pentaerythritoltetra (aminopropyl) polyoxyethylene) (PA). By capillary electrophoresis of the DNA fragments with this hydrogel, it is found that a homogenous structure of hydrogel which is more suitable for the DNA separation can be achieved when the molecular weight of PA is approximate to that of PS. DNA fragments smaller than 1500 bp can be well resolved in this hydrogel within 13 min. More than 100 consecutive runs can be carried out in such a dynamically coated capillary before performance begins to degrade. Notably, such hydrogel can realize separation of dsDNA up to single base pair resolution and same length of dsDNA with 1 bp difference.


Assuntos
Hidrogéis , Polietilenoglicóis , DNA , Eletroforese Capilar , Peso Molecular
11.
Anal Chim Acta ; 1178: 338789, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34482877

RESUMO

Electroosmotic flow (EOF) plays a pivotal role in optimization of capillary electrophoresis (CE) separations of (bio)molecules and (bio)particles. EOF velocity is directly related to analysis time, peak resolution and separation efficiency. Here, we report a concept of charged polymer coatings of the inner fused silica capillary wall, which allows anodic EOF with mobility ranging from 0 to ∼(30-40) × 10-9 m2V-1s-1. The capillary wall is modified by covalently bound cationic copolymer poly(acrylamide-co-(3-acrylamidopropyl)trimethylammonium chloride) (PAMAPTAC) containing variable ratio of the charged monomer in the 0-60 mol. % interval. The EOF mobility showed minor variability with composition of background electrolyte (BGE) and pH in the 2-10 interval. The coatings were evaluated by CE-UV and nanospray CE-MS in the counter-EOF arrangement for a series of basic drug molecules in acetic acid based acidic BGE. Tunable EOF velocity was demonstrated as a useful tool for optimization of peak resolution, separation efficiency and migration time of analytes. Electrostatic repulsion of positively charged capillary surface was shown as beneficial for suppression of analyte adsorption, notably for hydrophobic cationic analytes.


Assuntos
Eletro-Osmose , Eletroforese Capilar , Adsorção , Cátions , Polímeros
12.
Talanta ; 235: 122747, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517615

RESUMO

Microchip capillary electrophoresis (MCE) is a powerful technique for rapid separation; however, its acceptance in routine laboratories is still limited. Compromises caused by the efforts for solving different problems, such as reducing its cost of fabrication and ensuring high separation efficiency, undermine the competitiveness of this technology compared to other separation techniques. Contrary to the conventional pursuit of narrow microchannels, this study investigated the suitability of microchips with channels at the sub-millimeter level, targeting the simplification of the overall operation, cost reduction, and robustness improvement. To this effect, we considered the influence of pressurized flow and Joule heating on the separation. The suppression of pressurized flow with viscous solutions was confirmed through a combination of simulations and experimental results, indicating that the buffer viscosity was enough for successful separation. We fabricated channels of 200 µm × 230 µm using computer numerical controlled (CNC) machining and obtained theoretical plate numbers of 4.8 × 105 m-1 and 5.3 × 105 m-1 for fluorescein isothiocyanate (FITC) labeled small molecules and DNA fragments, respectively, with a buffer viscosity of 168 mPa s (0.5 % hydroxypropyl methylcellulose, HPMC). These values are comparable with that of narrow-bore microchips. Furthermore, we did not observe any deleterious effects with low-conductivity buffers. We investigated the rapid and highly sensitive detection of mycoplasma contamination and the real samples of circulating cell-free DNA (cfDNA), which gave a limit of detection (LOD) as low as 2.3 ng mL-1. Owing to the significant reduction in cost, ease of operation, and fast separation capabilities demonstrated in this work, MCE can be a viable alternative to the usual slab gel electrophoresis running in most biological laboratories.


Assuntos
Eletroforese em Microchip , DNA , Eletroforese Capilar , Derivados da Hipromelose , Limite de Detecção
13.
Se Pu ; 39(10): 1077-1085, 2021 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-34505429

RESUMO

After entering human blood circulation, small-molecule drugs interact extensively with various plasma proteins, such as human serum albumin and α1-acid glycoprotein. These interactions profoundly affect the distribution of drugs in vivo and the binding of drugs to targets, thus affecting the efficacy of drugs. In-depth investigation of drug-plasma protein interactions is of great significance for the optimization of drug properties, the development of new drugs, risk assessment, and combination therapy of drugs. Therefore, it is essential to develop highly efficient, sensitive, and accurate methods for elucidating drug-plasma protein interactions. Chromatography is a powerful tool with high throughput, high separation performance, and high sensitivity in the characterization of drug-protein interactions. High-performance affinity chromatography (HPAC) and capillary electrophoresis (CE) have been widely utilized in this field. These methods include the determination of the effects of the posttranslational modification of proteins on binding and the competitive binding of multiple drugs. In addition, various chromatographic methods are used to obtain interaction information such as the binding constant, binding-site number, and dissociation rate constant. In this review, the common strategies and recent advances in HPAC and CE in the study of drug-plasma protein interactions are briefly reviewed. The immobilization methods of proteins, the principles and applications of frontal analysis, zonal elution, ultrafast affinity extraction, peak profiling, and peak decay analysis are discussed for HPAC and affinity capillary electrophoresis (ACE) and capillary electrophoresis frontal analysis (CE-FA) for CE. HPAC relies on the fixation of proteins on the surfaces of chromatographic stationary phases by covalent linking or physical adsorption, followed by obtaining the drug-protein interaction information through a variety of chromatographic methods. In the frontal chromatography analysis, mobile phases with different concentrations of drugs are passed through the HPAC column to obtain different breakthrough times. The process can determine the number of drug binding sites and the binding constant of each site in the affinity protein with high accuracy. The zonal elution method can detect the drug binding sites on proteins using site-specific probes to determine whether there is competition between drugs and probes. The sample consumption and analysis time of the zonal elution method are much less than those in frontal chromatography analysis. The ultrafast affinity extraction method can inject complex samples, such as serum, into affinity columns to determine the free drug components. It can measure the combination and dissociation constants of drug-protein interactions by changing the chromatography flow rate. Peak profiling and peak decay analyses are both effective methods for investigating the dissociation of drugs and proteins. In CE analysis, the drug and protein samples are dissolved in an electrophoresis buffer, and their interactions are measured during electrophoresis with high accuracy and low sample consumption. However, the adsorption of proteins on the capillary wall can compromise CE performance. Common CE methods in drug-protein interaction analysis are ACE and CE-FA. ACE is usually performed by changing the effective mobility of drugs via the addition of different concentrations of proteins. This method has been widely used, and several variant techniques have been developed recently. CE-FA involves the sampling of a drug premixed at a known concentration with a target protein. Compared with other CE methods, CE-FA exhibits the unique advantages of high throughput, automatic online analysis, and the ability to determine high-order drug-protein interactions. Finally, the shortcomings of current chromatography methods are summarized, and the application prospects and development direction of chromatography technology in the field of drug-plasma protein interaction research are discussed.


Assuntos
Proteínas Sanguíneas , Preparações Farmacêuticas , Sítios de Ligação , Proteínas Sanguíneas/metabolismo , Cromatografia de Afinidade , Eletroforese Capilar , Humanos , Ligação Proteica
14.
J Pharm Biomed Anal ; 205: 114327, 2021 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-34479172

RESUMO

Lipid-oligonucleotides (LON) attract great interest as supramolecular scaffolds to improve the intracellular delivery of nucleic acids. Analytical characterization of LON assemblies is critical to formulation development, understanding in-vivo performance, as well as quality control. For this study, we selected LONs featuring different modifications on both oligonucleotide (with or without a G4 prone sequence) and lipid (mono or bis-alkyl chain covalently attached to the oligonucleotide sequence). Size exclusion chromatography (SEC) and, for the first time, capillary electrophoresis (CE) were investigated to study LON supramolecular self-assemblies. Results were correlated to those obtained with conventional physico-chemical characterization techniques i.e. gel electrophoresis, dynamic light scattering, and circular dichroism. In SEC, a separation between LON monomers and micelles was achieved in 5min on a TSK-gel G3000PW column at 70°C with 100% water, as mobile phase. CE conditions were optimized using a fused-silica capillary length of 10.0cm effective length at 15°C. Different background electrolytes were tested by varying the nature and the concentration of salts added. A sodium tetraborate buffer with 75mM NaCl appeared suitable to promote LON assembly. CE offers benefits to LON micelle analysis in terms of speed of analysis, high resolution, and low quantity of sample injected. Moreover, CE provides an appropriate tool to assess the impact of media of biological relevance on LON self-assembly. In this work, the key role of lipophilic tails and the formation of tetramolecular G-quadruplexes on the stability of LON micelles was confirmed.


Assuntos
Eletroforese Capilar , Oligonucleotídeos , Cromatografia em Gel , Lipídeos , Micelas
15.
Molecules ; 26(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361834

RESUMO

Chirality is one of the major issues in pharmaceutical research and industry. Capillary electrophoresis (CE) is an interesting alternative to the more frequently used chromatographic techniques in the enantioseparation of pharmaceuticals, and is used for the determination of enantiomeric ratio, enantiomeric purity, and in pharmacokinetic studies. Traditionally, optimization of CE methods is performed using a univariate one factor at a time (OFAT) approach; however, this strategy does not allow for the evaluation of interactions between experimental factors, which may result in ineffective method development and optimization. In the last two decades, Design of Experiments (DoE) has been frequently employed to better understand the multidimensional effects and interactions of the input factors on the output responses of analytical CE methods. DoE can be divided into two types: screening and optimization designs. Furthermore, using Quality by Design (QbD) methodology to develop CE-based enantioselective techniques is becoming increasingly popular. The review presents the current use of DoE methodologies in CE-based enantioresolution method development and provides an overview of DoE applications in the optimization and validation of CE enantioselective procedures in the last 25 years. Moreover, a critical perspective on how different DoE strategies can aid in the optimization of enantioseparation procedures is presented.


Assuntos
Eletroforese Capilar/métodos , Preparações Farmacêuticas/química , Preparações Farmacêuticas/isolamento & purificação , Humanos , Estereoisomerismo
16.
Anal Chem ; 93(34): 11843-11851, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34410102

RESUMO

A thermally reversible nanogel is used in capillary electrophoresis to create discrete regions for a galactosyltransferase reaction and separation. The ß1-4 galactosyltransferase enzyme, donor, and co-factor were patterned in the capillary. The substrate was driven through these zones and converted to galactosylated products, which were separated and identified. Using this capillary electrophoresis method, the degree of glycosylation was discernible for a pentasaccharide and for biantennary N-glycans. With the ability to distinguish between reaction products for which either one or two galactose residues were transferred, the capillary nanogel electrophoresis system was used to determine the Michaelis-Menten value, KM. For the ß1-4 galactosyltransferase, the KM value obtained for a pentasaccharide substrate was 1.23 ± 0.08 mM. Once KM was established, the enzyme/substrate ratio was evaluated to add a single galactose residue or to fully galactosylate a biantennary N-glycan. Additionally, capillary nanogel electrophoresis was adapted to transfer galactose residues to protein. The applicability of the method for real-time online modification of whole protein was demonstrated with the Herceptin glycoprotein. Complete retardation by Erythrina cristagalli lectin after enzymatic modification confirmed the addition of galactose residues to the Herceptin. This demonstrated the potential of the method to be used for online modification of other glycoproteins.


Assuntos
Galactose , Polissacarídeos , Eletroforese Capilar , Galactosiltransferases , Glicoproteínas , Nanogéis
18.
Molecules ; 26(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34443426

RESUMO

Proteomics and metabolomics are analytic tools used in combination with bioinformatics to study proteins and metabolites which contribute to describing complex biological systems. The growing interest in research concerning the resolution of these systems has stimulated the development of sophisticated procedures and new applications. This paper introduces the evolution of statistical techniques for the treatment of data, suggesting the possibility to successfully characterize the milk-whey syneresis process by applying two-dimensional correlation analysis (2DCOR) to a series of CE electropherograms referring to milk-whey samples collected during cheese manufacturing. Two cheese-making processes to produce hard cheese (Grana type) and fresh cheese (Crescenza) were taken as models. The applied chemometric tools were shown to be useful for the treatment of data acquired in a systematically perturbed chemical system as a function of time.


Assuntos
Ácidos/análise , Queijo/análise , Leite/química , Nitrogênio/análise , Soro do Leite/química , Animais , Eletroforese Capilar , Projetos Piloto
19.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361026

RESUMO

In the presented study, a capillary electrophoresis-mass spectrometry method combining high separation efficiency and sensitive detection has been developed and validated, for the first time, to quantify branched chain amino acids (valine, isoleucine, leucine) in commercial food and sport supplement samples and human plasma samples. The separations were performed in a bare fused silica capillary. The background electrolyte was composed of 500 mM formic acid with pH 2.0. The plasma sample pretreatment was realized by simple protein precipitation with acetonitrile. Injection of a short zone of highly basic electrolyte before the sample injection and application of the negative pressure on the separation were accompanied by enhanced resolution of the isobaric amino acids-isoleucine and leucine. The developed method was characterized by favorable validation parameters, such as linearity (r2 > 0.99), accuracy and precision, the limit of detection, lower limit of quantification, or robustness. These parameters were more than sufficient for the quantification of branched chain amino acids in various samples. The determined concentrations of branched chain amino acids in food and sports supplements were in very good agreement with the content declared by the manufacturer. The investigated concentrations of branched chain amino acids were in the range 294.68-359.24 µM for valine, 91.76-95.67 µM for isoleucine, and 196.78-251.24 µM for leucine. These concentrations fall within the physiological limits. The developed CE-MS/MS method represents a suitable alternative to traditional approaches used in branched chain amino acid quality control and bioanalysis.


Assuntos
Aminoácidos de Cadeia Ramificada/análise , Sangue/metabolismo , Suplementos Nutricionais , Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Adulto , Aminoácidos de Cadeia Ramificada/sangue , Análise Química do Sangue/métodos , Humanos , Masculino
20.
Anal Methods ; 13(34): 3845-3851, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34378552

RESUMO

Cobratide is a peptide drug extracted from the venom of Chinese cobra, and has been widely used in the clinical treatment of chronic, intractable and persistent pain. In a recent study, it was reported that it has the potential to treat COVID-19. In order to control the quality of commercial cobratide drugs, a protocol was established for the separation, identification and quantification of cobratide and its associated impurities, in which sheathless capillary electrophoresis-mass spectrometry (CE-MS) was used for identification and a rapid capillary electrophoresis-ultraviolet-visible detector (CE-UV) method was developed for accurate quantification. Separation conditions that affect the resolution and MS intensities of cobratide and its associated impurities were investigated, including pH value, concentration of background electrolyte (BGE), ratio of organic additive and sample solution. The optimized CE conditions (BGE: 50 mM NH4Ac, pH 4.0; sample solution: deionized water) were used for both sheathless CE-MS and CE-UV methods. Three associated impurities were separated and identified for the first time by sheathless CE-MS. Then, a rapid CE-UV method was validated and used for accurate quantification of cobratide and its associated impurities. The CE-UV method showed good linearity between concentration and corrected peak area of cobratide in the concentration range of 5.36-536.30 µg mL-1. The limit of quantification of the CE-UV method was 4.16 µg mL-1. The relative standard deviations of migration time were less than 1% for both intra-day and inter-day experiments, and those of corrected peak area were less than 5%. Finally, different cobratide drugs were analyzed to evaluate the batch-to-batch consistency. This established protocol combining sheathless CE-MS and CE-UV methods would provide useful information for both quality control and process analysis of peptide drugs.


Assuntos
COVID-19 , Eletroforese Capilar , Humanos , Espectrometria de Massas , Peptídeos , SARS-CoV-2
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...