Capillary electrophoresis-acid barrage stacking online enrichment method for highly sensitive determination of four isoflavones

A capillary electrophoresis-acid barrage stacking online enrichment method has been established to detect the four isoflavones which are Daidzein, Genistein, Formononetin, and Biochanin A. The proposed method was optimized using a single factor alternative method, and the optimal conditions obtained from the optimization were: the BGE was 25 mM borax and 2 mM β-cyclodextrin, the applied separation voltage was 20 kV, and the detection wavelength was 260 nm. The time ratio of the injection of sample and the injection of acid was 150 s:20 s, and the acid used was 250 mM acetic acid. The sample solvent used was 60% v/v acetonitrile. The established method had the enrichment factor of these four isoflavones at 24.5, 32.0, 29.2, and 33.7, respectively, LOD and LOQ are as low as nanograms per milliliter. Finally, the CE-acid barrage stacking method was successfully applied to the determination of four isoflavones in rat plasma and red clover extract, verifying the applicability and feasibility of the method.     

Modifying the physicochemical properties,solubility and foaming capacity of milk proteins by ultrasound-assisted alkaline pH-shifting treatment

This study investigated the effects of different treatment of alkaline pH-shifting on milk protein concentrate (MPC), micellar casein concentrate (MCC) and whey protein isolate (WPI) assisted by the same ultrasound conditions, including changes in the physicochemical properties, solubility and foaming capacity. The solubility of milk proteins had a significant increase with gradual enhancement of ultrasound-assisted alkaline pH-shifting (p < 0.05), especially for MCC up to 99.50 %. Also, treatment made a significant decline in the particle size of MPC and MCC, as well as the turbidity of the proteins (p < 0.05). The foaming capacity of MPC, MCC, and WPI was all improved, especially at pH 11, and at this pH, the milk protein also showed the highest surface hydrophobicity. The best foaming capacity at pH 11 was the result of the combined effect of particle size, potential, protein conformation, solubility, and surface hydrophobicity. In conclusion, ultrasound-assisted pH-shifting treatment was found to be effective in improving the physicochemical properties and solubility and foaming capacity of milk proteins, especially MCC, with promising application prospect in food industry.     

Ideal‐filter capillary electrophoresis: A highly efficient partitioning method for selection of protein binders from oligonucleotide libraries

Selection of affinity ligands for protein targets from oligonucleotide libraries currently involves multiple rounds of alternating steps of partitioning of protein‐bound oligonucleotides (binders) from protein‐unbound oligonucleotides (nonbinders). We have recently introduced ideal‐filter capillary electrophoresis (IFCE) for binder selection in a single step of partitioning. In IFCE, protein‐binder complexes and nonbinders move inside the capillary in the opposite directions, and the efficiency of their partitioning reaches 10⁹, i.e., only one of a billion molecules of nonbinders leaks through IFCE while all binders pass through. The condition of IFCE can be satisfied when the magnitude of the mobility of EOF is smaller than that of the protein‐binder complexes and larger than that of nonbinders. The efficiency of partitioning in IFCE is 10 million times higher than those of solid‐phase‐based methods of partitioning typically used in selection of affinity ligands for protein targets from oligonucleotide libraries. Here, we provide additional details on our justification for IFCE development. We elaborate on electrophoretic aspects of the method and define the theoretical range of EOF mobilities that support IFCE. Based on these theoretical results, we identify an experimental range of background electrolyte's ionic strength that supports IFCE. We also extend our interpretation of the results and discuss in‐depth IFCE's prospective in practical applications and fundamental studies.     

Coupling of CE‐MS for protein and peptide analysis

The review is focused on the latest developments in the analysis of proteins and peptides by capillary electrophoresis techniques coupled to mass spectrometry. First, the methodology and instrumentation are overviewed. In this section, recent progress in capillary electrophoresis with mass spectrometry interfaces and capillary electrophoresis with matrix‐assisted laser desorption/ionization is mentioned, as well as separation tasks. The second part is devoted to applications—mainly bottom‐up and top‐down proteomics. It is obvious that capillary electrophoresis with mass spectrometry methods are well suited for peptide and protein analysis (proteomic research) and it is described how these techniques are complementary and not competitive with the often used liquid chromatography with mass spectrometry methods.     

Recent trends in capillary and micro-chip electrophoretic instrumentation for field-analysis

Technical advances in the development of field-deployable capillary and microchip electrophoretic instruments and reports of their deployment between 2013 and 2017 were reviewed. Strategies and considerations in the design of the injection, separation and detection hardware, chemistry and associated infrastructure were discussed from an in-field perspective, with portability, robustness and automation/“ease of use” featuring as key requirements. Integration of functionality is important for adequate in-field performance. Progress was made towards the use of multiple channel devices for increased throughput and/or resolving power, mixing devices for on-line/in-line sample derivatization, battery operation and temperature control. The strengths and weaknesses of the various approaches described in the literature are discussed from the perspective of in-field operation. An overview of the applications of the field electrophoretic instruments is provided, including environmental science and planetary investigation.     

Capillary electrophoresis and ultra-high-performance liquid chromatography methods in clinical monitoring of creatinine in human urine: A comparative study

Creatinine is an important diagnostic marker and is also used as a standardization tool for the quantitative evaluation of exogenous/endogenous substances in urine. This study aimed at evaluating and comparing three analytical approaches, based on hyphenations of different separation [two-dimensional capillary isotachophoresis (CITP–CITP), capillary zone electrophoresis (CZE), ultra-high-performance liquid chromatography (UHPLC)] and detection [conductivity (CD), ultraviolet (UV), tandem mass spectrometry (MS/MS)] techniques, for their ability to provide reliable clinical data along with their suitability for the routine clinical use (cost, simplicity, sample throughput). The developed UHPLC–MS/MS, CITP–CITP–CD, and CZE–UV methods were characterized by favorable performance parameters, such as linearity (r ˃ 0.99), precision (relative standard deviation, 0.22–2.97% for the creatinine position in analytical profiles), and recovery (87.1–115.1%). Clinical data, obtained from the analysis of 24 human urine samples by a reference enzymatic method, were comparable with those obtained by the tested methods (Passing–Bablok regression and Bland–Altman analysis), approving their usefulness for the routine clinical use. In this context, the UHPLC–MS/MS method provides benefits of enhanced orthogonality/accuracy and high sample throughput (threefold shorter total analysis times than the CE methods), whereas advantages of the CE methods for routine labs are simplicity and low cost of both the instrumentation and measurements.     

Stepwise optimization and sensitivity improvement of green micellar electrokinetic chromatography method to simultaneously determine some fluoroquinolones and glucocorticoids present in various binary ophthalmic formulations

A sensitive micellar electrokinetic chromatography method is presented to simultaneously quantify ofloxacin, gatifloxacin, dexamethasone sodium phosphate and prednisolone acetate. The method has the advantages of being rapid, accurate, reproducible, ecologically acceptable and sensitive. The electrophoretic separation utilized 20 mm borate buffer as background electrolyte with pH 10.0 ± 0.1 and 50 mm sodium dodecyl sulfate as a micelle forming molecule. A capillary tube (50 μm i.d., 33 cm) of fused silica was used and on-column diode array detection at 243 nm for dexamethasone sodium phosphate and prednisolone acetate, and 290 nm for ofloxacin and gatifloxacin. Various factors were optimized such as the background electrolyte (type, concentration and pH), addition of sodium dodecyl sulfate and its concentration, detection wavelength, applied voltage and injection parameters. The studied drugs were efficiently separated in 6.2 min, at 20 kV with high resolution. The greenness of the method was estimated using an eco-scale tool and the presented method was found to have excellent green characteristics. The method was validated in conformance with International Conference on Harmonization guidelines, with acceptable accuracy, precision and selectivity. The suggested method can be employed for the economic analysis of the four drugs in dissimilar binary combinations of eye drops saving solvents and chemicals.     

New developments in Western blot technology

Western blotting is a highly valued method for protein identification and relative quantitation in complex samples. It combines size-based electrophoretic separation with immunoaffinity to identify specific proteins. This technique remains popular and has become a workhorse in biochemical research and clinical laboratories. Despite its utility and popularity, this method has many limitations including slow analysis, incompatibility with limited sample application, low throughput and low information content. Recently there has been significant success in improving different aspects of Western blotting. In this review, we provide an overview of the developments in the area of improving conventional Western blotting methods with a focus on recent developments in microfluidic Western blotting. We overview different separation platforms, and discuss studies on protein transfer methods as well as protein immobilization methods and chemistries. We also describe integrated miniaturized platforms that can perform rapid separations and immunodetections.     

Dual‐signal amplification strategy for ultrasensitive chemiluminescence detection of PDGF–BB in capillary electrophoresis

Many efforts have been made toward the achievement of high sensitivity in capillary electrophoresis coupled with chemiluminescence detection (CE‐CL). This work describes a novel dual‐signal amplification strategy for highly specific and ultrasensitive CL detection of human platelet‐derived growth factor–BB (PDGF–BB) using both aptamer and horseradish peroxidase (HRP) modified gold nanoparticles (HRP–AuNPs–aptamer) as nanoprobes in CE. Both AuNPs and HRP in the nanoprobes could amplify the CL signals in the luminol–H₂O₂ CL system, owing to the excellent catalytic behavior of AuNPs and HRP in the CL system. Meanwhile, the high affinity of aptamer modified on the AuNPs allows detection with high specificity. As proof‐of‐concept, the proposed method was employed to quantify the concentration of PDGF–BB from 0.50 to 250 fm with a detection limit of 0.21 fm. The applicability of the assay was further demonstrated in the analysis of PDGF–BB in human serum samples with acceptable accuracy and reliability. The result of this study exhibits distinct advantages, such as high sensitivity, good specificity, simplicity, and very small sample consumption. The good performances of the proposed strategy provide a powerful avenue for ultrasensitive detection of rare proteins in biological sample, showing great promise in biochemical analysis. Copyright © 2015 John Wiley & Sons, Ltd.     

Achiral and chiral analysis of duloxetine by chromatographic and electrophoretic methods,a review on the separation methodologies

Duloxetine (DLX) is a widely used antidepressant drug belonging to the class of selective serotonin and norepinephrine reuptake inhibitors (SNRIs); its efficacy has been demonstrated in the treatment of not only major depressive disorders but also diabetic neuropathic pain, generalized anxiety disorder, fibromyalgia or stress urinary incontinence. It is a chiral substance and is used in therapy in the form of the enantiopure S‐DLX, which is twice as active as R‐DLX. Several methods have been published for the achiral and chiral determination of DLX in pharmaceuticals, biological materials and environmental samples, the majority using liquid chromatography and capillary electrophoresis coupled with different detection techniques (UV detection, fluorescence, mass spectrometry). The aim of the current review is to provide a systematic survey of the analytical techniques used for the determination of DLX from different matrices.