Electrophoresis

The present paper deals with the evaluation of the stereoselective binding of antihistamines (brompheniramine, chlorpheniramine, hydroxyzine, orphenadrine and phenindamine), phenothiazines (promethazine and trimeprazine) and a local anesthetic (bupivacaine) to human plasma proteins. Since all of them are drugs highly bound to proteins, a methodology to determine the bound fraction of each drug enantiomer was proposed. This methodology includes the incubation of samples containing plasma and racemic drug, ultrafiltration of the mixture and the chiral separation of enantiomers in the bound drug fraction using affinity EKC (AEKC)‐partial filling technique and HSA as chiral selector. The results shown in this paper represent the first evidence of the enantioselective binding of some antihistamines such as brompheniramine, hydroxyzine, orphenadrine and phenindamine and the phenothiazines, promethazine and trimeprazine, to human plasma proteins. The binding of phenindamine to plasma presented the highest enantioselectivity (ES) (ES = 2.5) followed by trimeprazine (ES = 1.5) and promethazine (ES = 1.4).

Microchip‐based bead‐packed columns for electrochromatography are described for several types of stationary phases. Chromatography columns 2 mm in length were used for the separation of proteins and peptides by size‐ and ion‐exchange modes of separation, respectively. In size‐exclusion electrochromatograpgy, FITC‐IgG and FITC‐insulin were baseline resolved in less than 10 s, with efficiencies of up to 139,000 plates/m for FITC‐insulin. In strong cation‐exchange electrochromatography, a mixture of three fluorescently labeled peptides was baseline resolved in less than 40 s, with efficiencies up to 400,000 plates/m. The RSD for the analytes retention times were<3% in both size‐exclusion and ion‐exchange modes of separations. The use of a 1‐mm‐long reverse‐phase column for the semiquantitative evaluation of pharmaceutical formulations in drug solubility tests illustrates the use of this microfluidic chip‐based electrochromatographic approach to drug development.

Lab‐on‐a‐chip electrophoresis is becoming increasingly useful for protein analysis, thanks to recent developments in this field. This review is an update of the review we published at the start of 2008 [Peng, Y., Pallandre, A., Tran, N. T., Taverna, M., Electrophoresis 2008, 29, 156–177]. The superiority of polymers for the manufacture of analytical microchips has been confirmed. This trend implies several modifications to the processes previously used with glass/silicon chips and requires a better understanding of the interfacial phenomena of these materials. Significant progress in chip‐based techniques for protein analysis has been made in the last 2 years. In addition to advances in traditional electrokinetic modes, counter‐flow gradient focusing techniques have emerged as useful methods not only for separation, but also for the online preconcentration of samples. This review, with more than 175 references, presents recent advances and novel strategies for EOF measurement, surface treatment, sample pretreatment, detection and innovations relating to the different modes of separation.

The aim of the investigation was to determine whether there are specific global quantitative and qualitative changes in protein expression in heart tissue from patients with dilated cardiomyopathy (DCM) compared with ischaemic heart disease and undiseased tissue. Two‐dimensional (2‐D) polyacrylamide gel electrophoresis and computer analysis was used to study protein alteration in DCM biopsy material (n=28) compared with donor heart biopsy samples (n=9) and explanted hearts from individuals suffering from ischaemic heart disease (IHD; n = 21). A total of 88 proteins displayed decreased abundance in DCM versus IHD material while five proteins had elevated levels in the DCM group (p<0.01). The most prominent changes occurred in the contractile protein myosin light chain 2 and in a group of proteins identified as desmin. These changes do not appear to be artefactual degradation events occurring during sample processing. These proteins are not apparent in electrophoretic separations of vascular tissue or cultured endothelial cells, mesothelial cells or cardiac fibroblasts, which are clearly distinguishable from the 2‐D protein patterns of whole heart and of isolated cardiac myocytes and do not appear to reflect variations in the cellular composition of biopsy samples. The different protein patterns observed in cardiomyopathy showed no obvious relationship with New York Heart Association (NYHA) functional class or haemodynamic parameters. The study has demonstrated significant alterations in quantitative protein expression in the DCM heart which would have serious implications for myocyte function. These changes might be explained by altered protease activity in DCM which could exacerbate contractile dysfunction in the failing heart.

Reference two‐dimensional (2‐D) gels are presented for human breast ductal carcinoma and histologically normal tissue. Whole biopsy fragments were analyzed, including epithelial and nonepithelial components. Thirty‐five spots have been assigned by gel matching to the human liver SWISS‐2DPAGE reference map and/or to the human primary keratinocyte IPG map from the Danish Center for Human Genome. N‐terminal microsequencing was applied to confirm randomly chosen matching assignments and to identify six new spots. Protein expression profiles in ductal carcinoma and in normal breast tissue appeared to be similar, except for a pattern consisting of 32 spots, which were highly expressed in all carcinoma specimens, and less intense and occasionally undetectable in normal tissue. This difference was statistically significant. Assignment has been obtained for several spots, namely GRP94, GRP78, GRP75, mitochondrial HSP60, calreticulin, protein disulfide isomerase, peptidyl‐prolyl cis‐trans isomerase, collagen‐binding protein 2, fructose bisphosphate aldolase, glyceraldehyde‐3‐phosphate dehydrogenase, thioredoxin, cytochrome c oxidase VA subunit, tubulin β isoform and macrophage migration inhibitory factor (MIF). The cancer‐ and tissue‐specificity of the described pattern was assessed by matching to the Swiss‐2DPAGE human liver, hepatoma, lymphoma, erythroleukemia reference maps. The pattern of 32 spots was found to be indicative of epithelial neoplasia.

An approximate analytic expression is derived for the electrophoretic mobility of a weakly charged spherical soft particle (i.e., a hard particle covered with a weakly charged polyelectrolyte layer) on the basis of the general mobility expression for soft particles (Ohshima, H., J. Colloid Interface Sci. 2000, 228, 190–193). The obtained mobility expression, which reproduces various approximate results so far derived and gives some new mobility formulas, covers all types of weakly charged soft particles with arbitrary values of the thickness of polymer layer, the radius of the particle core, the electrophoretic softness, and the Debye length, including spherical polyelectrolytes with no particle core as well as spherical hard particles with no polyelectrolyte layer.

As a typical miniaturized analytical technique, CEC has attracted much attention because of its low sample and solvent consumption, high efficiency, high selectivity, high resolution, and fast speed. In this review, we mainly cover the development of capillary columns and detection techniques in the CEC since 2009. Herein, three types of capillary columns, namely, open‐tubular capillary columns, monolithic columns and packed columns, and several types of detectors are reviewed in detail. Moreover, a 2D separation system based on CEC is also reported.

Tryptic hydrolysis of β‐Lactoglobulin (β‐Lg) is attracting more and more attention due to the reduced allergenicity and the functionality of resulting hydrolysates. To produce hydrolysates in an economically viable way, immobilized trypsin reactors (IMTRs), based on polymethacrylate monolith with pore size 2.1 μm (N1) and 6 μm (N2), were developed and used in a flow‐through system. IMTRs were characterized in terms of permeability and enzymatic activity during extensive usage. N1 showed twice the activity compared with N2, correlating well with its almost two times higher amount of immobilized trypsin. N2 showed high stability over 18 cycles, as well as over more than 30 weeks during storage. The efficiency of IMTRs on hydrolyzing β‐Lg was compared with free trypsin, and the resulting hydrolysates were analyzed by MALDI‐TOF/MS. The final hydrolysis degree by N1 reached 9.68% (86.58% cleavage sites) within 4 h, while only around 6% (53.67% cleavage sites) by 1.5 mg of free trypsin. Peptides analysis showed the different preference between immobilized trypsin and free trypsin. Under the experimental conditions used in this study, the potential cleavage site Lys¹³⁵‐Phe¹³⁶ was resistant against the immobilized trypsin in N1.

Simultaneous electrochemically driven double anion transfer across liquid–liquid interfaces is demonstrated at a gold–gold junction electrode. In the presence of two closely spaced electrodes (generator and collector), anion uptake into the organic phase (oxidation) and anion expulsion into the aqueous phase (reduction) can be combined to result in a generator–collector anion transport system across the liquid–liquid interface. In this report we are employing a paired gold junction grown by electro‐deposition to ca. 5 μm gap size with the N,N‐diethyl‐N′,N′‐didodecyl‐phenylene‐diamine water immiscible redox liquid immobilized into the gap to demonstrate simultaneous perchlorate anion uptake and expulsion. The effects of redox liquid volume and scan rate on the magnitude of currents and two mechanistic pathways for ion transport are discussed in the context of micro‐electrophoretic processes.

Capillary electrophoresis methodology is presented for the routine analysis of a wide variety of seized drugs using the same capillary with dynamic coatings and multiple run buffers. The types of exhibits analyzed using diode array UV detection include phenethylamines, cocaine, oxycodone, heroin, lysergic acid diethylamide (LSD), opium, hallucinogenic mushrooms, and γ‐hydroxybutyrate‐γ‐butyrolactone (GHB‐GBL). Both qualitative and quantitative analyses are achieved using run buffers that contain additives that provide for secondary equilibrium and/or dynamic coating of the capillary. Dynamic coating of the capillary surface is accomplished by rapid flushes of 0.1 Nsodium hydroxide, water, buffer containing polycation coating reagent, and a buffer containing a polyanionic coating reagent (with or without cyclodextrin(s)) or a micelle coating reagent. Dynamic coating with a polyanionic coating reagent is used for the analysis of moderately basic seized drugs and adulterants. The use of cyclodextrin in the run buffer not only allows for chiral analysis but also greatly enhances separation selectivity for achiral solutes. A capillary dynamically coated with a micelle allows for the analysis of neutral, acidic, and weakly basic drugs (GHB, GBL and neutral, acidic, and weakly basic adulterants). Dynamic coating, which gives rise to a relatively high and robust electroosmotic flow at pH < 7, allows for rapid, precise and reproducible separations. For a wide variety of drugs, excellent linearity and migration time precision and good peak area precision (external and internal standard) is obtained. Quantitative results for synthetic mixtures are in good agreement with actual values. Screening for adulterants is greatly enhanced by the use of automated library searches.