Sami El Deeb

MEKC as a powerful growing analytical technique

This review summarizes the principle and the developments in MEKC in terms of separation power, sensitivity, and detection approaches more than 25 years after its appearance. Newly used surfactants are mentioned. Classical and new sample concentration techniques in MEKC are described. The different detection approaches in MEKC with advantages, limitations, and future prospects are also discussed. This review highlights the wider application of MEKC in different analytical fields. Various recent selected applications of this technique in different analytical fields are reported.

Recent advances in capillary electrophoretic migration techniques for pharmaceutical analysis (2013-2015)

This review updates and follows‐up a previous review by highlighting recent advancements regarding capillary electromigration methodologies and applications in pharmaceutical analysis. General approaches such as quality by design as well as sample injection methods and detection sensitivity are discussed. The separation and analysis of drug‐related substances, chiral CE, and chiral CE‐MS in addition to the determination of physicochemical constants are addressed. The advantages of applying affinity capillary electrophoresis in studying receptor–ligand interactions are highlighted. Finally, current aspects related to the analysis of biopharmaceuticals are reviewed. The present review covers the literature between January 2013 and December 2015.

Recent advances in affinity capillary electrophoresis for binding studies

The present review covers recent advances and important applications of affinity capillary electrophoresis (ACE). It provides an overview about various ACE types, including ACE-MS, the multiple injection mode, the use of microchips and field-amplified sample injection-ACE. The most common scenarios of the studied affinity interactions are protein-drug, protein-metal ion, protein-protein, protein-DNA, protein-carbohydrate, carbohydrate-drug, peptide-peptide, DNA-drug and antigen-antibody. Approaches for the improvements of ACE in term of precision, rinsing protocols and sensitivity are discussed. The combined use of computer simulation programs to support data evaluation is presented. In conclusion, the performance of ACE is compared with other techniques such as equilibrium dialysis, parallel artificial membrane permeability assay, high-performance affinity chromatography as well as surface plasmon resonance, ultraviolet, circular dichroism, nuclear magnetic resonance, Fourier transform infrared, fluorescence, MS and isothermal titration calorimetry.

Strategies in method development to quantify enantiomeric impurities using CE

The growing number of chiral new drug substances requires increasing efforts in developing enantioselective methods. According to International conference on Harmonization guidelines, one should quantify the enantiomeric impurity of 0.1% relative to the major constituent. Capillary electrophoresis has evolved into an important tool for the separation of chiral drugs. The common strategies consist of two steps: firstly, initial separation conditions are evaluated. This screening usually focuses on the selection of the appropriate chiral selector. In our study 22 neutral, anionic or cationic cyclodextrins were dissolved in phosphate buffer (pH 2.5, 50 mM, CD conc.: 2.0%). Then they were investigated for the separation of 14 chiral compounds. Secondly, the obtained initial conditions for the enantiomeric separation were optimized in terms of resolution and analysis time. In our approach, important optimized factors including the concentration of the chiral selector (1–10%), the pH of the buffer (2.0–9.0), and the percentage of organic modifier (0–15%) were studied.

Protein quantitation using various modes of high performance liquid chromatography.

Pharmaceuticals based on proteins (biologicals), such as monoclonal antibodies (mAb), attain more and more relevance since they were established as potent drugs in anticancer therapy or for the treatment of autoimmune based diseases. Due to their high efficiency it is essential to have accurate and precise methods for protein quantitation and the detection of protein aggregates, which in some cases may lead to adverse effects after application. Selectivity and precision of traditional protein quantification methods such as the Bradford assay or SDS-PAGE are insufficient for quality control (QC) purposes. In this work several HPLC separation modes, which can significantly improve these important parameters, were compared for their application in this field. High performance size exclusion (HP-SEC), strong anion exchange (SAX), weak cation exchange (WCX) as well as reversed phase chromatography are all already successfully applied in protein analysis. Good precision (SEC: <1.9%, SAX: <5%, RP: <2% and WCX: <3.5% - RSD% for peak areas day-to-day), high selectivity and low quantitation limits (<15μg/ml) for the model proteins ovalbumin, myoglobin and bovine serum albumin (BSA), respectively cytochrome c and lysozyme in the cation exchange mode, could be achieved. Consecutively, the four separation modes were compared to each other and to electrophoretic techniques in terms of precision, selectivity, analysis time, effort of sample and mobile phase preparation as well as separating capacity. Moreover, the analysis of an IgG1-type antibody was included in this study.

Recent methodological and instrumental development in MEKC

The review gives an update about the methodological and instrumental developments in micellar electrokinetic capillary chromatography as a type of CE analytical technique. Here, the last two years development of the technique are particularly presented. Recent approaches to improve sensitivity are discussed. Newly introduced concentration techniques and experimental methods for verification of the different mechanisms and processes of micellar electrokinetic chromatography analysis are highlighted. A theoretical model to explain changes in separation and electrophoretic mobility order of fully charged analytes are demonstrated. Modern approaches for improving compatibility of micellar electrokinetic capillary chromatography to mass spectrometry are also reported.

Monolithic Silica for Fast HPLC: Current Success and Promising Future

As an approach for fast HPLC, monolithic silica has proven to be highly effective. It is especially successful for routinely obtaining fast isocratic HPLC analyses of small drug molecules. The low cost of monolithic compared with other approaches, such as UPLC, makes it more convenient for everyday application. It is also the more developed and widely applied technique compared with superficially porous particles. It offers the possibility for gaining high plate numbers through column coupling, but not at the expense of run time if a proper flow program is subsequently applied. Good precision and batch reproducibility are now achieved with commercially available monolithic silica columns. The application of monolithic silica columns is already well developed in various fields. It invades the field of bio-analysis and proteomics. Hundreds of analytical methods have already been successfully transferred to or developed on monolithic silica columns. An updated strategy based on Snyder’s method for rapid method development using monolithic column has been provided. However, more is still to be expected from monolithic silica in term of chemistry variation, application, and instrument compatibility. The future of monolithic silica is promising when considering the high demand for fast chromatographic analysis.

A comprehensive platform to investigate protein–metal ion interactions by affinity capillary electrophoresis

In this work, the behavior of several metal ions with different globular proteins was investigated by affinity capillary electrophoresis. Screening was conducted by applying a proper rinsing protocol developed by our group. The use of 0.1M EDTA in the rinsing solution successfully desorbs metal ions from the capillary wall. The mobility ratio was used to evaluate the precision of the method. Excellent precision for repeated runs was achieved for different protein metal ion interactions (RSD% of 0.05-1.0%). Run times were less than 6 min for all of the investigated interactions. The method has been successfully applied for the interaction study of Li(+), Na(+), Mg(2+), Ca(2+), Ba(2+), Al(3+), Ga(3+), La(3+), Pd(2+), Ir(3+), Ru(3+), Rh(3+), Pt(2+), Pt(4+), Os(3+), Au(3+), Au(+), Ag(+), Cu(1+), Cu(2+), Fe(2+), Fe(3+), Co(2+), Ni(2+), Cr(3+), V(3+), MoO4(2-) and SeO3(2-) with bovine serum albumin, ovalbumin, β-lactoglobulin and myoglobin. Different interaction values were obtained for most of the tested metal ions even for that in the same metal group. Results were discussed and compared in view of metal and semimetal groups interaction behavior with the tested proteins. The calculated normalized difference of mobility ratios for each protein-metal ion interaction and its sign (positive and negative) has been successfully used to detect the interaction and estimate further coordination of the bound metal ion, respectively. The comprehensive platform summarizes all the obtained interaction results, and is valuable for any future protein-metal ion investigation.

Repeatability of monolithic HPLC columns while using a flow program

Fast HPLC methods are becoming more and more important. Using monolithic HPLC columns for fast separations, a flow program can be applied for further decrease in the total run time. An interesting issue was whether the flow program affects repeatability. The investigated method was a generic assay for the oral antidiabetic drugs glibenclamide and glimepiride in the presence of two of their degradation products. A flow program ranging from 5.0 to 9.9 mL/min had been set up to decrease the run time to approximately 1.7 min. Within-day RSD% (n = 40) for both retention times and peak areas were less than 1%. At flow rates higher than 7 mL/min, repeatability was impaired to some extent. It became mainly noticeable through the day-to-day precision (n = 60) which showed RSD% up to 2%. However, further investigations indicated that this was rather related to pump inefficiency at high flow rates than to the flow program as such. Presuming the use of appropriate equipment, qualified for high flow rates, the application of a flow program for shortening the run time is absolutely reasonable and does not affect repeatability.

Optimization of affinity capillary electrophoresis for routine investigations of protein–metal ion interactions

To facilitate the implementation of affinity capillary electrophoresis into routine binding screening studies of proteins with metal ions, method acceleration, transfer and precision improvement were investigated. Affinity capillary electrophoresis was accelerated by using shorter capillaries, employing lower sample concentrations and smaller injection volumes. Intra- and inter-instrument method transfers were investigated considering the temperature setting of the capillary cooling system. For intra-instrument method transfer, similar results were obtained when transferring a method from a long (62 cm) to a short (31 cm) capillary. The analysis time was reduced from 9 to 4 min. In case of inter-instrument method transfer, interaction results showed small variation on the capillary electrophoresis instrument with inefficient capillary cooling system. Binding measurement precision was enhanced by slightly pushing the sample above the beginning of the capillary. Changing the buffer vials after each 30 runs and employing extra flushing after each 60 subsequent runs further enhanced the precision. The use of 0.1 molar ethylenediaminetetraacetic acid in the rinsing solution successfully desorbs the remaining metal ions from the capillary wall. Excellent precision for apparent mobility ratio measurements was achieved for different protein-metal ion interactions (relative standard deviation of 0.16-0.89%, 15 series, 12 runs for each).