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1.
Pan Afr Med J ; 48: 10, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38946743

RESUMO

Introduction: the utility of glycated haemoglobin (HbA1c) for the diagnosis and monitoring of diabetes in sub-Saharan Africa is uncertain due to limited data on the performance of the available HbA1c assay methods in this population, which has a high prevalence of haemoglobin variants. We aimed to compare the diagnostic accuracy of the major HbA1c methodologies (Boronate Affinity, Capillary Electrophoresis, High Performance Liquid Chromatography, Immunoassay) in an African population, and assess the impact of the common haemoglobin variant HbAS (sickle cell trait). Methods: whole blood samples were obtained from 182 individuals living with type 2 diabetes in Uganda. HbA1c values for each method were compared to average glucose measured over 14 days by continuous glucose monitoring (CGM). To determine concordance, the three HbA1c assay methods were compared to the capillary electrophoresis method. Results: there was a strong correlation between CGM average glucose levels and all four HbA1c methodologies (r=0.81-0.89) which did not differ in those with and without HbAS (present in 37/182 participants). The presence of HbAS did not alter the relationship between HbA1c and CGM glucose for any assay (p for interaction >0.2 for all methods). Diagnostic accuracy for CGM average glucose thresholds of 7 and 10mmol/L was similar across methods (area under the receiver operating characteristic curve 0.80-0.84 and 0.76-0.84 respectively). The maximum bias between the HbA1c assay methodologies was 2 mmol/mol (2.07%). Conclusion: all major HbA1c technologies offer accurate and comparable HbA1c measurement even in this population with high prevalence of haemoglobin variants.


Assuntos
Glicemia , Diabetes Mellitus Tipo 2 , Eletroforese Capilar , Hemoglobinas Glicadas , Sensibilidade e Especificidade , Humanos , Hemoglobinas Glicadas/análise , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/sangue , Eletroforese Capilar/métodos , Feminino , Glicemia/análise , Masculino , Pessoa de Meia-Idade , Cromatografia Líquida de Alta Pressão/métodos , Uganda , Adulto , Imunoensaio/métodos , Imunoensaio/normas , Automonitorização da Glicemia/métodos , Idoso , Hemoglobinas Anormais/análise
2.
Clin Lab ; 70(7)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38965958

RESUMO

BACKGROUND: New hemoglobin (Hb) variants are constantly being updated as assays are developed and the testing population expands. Here, we report a novel Hb variant, named Hb Guigang. METHODS: Hemoglobin (Hb) analysis was analyzed by capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). Glycated hemoglobin was performed by CE and HPLC. Routine genetic analysis was done with Gap-PCR and PCR-reverse dot-blot hybridization. The hemoglobin variant was identified by Sanger sequencing. RESULTS: CE of three cases showed the presence of Hb variants in Zone 5 and Zone 12, respectively. HPLC indicated an elevated P3 peak, suggesting the possible presence of the Hb variant. Hb A1c was measured by CE and HPLC, and the results were 6.7% and 4.76%, respectively. Sanger sequencing confirmed an AAG˃AAT mutation at codon 90 of the HBA1 gene. This mutation was reported for the first time, and we named it Hb Guigang based on the proband's place of residence. CONCLUSIONS: Hb Guigang with normal hematological parameters was separated and quantified by CE, whereas HPLC suggested that Hb Guigang co-eluted with the P3 peaks and could not be quantified.


Assuntos
Eletroforese Capilar , Hemoglobinas Glicadas , Hemoglobinas Anormais , alfa-Globinas , Humanos , Hemoglobinas Anormais/genética , alfa-Globinas/genética , Cromatografia Líquida de Alta Pressão , Hemoglobinas Glicadas/análise , Hemoglobinas Glicadas/metabolismo , Masculino , Feminino , Mutação , Análise Mutacional de DNA , Adulto
3.
J Zoo Wildl Med ; 55(2): 330-340, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38875190

RESUMO

The white stork (Ciconia ciconia) is a ciconiiform species widely represented in zoological institutions. Plasma protein electrophoresis is widely used in avian patients for assessment of inflammatory conditions, but reference intervals for this testing modality are lacking for the white stork. The two main electrophoretic methods are agarose gel electrophoresis (AGE) and capillary zone electrophoresis (CZE). This study assessed fresh plasma samples of healthy adult white storks (n = 30). Statistical analyses were performed to evaluate agreement between AGE and CZE. Typical electrophoretic fractions were obtained from both methods (prealbumin, albumin, α1, α2, ß, γ1, and γ2). The AGE and CZE methods were not equivalent for determining major electrophoretic fractions (except ß-globulins) and albumin:globulin ratio on plasma samples. An additional prealbumin fraction was seen with CZE. Reference intervals were established for each method as the smallest n group was 27 individuals for a given value; most values had normal distribution, and robust or parametric methods were used on the data.


Assuntos
Aves , Proteínas Sanguíneas , Eletroforese em Gel de Ágar , Eletroforese Capilar , Animais , Valores de Referência , Aves/sangue , Proteínas Sanguíneas/análise , Eletroforese Capilar/veterinária , Eletroforese Capilar/métodos , Eletroforese em Gel de Ágar/veterinária , Feminino , Eletroforese das Proteínas Sanguíneas/veterinária , Eletroforese das Proteínas Sanguíneas/métodos , Masculino
4.
Int J Biol Macromol ; 272(Pt 1): 132624, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38838594

RESUMO

In this work, the interaction of chondroitin sulfate (CS) and dermatan sulfate (DS) with plant lectins was studied by affinity capillary electrophoresis (ACE), surface plasmon resonance (SPR) technology, molecular docking simulation, and circular dichroism spectroscopy. The ACE method was used for the first time to study the interaction of Ricinus Communis Agglutinin I (RCA I), Wisteria Floribunda Lectin (WFA), and Soybean Agglutinin (SBA) with CS and DS, and the results were in good agreement with those of the SPR method. The results of experiments indicate that RCA I has a strong binding affinity with CS, and the sulfated position does not affect the relationship, but the degree of sulfation can affect the combination of RCA I with CS to some extent. However, the binding affinity with DS is very weak. This study lays the foundation for developing more specialized analysis methods for CS and DS based on RCA I.


Assuntos
Sulfatos de Condroitina , Dermatan Sulfato , Simulação de Acoplamento Molecular , Lectinas de Plantas , Ligação Proteica , Sulfatos de Condroitina/química , Dermatan Sulfato/química , Dermatan Sulfato/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Ressonância de Plasmônio de Superfície , Aglutininas/química , Aglutininas/metabolismo , Dicroísmo Circular , Eletroforese Capilar
5.
J Sep Sci ; 47(11): e2400174, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38867483

RESUMO

The review provides an overview of recent developments and applications of capillary electromigration (CE) methods for the determination of important physicochemical parameters of various (bio)molecules and (bio)particles. These parameters include actual and limiting (absolute) ionic mobilities, effective electrophoretic mobilities, effective charges, isoelectric points, electrokinetic potentials, hydrodynamic radii, diffusion coefficients, relative molecular masses, acidity (ionization) constants, binding constants and stoichiometry of (bio)molecular complexes, changes of Gibbs free energy, enthalpy and entropy and rate constants of chemical reactions and interactions, retention factors and partition and distribution coefficients. For the determination of these parameters, the following CE methods are employed: zone electrophoresis in a free solution or in sieving media, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography. In the individual sections, the procedures for the determination of the above parameters by the particular CE methods are described.


Assuntos
Eletroforese Capilar , Proteínas/análise , Proteínas/química , Termodinâmica , Focalização Isoelétrica/métodos , Peso Molecular , Humanos
6.
J Sep Sci ; 47(11): e2400170, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38863084

RESUMO

The glycomic analysis holds significant appeal due to the diverse roles that glycans and glycoconjugates play, acting as modulators and mediators in cellular interactions, cell/organism structure, drugs, energy sources, glyconanomaterials, and more. The glycomic analysis relies on liquid-phase separation technologies for molecular purification, separation, and identification. As a miniaturized form of liquid-phase separation technology, microscale separation technologies offer various advantages such as environmental friendliness, high resolution, sensitivity, fast speed, and integration capabilities. For glycan analysis, microscale separation technologies are continuously evolving to address the increasing challenges in their unique manners. This review discusses the fundamentals and applications of microscale separation technologies for glycomic analysis. It covers liquid-phase separation technologies operating at scales generally less than 100 µm, including capillary electrophoresis, nanoflow liquid chromatography, and microchip electrophoresis. We will provide a brief overview of glycomic analysis and describe new strategies in microscale separation and their applications in glycan analysis from 2014 to 2023.


Assuntos
Eletroforese Capilar , Glicômica , Polissacarídeos , Glicômica/métodos , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Polissacarídeos/análise , Humanos , Cromatografia Líquida , Eletroforese em Microchip/métodos
7.
Se Pu ; 42(6): 590-598, 2024 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-38845520

RESUMO

Fluorescent whitening agents (FWAs) are dyes that emit visible blue or blue-purple fluorescence upon ultraviolet-light absorption. Taking advantage of light complementarity, FWAs can compensate for the yellow color of many substances to achieve a whitening effect; thus, they are used extensively in various applications. FWAs are generally stable, but their presence in the environment can lead to pollution and accumulation in the body through the food chain. Recent studies have revealed that some types of FWAs, such as coumarin-based FWAs, may exhibit photo-induced mutagenic effects that can trigger allergic reactions in humans and even pose carcinogenic risks. Hence, the development of an accurate and highly sensitive method for detecting FWAs in food-related samples is a crucial endeavor. Owing to the high polarity and structural similarity of FWAs, the accurate determination of these substances in complex food samples requires an analytical method that offers both efficient separation and sensitive detection. Capillary electrophoresis (CE) exhibits essential features such as high separation efficiency, short analysis times, very small sample injection requirements, minimal use of organic solvents, and simple operation. Thus, it is often used as an effective alternative to liquid chromatographic techniques. Over the past few decades, electrospray ionization mass spectrometry (ESI-MS) has been utilized as a highly sensitive and accurate detection method in numerous chemical analytical fields because it enables the analysis of molecular structures. By combining the high separation efficiency of CE with the high sensitivity of ESI-MS, a powerful tool for identifying and quantifying trace amounts of FWAs in food samples may be obtained. In this study, we present a method based on sheathless CE coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS) for the simultaneous detection of six trace FWAs in flour. In the proposed method, the CE separation device is directly coupled to the mass spectrometer through a sheathless interface without the need for a sheath liquid for electric contact, thereby avoiding the dilution of the analytes and improving detection sensitivity. Various conditions that could affect extraction recovery, separation efficiency, and detection sensitivity were evaluated and optimized. The FWAs were effectively extracted from the sample matrix with reduced matrix effects by ultrasonic-assisted extraction at a temperature of 30 ℃ for 20 min using CHCl3-MeOH (3∶2, v/v) as the extraction solvent. The extract was centrifuged, dried under N2, and reconstituted in CHCl3-MeOH (1∶4, v/v) for subsequent analysis. During the detection process, the CE device was coupled to the ESI-MS/MS instrument via a highly sensitive porous spray needle, which served as the sheathless electrospray interface. The target FWAs were scanned in positive-ion mode (ESI+) to ensure the stability and intensity of the obtained signals. Additionally, multiple-reaction monitoring (MRM) mode and MS/MS analysis were used to simultaneously quantify the six targets with high selectivity. The developed sheathless CE-ESI-MS/MS method detected the FWAs with high sensitivity over wide linear ranges with low method limits of detection (0.04-0.67 ng/g). The recoveries of the six target FWAs at three spiked levels were between 77.5% and 97.2%, with good interday (RSD≤11.5%) and intraday (RSD≤10.2%) precision. Analyses of the six target FWAs in eight commercial flour samples were performed using this method, and four positive samples were identified. These results demonstrate that the proposed CE-ESI-MS/MS method is a promising strategy for the determination of trace FWAs in complex food sample matrices with efficient separation and high sensitivity.


Assuntos
Eletroforese Capilar , Farinha , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Eletroforese Capilar/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Farinha/análise , Corantes Fluorescentes/química , Contaminação de Alimentos/análise
8.
Anal Chem ; 96(27): 10969-10977, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38938066

RESUMO

Sodium dodecyl sulfate capillary gel electrophoresis is one of the frequently used methods for size-based protein separation in molecular biology laboratories and the biopharmaceutical industry. To increase throughput, quite a few multicapillary electrophoresis systems have been recently developed, but most of them only support fluorescence detection, requiring fluorophore labeling of the sample proteins. To avoid the time-consuming derivatization reaction, we developed an on-column labeling approach utilizing propidium iodide for the first time in SDS-CGE of proteins, a dye only used before for nucleic acid analysis. As a key ingredient of the gel-buffer system, the oppositely migrating positively charged propidium ligand in migratio complexes with the SDS-proteins, therefore, supports in situ labeling during the electrophoretic separation process, not requiring any extra pre- or postcolumn derivatization step. A theoretical treatment is given to shed light on the basic principles of this novel online labeling process, also addressing the influence of propidium iodide on the electroosmotic flow, resulting in reduced retardation. The concept of propidium labeling in SDS-CGE was first demonstrated using a commercially available protein sizing ladder ranging from 6.5 to 200 kDa with different isoelectric points and post-translational modifications. Considering the increasing number of protein therapeutics on the market next, we focused on the labeling optimization of a therapeutic monoclonal antibody and its subunits, including the addition of the nonglycosylated heavy chain. Peak efficiency and resolution were compared between noncovalent and covalent labeling. The effect of ligand concentration on the effective and apparent electrophoretic mobility, the resulting peak area, and the resolution were all evaluated in view of the theoretical considerations. The best detection sensitivity for the intact monoclonal antibody was obtained by using 200 µg/mL propidium iodide in the separation medium (LOD 2 µg/mL, 1.35 × 10-8 M) with excellent detection linearity over 3 orders of magnitude. On the other hand, the resolution between the biopharmaceutical protein test mixture components containing the intact and subunit fragments of the therapeutic monoclonal antibody was very good in the ligand concentration range of 50-200 µg/mL, but using the local maximum at 100 µg/mL for the nonglycosylated/glycosylated heavy chain pair is recommended. The figures of merit, including precision, sensitivity, detection linear range, and resolution for a sample mixture in hand, can be optimized by varying the propidium iodide concentration in the gel-buffer system, as demonstrated in this paper.


Assuntos
Eletroforese Capilar , Corantes Fluorescentes , Propídio , Proteínas , Eletroforese Capilar/métodos , Corantes Fluorescentes/química , Proteínas/química , Proteínas/análise , Propídio/química , Dodecilsulfato de Sódio/química
9.
Analyst ; 149(14): 3793-3802, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38847183

RESUMO

Benefitting from the rapid evolution of artificial intelligence and structural biology, an expanding collection of high-resolution protein structures has greatly improved our understanding of protein functions. Yet, proteins are inherently flexible, and these static structures can only offer limited snapshots of their true dynamic nature. The conformational and functional changes of calmodulin (CaM) induced by Ca2+ binding have always been a focus of research. In this study, the conformational dynamics of CaM and its complexes were investigated using a mobility capillary electrophoresis (MCE) and native mass spectrometry (native MS) based method. By analyzing the ellipsoidal geometries of CaM in the solution phase at different Ca2+ concentrations, it is interesting to discover that CaM molecules, whether bound to Ca2+ or not, possess both closed and open conformations. Moreover, each individual CaM molecule actively "jumps" (equilibrium exchange) between these two distinct conformations on a timescale ranging from milli- to micro-seconds. The binding of Ca2+ ions did not affect the structural dynamics of CaM, while the binding of a peptide ligand would stabilize CaM, leading to the observation of a single, compact conformation of the resulting protein complex. A target recognition mechanism was also proposed based on these new findings, suggesting that CaM's interaction with targets may favor a conformational selection model. This enriches our understanding of the binding principles between CaM and its numerous targets.


Assuntos
Cálcio , Calmodulina , Eletroforese Capilar , Espectrometria de Massas , Calmodulina/química , Calmodulina/metabolismo , Eletroforese Capilar/métodos , Cálcio/química , Cálcio/metabolismo , Espectrometria de Massas/métodos , Conformação Proteica , Ligação Proteica , Animais
10.
Clin Chim Acta ; 560: 119749, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38796052

RESUMO

Thalassemia is one of the most common and damaging monogenic diseases in the world. It is caused by pathogenic variants of α- and/or ß-globin genes, which disrupt the balance of these two protein chains and leads to α-thalassemia or ß-thalassemia, respectively. Patients with α-thalassemia or ß-thalassemia could exhibit a severe phenotype, with no simple and effective treatment. A three-tiered strategy of carrier screening, prenatal diagnosis and newborn screening has been established in China for the prevention and control of thalassemia, of which the first two parts have been studied thoroughly. The implementation of neonatal thalassemia screening is lagging, and the effectiveness of various screening programs has not yet been demonstrated. In this study, hemoglobin capillary electrophoresis (CE), hotspot testing method, and third-generation sequencing (TGS) were used in the variant detection of 2000 newborn samples, to assess the efficacy of these methods in neonatal thalassemia screening. Compared with CE (249, 12.45 %) and hotspot analysis (424, 21.2 %), CATSA detected the largest number of thalassemia variants (535, 26.75 %), which included 24 hotspot variants, increased copy number of α-globin gene, rare pathogenic variants, and three unreported potentially disease-causing variants. More importantly, CATSA directly determined the cis-trans relationship of variants in three newborns, which greatly shortens the clinical diagnosis time of thalassemia. CATSA showed a great advantage over other genetic tests and could become the most powerful technical support for the three-tiered prevention and control strategy of thalassemia.


Assuntos
Alelos , Triagem Neonatal , Talassemia , Humanos , Recém-Nascido , Talassemia/genética , Talassemia/diagnóstico , Eletroforese Capilar , alfa-Globinas/genética , Sequenciamento de Nucleotídeos em Larga Escala
11.
Anal Methods ; 16(23): 3675-3683, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38804529

RESUMO

Determination of digoxin through in-capillary derivatisation based on the formation of o-tolyl- and 2-naphthyl-anionic boronate esters in combination with large volume sample stacking-capillary electrophoresis is proposed. The derivatisation reaction was performed at basic pH values to obtain compounds with a charge and chromophore group during the stacking process. After stacking, the species were separated and detected at 225 nm using p-nitrophenol as an internal standard. Stacking and derivatisation parameters such as pre-concentration time, preconcentration voltage and injection time (relation between the analyte and the derivatisation agent) were evaluated using a Box-Behnken design. Under optimal conditions, the proposed method exhibits a linear range of 1.08-50.00 µM with a limit of detection of 0.36 µM; additionally, adequate repeatability and reproducibility was obtained (%RSD ≤ 5.0%). The methodology was validated by comparing it to an HPLC-UV established methodology and was successfully applied for the determination of digoxin in pharmaceutical tablets and blood serum samples, showing a positive performance for these matrices.


Assuntos
Ácidos Borônicos , Digoxina , Eletroforese Capilar , Digoxina/sangue , Digoxina/análise , Digoxina/química , Eletroforese Capilar/métodos , Ácidos Borônicos/química , Humanos , Ésteres/química , Limite de Detecção , Reprodutibilidade dos Testes , Comprimidos
12.
Se Pu ; 42(5): 401-409, 2024 Apr 08.
Artigo em Chinês | MEDLINE | ID: mdl-38736383

RESUMO

This paper serves as an annual review of capillary electrophoresis (CE) technology for 2023. The journals were selected based on their impact factor (IF), a universally recognized academic performance metric, combined with experimental work closely related to CE technology, to facilitate the rapid acquisition of significant research and application advancements in CE technology in 2023. A thematic search of the ISI Web of Science database yielded 669 research papers on CE technology published in 2023. This review highlights five experimental papers published in journals with IFs greater than 10.0, including Nature Communications, Nucleic Acids Research, Engineering, Journal of Medical Virology, and Carbohydrate Polymers, and 31 experimental papers from representative journals with IFs between 5.0 and 10.0, such as Analytical Chemistry, Analytica Chimica Acta, Talanta, and Food Chemistry. It also provides an overview of experimental research in journals with focused reporting on CE technology but with IFs less than 5.0, such as Journal of Chromatography A and Electrophoresis, as well as significant experimental research from key domestic Chinese core journals (Peking University). In 2023, all the latest scientific advancements reported in journals with an IF greater than 10.0 utilized previously reported CE methods, offering new breakthroughs for the promotion and application of CE technology. Additionally, new applications of CE in conjunction with mass spectrometry remained a hot topic. An increase in reports on the hardware aspects of CE, such as 3D printing and underwater systems, and significant breakthroughs in the analysis of non-solution samples, such as solid particles, cell vesicles, cells, viruses, and bacteria, was noted. CE is advantageous for the analysis of drugs and their components. In Chinese journals, the number of papers on CE applications exceeded that in previous years, with particular focus on the field of printing for new applications.


Assuntos
Eletroforese Capilar , Eletroforese Capilar/métodos
13.
Clin Lab ; 70(5)2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38747917

RESUMO

BACKGROUND: Hyperthyroidism can lead to diverse hematological disorders, such as microcytosis and a mild increase in hemoglobin A2 fraction. METHODS: This study reported a 31-year-old woman of Moroccan origin recently diagnosed with Graves' disease. Her blood tests revealed microcytosis, hypochromia, and a normal ferritin level. A phenotypic analysis of hemo-globin was performed using two techniques: capillary electrophoresis and reversed-phase high performance liquid chromatography. RESULTS: Both techniques indicated a slight increase in hemoglobin A2 level. These results initially suggested het-erozygous beta-thalassemia, eventually correlating with the concurrent presence of Graves' disease, as evidenced by the normalization of hemoglobin A2 level following treatment. CONCLUSIONS: This case highlights the importance of having clinical, biological, and therapeutic data for a relevant interpretation of a phenotypic hemoglobin study.


Assuntos
Doença de Graves , Hemoglobina A2 , Humanos , Doença de Graves/sangue , Doença de Graves/diagnóstico , Doença de Graves/complicações , Feminino , Adulto , Hemoglobina A2/análise , Talassemia beta/sangue , Talassemia beta/complicações , Talassemia beta/diagnóstico , Eletroforese Capilar/métodos , Cromatografia Líquida de Alta Pressão , Fenótipo
14.
Nat Commun ; 15(1): 3847, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38719792

RESUMO

The development of reliable single-cell dispensers and substantial sensitivity improvement in mass spectrometry made proteomic profiling of individual cells achievable. Yet, there are no established methods for single-cell glycome analysis due to the inability to amplify glycans and sample losses associated with sample processing and glycan labeling. In this work, we present an integrated platform coupling online in-capillary sample processing with high-sensitivity label-free capillary electrophoresis-mass spectrometry for N-glycan profiling of single mammalian cells. Direct and unbiased quantitative characterization of single-cell surface N-glycomes are demonstrated for HeLa and U87 cells, with the detection of up to 100 N-glycans per single cell. Interestingly, N-glycome alterations are unequivocally detected at the single-cell level in HeLa and U87 cells stimulated with lipopolysaccharide. The developed workflow is also applied to the profiling of ng-level amounts (5-500 ng) of blood-derived protein, extracellular vesicle, and total plasma isolates, resulting in over 170, 220, and 370 quantitated N-glycans, respectively.


Assuntos
Eletroforese Capilar , Glicômica , Espectrometria de Massas , Polissacarídeos , Análise de Célula Única , Humanos , Eletroforese Capilar/métodos , Polissacarídeos/metabolismo , Polissacarídeos/sangue , Análise de Célula Única/métodos , Células HeLa , Espectrometria de Massas/métodos , Glicômica/métodos , Proteômica/métodos , Vesículas Extracelulares/metabolismo , Lipopolissacarídeos , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/metabolismo
15.
Anal Chim Acta ; 1310: 342718, 2024 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-38811137

RESUMO

BACKGROUND: Dried blood spot (DBS) sampling on cellulose cards suffers from varying blood haematocrit levels and from chromatographic effects, which have a direct impact on quantitative DBS analyses. Commercial volumetric microsampling devices were, therefore, introduced to mitigate these effects, however, these devices are not compatible with automated DBS processing systems and must be processed manually. RESULTS: Capillary electrophoresis (CE) instruments use fused-silica (FS) capillaries for precise and accurate liquid handling as well as for injection, separation, and quantitative analyses of liquid samples. These inherent features of an Agilent 7100 CE instrument were employed for the automated processing (elution and homogenization) of DBSs collected by hemaPEN® volumetric devices (2.74 µL of capillary blood per spot). The hemaPEN® samples were processed directly in CE vials by consecutive transfers of 56 µL of methanol and 14 µL of deionized water through the FS capillary in a sequence of 39 DBSs with repeatability of the liquid transfers better than 1.4 %. The resulting DBS eluates were homogenized by a quick air flush through the capillary and analyzed by the same capillary and CE instrument. Creatinine was selected as a clinically relevant model analyte and its endogenous concentrations in DBSs were determined by CE with capacitively coupled contactless conductivity detection (CE-C4D) in a background electrolyte solution consisting of 50 mM acetic acid and 0.1 % (v/v) Tween 20 (pH 3.0). The overall repeatability of the automated DBS processing and CE-C4D analyses of 39 DBSs was ≤7.1 % (peak areas) and ≤0.6 % (migration times), the calibration curve was linear in the 25-500 µM range (R2 = 0.9993) and covered all endogenous blood creatinine levels, the limit of detection was 5.0 µM, and sample throughput was >12 DBSs per hour. DBS ageing for 60 days and varying blood haematocrit levels (20-70 %) did not affect creatinine quantitative results (≤6.9 % for peak areas). Inter-capillary and inter-instrument repeatability was ≤7.7 % (peak areas) and ≤3.4 % (migration times) and demonstrated an excellent transferability of the proposed analytical concept among laboratories. SIGNIFICANCE AND NOVELTY: This contribution is the first-ever report on the use of a single off-the-shelf analytical instrument for fully automated analyses of DBSs collected by commercial volumetric microsampling devices and holds great promise for future unmanned quantitative DBS analyses.


Assuntos
Teste em Amostras de Sangue Seco , Eletroforese Capilar , Teste em Amostras de Sangue Seco/métodos , Teste em Amostras de Sangue Seco/instrumentação , Humanos , Eletroforese Capilar/métodos , Automação , Creatinina/sangue
16.
Anal Chim Acta ; 1311: 342736, 2024 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-38816165

RESUMO

BACKGROUND: Capillary electrophoresis is a powerful analytical method featured with high separation efficiency, minimal sample requirements, and reduced organic solvents consumption. However, its low sensitivity hinders its wide application in determination of trace analytes especially for the weakly ionized hydrophobic compounds. Offline and Online capillary electrophoresis stacking methods are more favored to enhance detection sensitivity of analytes. The determination of two sesquiterpenes and an alkaloid from the dried root of Lindera aggregata merged as an example for developing a simple, sensitive and green method for the simultaneous determination of two hydrophobic compounds in complicated matrix samples. RESULTS: An offline-online capillary electrophoresis stacking strategy by integrating micro matrix solid phase dispersion with field-amplified sample stacking and micelle to cyclodextrin stacking has been developed for the simultaneous determination of dehydrocostus lactone, linderane, norisoboldine in complex matrices. The optimized parameters were set at 65 mM sodium dihydrogen phosphate, 35 % methanol, 180 s for sample injection and 210 s for cyclodextrin injection, 20 mM sodium dodecyl sulfate of sample matrix for online stacking; 1:1 sample to MCM-48, 180 s grinding time, and 1000 µL of 20 mM sodium dodecyl sulfate elution for offline procedure. Under the optimum conditions, the method showed good linearity with correlation coefficients (R2 ≥ 0.9927), low limits of detection within the range of 25-50 ng mL-1, satisfactory repeatability and reproducibility below 3.98 %, and acceptable recoveries between 94 % and 97 %. The developed method was successfully applied to two real samples, the root of L. aggregata and rat feces. SIGNIFICANCE: Sodium dodecyl sulfate is firstly used as an eluent in micro matrix solid phase dispersion and plays a dual role throughout the analytical procedure, including extraction solvent in sample preparation and micelle pseudophase during online stacking. It brings great procedure convenience to the method. The sensitivity of this method can improve up to 1283-folds compared with the normal mode. Moreover, the overall strategy indicates satisfied green potential evaluated by greenness assessment tools.


Assuntos
Eletroforese Capilar , Interações Hidrofóbicas e Hidrofílicas , Dodecilsulfato de Sódio , Eletroforese Capilar/métodos , Dodecilsulfato de Sódio/química , Animais , Ratos , Química Verde , Limite de Detecção , Ciclodextrinas/química , Sesquiterpenos/análise , Alcaloides/análise , Raízes de Plantas/química
17.
J Sep Sci ; 47(11): e2400092, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38819776

RESUMO

This paper presents an approach based on triple injection capillary zone electrophoresis for identification of monoclonal antibodies. The analyte to be identified is injected between two zones of a known reference. The distances between the reference zones (plug I and III) and the target zone (plug II) are adjusted by partial electrophoresis of the first and second injection plugs. The full migration time of the target analyte is calculated from the observed migration time by considering the migration times of the reference in the first and third injection plugs. The relative migration time, that is, the ratio between the full migration time of the analyte and the migration time of the reference in the third injection plug provides the basis for identification. Here, eight monoclonal antibodies, including a pair of biosimilars, were used interchangeably as both analyte and reference to investigate potential of the method. The relative migration time for a preliminary positive identification were found to vary between 0.994 and 1.006 (1.000 ± 0.006, p = 95%). Beside the relative migration time, isoform distribution, peak profiles, and early migrating peaks, originating from components in the pharmaceutical formulations, were successfully used to verify the identity of all tested monoclonal antibodies.


Assuntos
Anticorpos Monoclonais , Eletroforese Capilar , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química
18.
J Chromatogr A ; 1727: 464990, 2024 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-38744188

RESUMO

An approach for the controllable separation and concentration of nucleic acid using a circular nonuniform electric field was proposed and developed. Using six different lengths of DNA molecules as standard samples, the distribution of the gradient electric field was increased from the outer circular electrode to the inner rod-shaped electrode, contributing to the migration of DNA molecules at a velocity gradient towards the region with the strongest inner electric field. The DNA molecules were arranged in a distribution of concentric circles that aligned with the distribution of concentric equipotential lines. The concentration of DNA multiplied with the alternation of radius. As a result, this platform allowed simultaneous DNA separation, achieving a resolution range of 1.17-3.03 through an extended electrophoresis time, resulting in enhanced concentration factors of 1.08-6.27. Moreover, the manipulation of the relative height of the inner and outer electrodes enabled precise control over the distribution and the deflection degree of electric field lines, leading to accurate control over DNA deflection.


Assuntos
DNA , DNA/isolamento & purificação , DNA/análise , DNA/química , Eletrodos , Eletricidade , Eletroforese Capilar/métodos
19.
J Sep Sci ; 47(9-10): e2300668, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38699940

RESUMO

Saccharides and biocompounds as saccharide (sugar) complexes have various roles and biological functions in living organisms due to modifications via nucleophilic substitution, polymerization, and complex formation reactions. Mostly, mono-, di-, oligo-, and polysaccharides are stabilized to inactive glycosides, which are formed in metabolic pathways. Natural saccharides are important in food and environmental monitoring. Glycosides with various functionalities are significant in clinical and medical research. Saccharides are often studied with the chromatographic methods of hydrophilic interaction liquid chromatography and anion exchange chromatograpy, but also with capillary electrophoresis and mass spectrometry with their on-line coupling systems. Sample preparation is important in the identification of saccharide compounds. The cases discussed here focus on bioscience, clinical, and food applications.


Assuntos
Eletroforese Capilar , Espectrometria de Massas , Humanos , Carboidratos/química , Cromatografia Líquida , Interações Hidrofóbicas e Hidrofílicas
20.
Anal Chem ; 96(21): 8763-8771, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38722793

RESUMO

Proteomics analysis of mass-limited samples has become increasingly important for understanding biological systems in physiologically relevant contexts such as patient samples, multicellular organoids, spheroids, and single cells. However, relatively low sensitivity in top-down proteomics methods makes their application to mass-limited samples challenging. Capillary electrophoresis (CE) has emerged as an ideal separation method for mass-limited samples due to its high separation resolution, ultralow detection limit, and minimal sample volume requirements. Recently, we developed "spray-capillary", an electrospray ionization (ESI)-assisted device, that is capable of quantitative ultralow-volume sampling (e.g., pL-nL level). Here, we developed a spray-capillary-CE-MS platform for ultrasensitive top-down proteomics analysis of intact proteins in mass-limited complex biological samples. Specifically, to improve the sensitivity of the spray-capillary platform, we incorporated a polyethylenimine (PEI)-coated capillary and optimized the spray-capillary inner diameter. Under optimized conditions, we successfully detected over 200 proteoforms from 50 pg of E. coli lysate. To our knowledge, the spray-capillary CE-MS platform developed here represents one of the most sensitive detection methods for top-down proteomics. Furthermore, in a proof-of-principle experiment, we detected 261 ± 65 and 174 ± 45 intact proteoforms from fewer than 50 HeLa and OVCAR-8 cells, respectively, by coupling nanodroplet-based sample preparation with our optimized CE-MS platform. Overall, our results demonstrate the capability of the modified spray-capillary CE-MS platform to perform top-down proteomics analysis on picogram amounts of samples. This advancement presents the possibility of meaningful top-down proteomics analysis of mass-limited samples down to the level of single mammalian cells.


Assuntos
Eletroforese Capilar , Proteômica , Eletroforese Capilar/métodos , Proteômica/métodos , Humanos , Escherichia coli/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas/métodos
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