Optimization of a nano‐enzymatic reactor for on‐line tryptic digestion of polypeptide conjugates by capillary electrophoresis

This work aims at studying the optimization of an on‐line capillary electrophoresis (CE)‐based tryptic digestion methodology for the analysis of therapeutic polypeptides (PP). With this methodology, a mixture of surrogate peptide fragments and amino acid were produced on‐line by trypsin cleavage (enzymatic digestion) and subsequently analyzed using the same capillary. The resulting automation of all steps such as injection, mixing, incubation, separation and detection minimizes the possible errors and saves experimental time. In this paper, we first study the differents parameters influencing PP cleavage inside the capillary (plug length, reactant concentration, incubation time, diffusion and electrophoretic plugs mixing). In a second part, the optimization of the electrophoretic separation conditions of generated hydrolysis products (nature, pH and ionic strength (I) of the background electrolyte (BGE)) is described. Using the optimized conditions, excellent repeatability was obtained in terms of separation (migration times) and proteolysis (number of products from enzymatic hydrolysis and corresponding amounts) demonstrating the robustness of the proposed methodology.     

Resolution of overlapped peaks of amino acid derivatives in capillary electrophoresis using multivariate curve resolution based on alternating least squares

The application of chemometric techniques to the resolution of overlapped peaks in capillary electrophoresis (CE) is described. When a physical separation can not be completely accomplished, chemometrics might still resolve the determination of the analytes mathematically. CE with diode array detection can provide a large amount of data consisting of spectra registered over time. In this study, the capillary electrophoretic separation of 1,2-naphthoquinone-4-sulfonate derivatives of amino acids is studied. Most of the common amino acid derivatives can be separated at 30 kV in a fused-silica capillary by using a 40 mM sodium tetraborate + isopropanol (3:1 v/v) solution as background electrolyte. However, peaks of certain derivatives (Phe, His, Leu and Ile) still overlap. A multivariate curve resolution method based on an alternating least squares optimization procedure is used for the resolution of the overlapped electrophoretic peaks. The method takes advantage of spectral and electrophoretic differences of analytes to recover their pure electrophoretic and spectral profiles. In addition, each analyte in the mixture can be quantified using the corresponding standards.     

In-capillary derivatization and analysis of amino acids,amino phosphonic acid-herbicides and biogenic amines by capillary electrophoresis with laser-induced fluorescence detection

This paper describes a general approach for the in-capillary derivatization of amino compounds and the subsequent sensitive determination of the derivatives by micellar electrokinetic chromatography (MEKC) or capillary zone electrophoresis (CZE) with laser-induced fluorescence (LIF) detection. Amino acids, biogenic amines and amino phosphonic acid-herbicides were chosen as model analytes to evaluate the analytical potential of this approach. Fulfilment of the in-capillary reaction of the analytes using LIF detection hinged on the excellent labeling chemistry of 5-(4,6-dichloro-s-triazin-2-ylamino)fluorescein (DTAF) and the good resolution achieved in the separation of derivatized analytes. Careful optimization of the electrophoretic conditions in the mixing step of this protocol allowed the determination of amino acids, biogenic amines and phosphorus-containing amino acid-herbicides with concentration limits of detection at the nug/L level and relative standard deviations from 3.5 to 5.8%. The whole analysis is carried out within 20 min, resulting in a very simple, fast and practical approach for the fully automated analysis of amino acids and related compounds in low-volume and low-concentration samples.     

Investigation of the ion-exchange properties of methacrylate-based mixed-mode monolithic stationary phases employed as stationary phases in capillary electrochromatography

The potential of methacrylate-based mixed-mode monolithic stationary phases bearing sulfonic acid groups for the separation of positively charged analytes (alkylanilines, amino acids, and peptides) by capillary electrochromatography (CEC) is investigated. The retention mechanism of protonated alkylanilines as positively charged model solutes on these negatively charged mixed-mode stationary phases is investigated by studying the influence of mobile phase and stationary phase parameters on the corrected retention factor which was calculated by taking the electrophoretic mobility of the solutes into consideration. It is shown that both solvophobic and ion-exchange interactions contribute to the retention of these analytes. The dependence of the corrected retention factor on (1) the concentration of the counter ion ammonium and (2) the number of methylene groups in the alkyl chain of the model analytes investigated shows clearly that a one-site model (solvophobic and ion-exchange interactions take place simultaneously at a single type of site) has to be taken to describe the retention behaviour observed. Comparison of the CEC separation of these charged analytes with electrophoretic mobilities determined by open-tubular capillary electrophoresis shows that mainly chromatographic interactions (solvophobic and ion-exchange interactions) are responsible for the selectivity observed in CEC, while the electrophoretic migration of these analytes plays only a minor role.     

Parameters influencing separation and detection of anions by capillary electrophoresis

Electrolyte composition is critical in optimizing separation and detection of ions by capillary electrophoresis. The parameters which must be considered when designing an electrolyte system for capillary electrophoresis include electrophoretic mobility of electrolyte constituents and analytes, detection mode, and compatibility of electrolyte constituents with one another. An electrolyte system based on pyromellitic acid is well suited for use with indirect photometric detection, and provides excellent separations of anions. The ability to modify the electrophoretic mobility of pyromellitic acid as a function of ph provides flexibility in matching electrophoretic mobilities of analytes. Additionally, the use of alkyl amines as electroosmotic flow modifiers allows the rapid separation of anions by reversing the direction of electroosmotic flow in a fused-silica capillary. The optimization of a capillary electrophoresis electrolyte for anion analysis is also discussed in terms of pH, ionic strength and applied voltage. The effect of organic solvent on separation selectivity is also discussed.     

Sensitive amperometric detection for capillary electrophoresis of phenol carbamates with in‐line thermal hydrolysis strategy

A strategy on amperometric detection for CZE of phenol carbamates as model analytes with a facile in‐line thermal hydrolysis was presented, in which a thermal hydrolysis, subsequent CZE separation and final column‐end amperometric detection were accomplished in an intact capillary. Key parameters of hydrolysis dynamics of carbamates and electrochemical detection of the hydrolysates were studied, as well as electrophoretic conditions. Under the optimal conditions, the capillary was utilized as chambers for in situ hydrolysis, CZE separation, and electrochemical detection. The successive separation of hydrolysates of five carbamates (propoxur, carbofuran, 3‐OH‐carbofuran, carbaryl and bendiocarb) were achieved within 17 min. Applied to vegetable samples, the recoveries of carbamates fortified at 0.02 and 0.05 mg/kg were ranging in 88–107.2 and 86.3–107.3%, respectively. The success in the implementation of such a scheme resulted in a simple instrument as compared with those current analytical methods with post‐column derivization or pre‐column hydrolysis, or online enrichment in chip, respectively. This protocol might possess a potential utility for the sensitive amperometric detection of phenol carbamates.     

Assay of protein drug substances present in solution mixtures by fluorescamine derivatization and capillary electrophoresis.

A method is described to enhance the resolution and detection sensitivity of proteins, peptides, and amino acids in capillary electrophoretic analysis of solution mixtures. The method consists of derivatizing the analytes with fluorescamine, which is normally used as a fluorogenic reagent for compounds containing a reactive primary amine functional group, and then using the derivative as an ultraviolet chromophore to enhance detection sensitivity (measured at 280 nm) in capillary electrophoresis. The results demonstrated a significant improvement in the separation and detection sensitivity of the derivatized analytes as compared to their underivatized counterparts. The use of chromophores, such as fluorescamine, in capillary electrophoresis facilitates the analysis of components of solution mixtures, such as pharmaceutical formulations, that could not be resolved and/or detected by conventional capillary electrophoresis procedures.     

Optimization of a capillary zone electrophoresis–contactless conductivity detection method for the determination of nisin

Determination of natural preservatives using electrophoretic or chromatographic techniques in fermented milk products is a complex task due to the following reasons: (i) the concentrations of the analytes can be below the detection limits, (ii) complex matrix and comigrating/coeluting compounds in the sample can interfere with the analytes of the interest, (iii) low recovery of the analytes, and (iv) the necessity of complex sample preparation. The aim of this study was to apply capillary zone electrophoresis coupled with contactless conductivity detection for the separation and determination of nisin in fermented milk products. In this work, separation and determination of natural preservative–nisin in fermented milk products is described. Optimized conditions using capillary zone electrophoresis coupled with capacitance‐to‐digital technology based contactless conductivity detector and data conditioning, which filter the noise of the electropherogram adaptively to the peak migration time, allowed precise, accurate, sensitive (limit of quantification: 0.02 μg/mL), and most importantly requiring very minute sample preparation, determination of nisin. Sample preparation includes following steps: (i) extraction/dilution and (ii) centrifugation. This method was applied for the determination of nisin in real samples, i.e. fermented milk products. The values of different nisin forms were ranging from 0.056 ± 0.003 μg/mL to 9.307 ± 0.437 μg/g.     

Coupling of capillary electrophoresis with reaction detection for the on-line evaluation of radical scavenging activity of analytes

The main task of this work was to create a rapid, simple and less material and time consuming method involving capillary electrophoretic separation in order to separate analytes and evaluate antioxidant activity within a single analytical run. Several interfaces were developed and used to couple CE to the reaction detector. The method developed enables simultaneous electrophoretic separation and evaluation of antioxidant activity of each separated compound in the mixture. The analysis was performed using DPPH (2,2-diphenyl-1-picrylhydrazyl) as synthetic radical reagent. The on-line capillary electrophoresis-reaction (antioxidant activity) detection method can be used for a rapid evaluation of individual antioxidants in complex mixtures, particularly extracts of natural products. Possibility to evaluate radical scavenging activity of extracts of natural products components is demonstrated on an example of aqueous propolis extract. Four phenolic acids where separated and their radical scavenging activity was on-line evaluated.     

Efficient Ring Opening of Aziridines with Carboxylic Acids

An efficient ring cleavage of aziridines with acids has been studied in the absence of any catalyst. The hydrolysis of the products, amino esters, leads to the corresponding amino alcohols. The reaction has been extended to chiral cycloalkyl aziridines, leading to the formation of diastereomers. After separation, these diastereomers have been converted to optically pure amino alcohols in two steps.     

Separation of twenty underivatized essential amino acids by capillary zone electrophoresis with contactless conductivity detection

Twenty underivatized essential amino acids were separated using capillary zone electrophoresis and consequently detected with contactless conductivity detection (CCD). A simple acidic background electrolyte (BGE) containing 2.3 M acetic acid and 0.1% w/w hydroxyethylcellulose (HEC) allowed the electrophoretic separation and sensitive detection of all 20 essential amino acids in their underivatized cationic form. The addition of HEC to the BGE suppressed both, electroosmotic flow and analyte adsorption on the capillary surface resulting in an excellent migration time reproducibility and a very good analyte peak symmetry. Additionally, the HEC addition significantly reduced the noise and long-term fluctuations of the CCD baseline. The optimized electrophoretic separation method together with the CCD was proved to be a powerful technique for determination of amino acid profiles in various natural samples, like beer, yeast, urine, saliva, and herb extracts.     

Gas chromatography electrospray ionization mass spectrometry analysis of trimethylsilyl derivatives

A method based on the analysis of trimethylsilyl (TMS) derivatives by capillary gas chromatography electrospray ionization mass spectrometry (GC–ESI/MS) was proposed. To improve separation, analytes were derivatized to their TMS derivative. During ESI analysis, TMS derivatives may hydrolyze back to their polar native form and are thus suitable for ESI analysis. Several types of analytes were studied to investigate the potential of the approach. Not all TMS derivatives hydrolyzed back to their native form as anticipated. Incomplete hydrolysis was observed for TMS‐organic acids and TMS‐nonchlorinated phenols. For TMS‐chlorophenols, the observation of only the [M ? H]^? ion suggested that these phenols were hydrolyzed back to their native form. For TMS‐beta agonists, the hydrolysis rate was low; therefore, the hydrolysis product was not detected. Both TMS‐chlorophenols and TMS‐beta agonists provide a sensitivity in the range of low parts per billion (0.25–5 ng/ml and 0.5–10 ng/ml respectively). Copyright © 2016 John Wiley & Sons, Ltd.     

Nonaqueous capillary electrophoresis of dextromethorphan and its metabolites

This study deals with the nonaqueous capillary electrophoretic separation of dextromethorphan and its metabolites using a methanolic background electrolyte. The optimization of separation conditions was performed in terms of the resolution of dextromethorphan and dextrorphan and the effect of separation temperature, voltage, and the characteristics of the background electrolyte were studied. Complete separation of all analytes was achieved in 40 mM ammonium acetate dissolved in methanol. Hydrodynamic injection was performed at 3 kPa for 4 s. The separation voltage was 20 kV accompanied by a low electric current. The ultraviolet detection was performed at 214 nm, the temperature of the capillary was 25°C. These conditions enabled the separation of four analytes plus the internal standard within 9 min. Further, the developed method was validated in terms of linearity, sensitivity, and repeatability. Rat liver perfusate samples were subjected to the nonaqueous capillary electrophoretic method to illustrate its applicability.     

Micellar electrokinetic capillary chromatographic separation and fluorescent detection of amino acids derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole

Another method has been developed for the separation of amino acids (1 min derivatization plus 22 min separation) by micellar electrokinetic capillary chromatography (MECC) with laser-induced fluorescence detection. Interestingly enough, such work has never been performed on essential amino acids derivatized by 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). Fifteen L-amino acid standards were labelled with NBD-F at 60 degrees C for 1 min, and separated in a buffer system containing 20 mM borate, 25 mM sodium cholate, 10 mM Brij 35 and 2.5% methanol. Methanol was employed to expand the MECC migration time window; whereas Brij 35 was used to improve the fluorescence intensity of amino acid derivatives. This method also indicates that bile salt is effective for MECC separation of ionic analytes. Surprising though, improvements in resolution, sensitivity and speed for amino acids analysis are obtained in this work, which are not initially apparent in just employing another derivatizing reagent. Under optimal conditions, 15 amino acids were separated in a short 22 min analysis time, the shortest ever reported, and detection limits of nanomolar concentration and attomole mass were obtained. Furthermore, RSDs of migration time and peak height are better than 1% and 1.8%, respectively, again the smallest ever reported in the literature.     

In-capillary derivatization and laser-induced fluorescence detection for the analysis of organophosphorus pesticides by micellar electrokinetic chromatography

We developed a rapid and sensitive method using in-capillary derivatization and laser-induced fluorescence (LIF) detection for the fully automated analysis of organophosphorus pesticides (OPPs), including glufosinate, aminomethylphosphonic acid (AMPA) and glyphosate by micellar electrokinetic chromatography (MEKC). The potential of 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as in-capillary derivatization reagent is described for the first time. The unique feature of this MEKC method is the capillary being used as a small reaction chamber. In in-capillary derivatization, the sample and reagent solutions were injected directly into the capillary by tandem mode, followed by an electrokinetic step to enhance the mixing efficiency of analytes and reagent plugs in accordance with their different electrophoretic mobilities. Standing a specified time for reaction, the derivatives were then immediately separated and determined. Careful optimization of the derivatization and separation conditions allowed the determination of glufosinate, AMPA and glyphosate with detection limits of 2.8, 3.6 and 32.2 ng/mL, respectively. These detection limits were comparable to those of 1.4, 1.9 and 23.8 ng/mL obtained from conventional pre-capillary derivatization. Furthermore, repeatability better than 0.40% for migration time and 3.4% for peak area, as well as shorter migration time, was obtained. The method was successfully applied to the analysis of spiked river water sample with satisfactory results.     

Stacking, derivatization, and separation by capillary electrophoresis of amino acids from cerebrospinal fluids

This paper describes the in-column derivatization, stacking, and separation of amino acids by CE in conjunction with light-emitting diode-induced fluorescence using naphthalene-2,3-dicarboxaldehyde (NDA). According to the relative electrophoretic mobilities and the migration direction in tetraborate solution (pH 9.3), the injection order is cyanide, then amino acids, then NDA. Once poly(ethylene oxide) (PEO) migrates through the capillary under EOF, the amino acid.NDA derivatives, amino acids, and CN- ions migrating against the EOF enter the PEO zone. As a result of increases in viscosity and possible interactions with PEO molecules, the reagents/analytes slow down such that they become stacked at the boundary. In comparison with the off-column approach to the analysis of amino acids, our proposed method provides a lower degree of interference from polymeric NDA compounds and other side products. As a result, the plot of the peak height as a function of gamma-aminobutyric acid (GABA) concentration is linear over the range from 10(-5) to 10(-8) M, with the LOD being 4 nM. We demonstrate the diagnostic potential of this approach for the determination of amino acids, including GABA and glutamine, in biological samples through the analysis of large volumes of cerebral spinal fluids without the need for sample pretreatment.     

Simultaneous analysis of biologically active pyridines in pharmaceutical formulations by capillary zone electrophoresis

A capillary zone electrophoretic method using UV detection is developed for the analysis of four biological active pyridines [i.e., nicotine (NIC), cotinine (COT), nicotinic acid (NA), and nicotinamide (NM)]. The separation of the pyridines is achieved in 25 mM sodium dihydrogen phosphate (pH 2.1) using a fused-silica capillary with an effective length of 56 cm and an inner diameter of 50 μm (extended light path), hydrodynamic injection at 50 mbar for 10 s, a temperature of 25°C, applied voltage of 30 kV, and UV detection at 260 nm. These conditions provide baseline separation of all the analytes [resolution (R(s)) > 3.6] in 9.4 min with good linearity (r(2) > 0.998, in ranges of 50-600 μg/mL for NIC, 8-160 μg/mL for NM, and 10-200 μg/mL for COT and NA), precision (relative standard deviation <2.04%), recovery (96.4-101.6%), limits of detection (<3.0 μg/mL), and quantitation (<10 μg/mL). The method is robust upon the alterations of pH of BGE, separating voltage, and injection time [the RSDs of the relative migration time (migration time of the analyte/migration time of the internal standard) and resolution <3.26%]. The method is efficient, reliable, and simple for the routine analysis of NIC, NA, and NM in various products such as gum and tablets and can be applied to determine COT in thermal degradation of NIC gum.     

An ionizable chromophoric reagent for the analysis of primary amine-containing drugs by capillary electrophoresis

We found that ofloxacin acyl chloride is a potential chromophoric reagent for labeling amino analytes for capillary electrophoresis. Ofloxacin acyl chloride has a tertiary amino function in its structure and the derivatives from ofloxacin acyl chloride reacting with amino analytes can be ionized by an acid treatment and analyzed by simple capillary zone electrophoresis. Ofloxacin acyl chloride was used to derivatize model analytes (without chromophore) of amantadine (amino drug), tranexamic acid (non-protein amino carboxylic acid), glycine, and methionine (protein amino acids). The resulting derivatives were analyzed by capillary zone electrophoresis with ultraviolet detection (300 nm). The detection limits of the analytes studied were in the range of 1.0-2.5 microM (S/N = 3, injection 3 s). The precision (relative standard deviation) and accuracy (relative error) of the method for intra- and inter-day analyses of the analytes were respectively below 4.5% and 3.9%. Application of the method to the analysis of tranexamic acid in plasma proved feasible.     

Method development and validation for the simultaneous determination of four coumarins in Saussurea superba by capillary zone electrophoresis

A capillary zone electrophoretic method has been developed for the determination of four coumarins--skimmin, scopolin, scopoletin, and umbelliferone-in Saussurea superba with UV detection at 254 nm. The capillary temperature was kept constant at 25 degrees C. Effects of buffer pH, electrolyte concentration, organic modifier, and applied voltage on migration behavior were studied systematically. The optimum conditions for separation were achieved by using 30 mM borate buffer at pH 9.02 containing 15% (v/v) methanol as the electrolyte and 25 kV as the applied voltage. For all analytes a good linear regression relationship (r > 0.999) was obtained between peak area and concentration over a relatively wide range. The method was validated for repeatability, precision, and accuracy. The validated method was successfully applied to the simultaneous determination of the four analytes in S. superba.