Separation and determination of enkephalin-related peptides using capillary electrophoresis

CZE with UV-absorption detection has been used for the separation and determination of enkephalin-related peptides. The experimental conditions, such as pH and concentration of running buffer, applied voltage, injection method, and time, were investigated in detail. Excellent separation efficiency could be obtained for ten enkephalin-related peptides with a 50 microm (ID) x 58 cm capillary using sodium dihydrogen phosphate as the running buffer (pH 3.11) when 20 kV of applied voltage was used. The concentration detection limits were found to be in the range of 0.31-1.94 microg/mL (defined as S/N = 3). The proposed method has been applied to analyze the spiked cerebrospinal fluid (CSF) sample, and the results showed that CZE is a powerful technique for separation and detection of the above biological peptides.     

Fluorescein thiocarbamyl amino acids as internal standards for migration time correction in capillary sieving electrophoresis

A number of algorithms have been developed to correct for migration time drift in capillary electrophoresis. Those algorithms require identification of common components in each run. However, not all components may be present or resolved in separations of complex samples, which can confound attempts for alignment. This paper reports the use of fluorescein thiocarbamyl derivatives of amino acids as internal standards for alignment of 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ)-labeled proteins in capillary sieving electrophoresis. The fluorescein thiocarbamyl derivative of aspartic acid migrates before FQ-labeled proteins and the fluorescein thiocarbamyl derivative of arginine migrates after the FQ-labeled proteins. These compounds were used as internal standards to correct for variations in migration time over a two-week period in the separation of a cellular homogenate. The experimental conditions were deliberately manipulated by varying electric field and sample preparation conditions. Three components of the homogenate were used to evaluate the alignment efficiency. Before alignment, the average relative standard deviation in migration time for these components was 13.3%. After alignment, the average relative standard deviation in migration time for these components was reduced to 0.5%.     

Analysis of the performance of antibody capture methods using fluorescent peptides with capillary zone electrophoresis with laser-induced fluorescence

A method to analyze the performance of an antibody capture method using fluorescent peptides by capillary zone electrophoresis using laser-induced fluorescence (CZE-LIF) for detection has been developed. Fluorescent peptides from the prion protein were synthesized and the corresponding antibodies were produced in rabbits against these peptides. The antibodies were used to capture the fluorescent peptides. The antibodies were then bound to protein A Sepharose. After elution, the amount of fluorescent peptide that was captured vs. the total amount placed in the assay was evaluated by CZE-LIF. Of the three peptides used in this evaluation, it was found that the recovery was approximately 25-35%. When the abnormal prion protein was prepared from scrapie-infected brain samples from hamsters and a sheep using the previously described extraction method and this method, the amount of abnormal prion protein that was measured in the fluorescence immunoassay correlated with amounts estimated from Western blot. We conclude that this method can be used to detect abnormal prion protein in a tissue sample.     

Modelling the electrophoretic migration behaviour of peptides and glycopeptides from glycoprotein digests in capillary electrophoresis-mass spectrometry

In this study, the classical semiempirical relationships between the electrophoretic mobility and the charge-to-mass ratio (m_e vs. q/M^α) were used to model the migration behaviour of peptides and glycopeptides originated from the digestion of recombinant human erythropoietin (rhEPO), a biologically and therapeutically relevant glycoprotein. The Stoke’s law (α = 1/3), the classical polymer model (α = 1/2) and the Offord’s surface law (α = 2/3) were evaluated to predict migration of peptides and glycopeptides, with and without sialic acids (SiA), in rhEPO digested with trypsin and trypsin–neuraminidase. The Stoke’s law resulted in better correlations for the set of peptides used to evaluate the models, while glycopeptides fitted better with the classical polymer model. Once predicted migration times with both models, it was easy to simulate their separation electropherogram. Results were later validated predicting migration and simulating separation of a different set of rhEPO glycopeptides and also human transferrin (Tf) peptides and glycopeptides. The excellent agreement between the experimental and the simulated electropherograms with rhEPO and Tf digests confirmed the potential applicability of this simple strategy to predict, in general, the peptide–glycopeptide electrophoretic map of any digested glycoprotein.     

On-line preconcentration capillary electrophoresis for purity profiling of synthetic peptides

In this work, an on-line preconcentration capillary electrophoresis method was optimized and evaluated for the purity control of the biologically active synthetic peptide fas-F (a 28-residue long fragment of fasciculin-1) and applied for the purity profiling of angiotensin I, oxytocin, bradykinin and luteinizing hormone releasing hormone. The laboratory-made device of the analyte concentrator cartridge consisted of a fused-silica capillary piece (150 μm i.d.×8 mm in length) packed with silica-based C18 reversed-phase chromatographic material and coupled on-line near the inlet of the separation capillary (bare fused-silica capillary, 75 μm from the concentrator to the detector). Separation of impurities present in assayed samples was achieved by using 25 mM potassium dihydrogen phosphate, pH 3.5, as running buffer and a mixture of acetonitrile: running buffer, 75:25 (v/v), as elution buffer. The intra-day relative standard deviation (R.S.D.) values for migration times ranged from 3.4 to 4.2 and 2.2-2.6% for peak areas. The inter-day R.S.D. values were 5.6-7.1 and 4.6-5.1% for migration times and peak areas, respectively. The impurity profiles obtained for each peptide were compared by CZE and on-line preconcentration CE. The proposed method allowed the preconcentration and separation of impurities with greater selectivity and higher sensitivity (100-200-fold) with respect to capillary zone electrophoresis without on-line preconcentration.     

Overview of the status and applications of capillary electrophoresis to the analysis of small molecules

The status of capillary electrophoresis (CE) in the analysis of small molecules is reviewed and summarised with the illustrative use of recent literature references. Examples are cited in this review which demonstrate that CE is now a recognised and established technique in many industries, law courts and government regulatory agencies. Each of the principal areas of CE application in small molecule analysis are covered in sections which highlight the recent developments and possibilities within that area. Application areas include the analysis of pharmaceuticals, agrochemicals, chiral separations, and forensics is covered. This is an update to a previous review article [J. Chromatogr. A 856 (1999) 443] and covers papers published between 1999 and 2002. Technical developments and improvements, such as the advent of capillary array instrumentation for increased sample throughput, and improved detection options are described. Overall it is concluded that CE has become a recognised and established technique in many areas and is still within a period of development of both instrumentation and application which will continue to expand usage.     

A sulfonated capillary that gives reproducible migration times for capillary zone electrophoresis and micellar electrokinetic chromatography

To obtain reproducible migration times and rapid analyses of analytes, sulfonate groups were chemically introduced to the inner wall of untreated fused-silica capillary with 2-(4-chlorosulfonylphenyl)ethyltrichlorosilane. The sulfonated capillary showed relatively constant electroosmotic mobility which was greater than that obtained by an untreated fused-silica capillary over the pH range studied (pH 2-9). In both CZE and MEKC, the RSDs of the migration times of analytes with the sulfonated capillary were less than 0.2% which were significantly lower than those obtained with an untreated fused-silica capillary (0.5-3.5%). When BGE were set at pH 7.0 for CZE and MEKC, the analysis times with the sulfonated capillary were about half those obtained with an untreated fused-silica capillary. These results indicate that the sulfonated capillary can provide highly reproducible and rapid analyses in CE.     

Advances in chiral separations of small peptides by capillary electrophoresis and chromatography

Many chemical and biological processes are controlled by the stereochemistry of small polypeptides (di‐, tri‐, tetra‐, penta‐, hexapeptides, etc). The biological importance of peptide stereoisomers is of great value. Therefore, the chiral resolution of peptides is an important issue in biological and medicinal sciences and drug industries. The chiral resolutions of peptide racemates have been discussed with the use of capillary electrophoresis and chromatographic techniques. The various chiral selectors used were polysaccharides, cyclodextrins, Pirkle types, macrocyclic antibiotics, crown ethers, imprinted polymers, etc. The stereochemistry of dipeptides is also discussed. Besides, efforts are made to explain the chiral recognition mechanisms, which will be helpful in understanding existing and developing new stereoselective analyses. Future perspectives of enantiomeric resolution are also predicted. Finally, the review concludes with the demand of enantiomeric resolution of all naturally occurring and synthetic peptides.     

Determination of protein-ligand affinity constants from direct migration time in capillary electrophoresis

A simple method to calculate dissociation constants for protein-ligand interactions by partial-filling capillary electrophoresis is demonstrated. The method uses raw migration time data for the ligand and needs only additional information about capillary inner radius and the absolute amount of protein loaded. A theoretical study supported by experimental data also demonstrates that the retention of analyte in affinity capillary electrophoresis (ACE) using the partial-filling technique depends linearly on the absolute amount of selector added but is independent of both selector zone length and selector mobility. Factors such as field strength and electroosmotic flow are also cancelled out if they are kept constant. The theory is confirmed and the usefulness of the method is demonstrated by enantioseparations using alpha-acid glycoprotein (AGP) and cellulase (Cel 7A) as chiral selectors.     

Field-amplified on-line sample stacking for determination of carnosine-related peptides by capillary electrophoresis

An on-line sample stacking method, namely field-amplified sample injection, has been developed for the separation and determination of carnosine, anserine, and homocarnosine by capillary electrophoresis. Using electrokinetic injection, about 130- to 160-fold improvement of sensitivity was achieved without loss of separation efficiency when compared to conventional sample injection. For conventional injection, the samples were dissolved in running buffer and then hydrodynamically injected for 10 s (3.45 kPa). Various parameters affecting separation and sample stacking were optimized. Under optimum conditions, linear responses were obtained over two orders of magnitude and the detection limits (defined as S/N = 3) of carnosine, anserine, and homocarnosine were 1.5 x 10(-8) to 1.6 x 10(-8) mol/L.     

Evaluation of the electrophoretic behaviour of opioid peptides Separation by capillary electrophoresis-electrospray ionization mass spectrometry

A general equation established in a previous study was used to model the electrophoretic mobility of a series of opioid peptides as a function of pH of the separation electrolyte. The concordance between the predicted and the experimental electrophoretic mobilities was excellent and the optimum pH for the separation of the modelled compounds could be predicted from a limited amount of experimental data. The equations were also useful for the accurate determination of the ionization constants of the polyprotic analytes. It was also demonstrated that if ionization constant values are known, the CE separations of the studied peptides can easily be predicted taking into account the classical semiempirical relationships between electrophoretic mobility and charge-to-mass ratio (m_e versus q/M^α). The separations simulated considering the accurate charge-to-mass ratios of each peptide at a certain pH value were in good agreement with the experimental results.Once an optimum separation pH value and a running buffer compatible with electrospray mass spectrometry (ESI) detection were selected, a method for the separation and characterization of this series of analytes by capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS) was established using a commercial sheath-flow interface. Method validation was performed in order to prove the suitability of the proposed method for quantitative analysis. Thus, quality parameters, such as repeatability, reproducibility, limits of detection and linearity were determined.     

Application of capillary electrophoresis in migration studies of food contact materials – part 1: Substituted phenolic additives

A method has been developed to determine 11 phenolic antioxidants in the food simulants distilled water, 3% acetie acid, and 15% ethanol, using; micellar capillary electrophoresis (MCE). All the phenols could he analyzed within 35 min. The analytical recovery from spiked simulants was 80 to 119% except for 2,6-di-tert-butyl-4hydroxytoluenc (BHT) and octyl gallate, which could not be recovered from 3% acetic acid simulant. Calibration graph correlation coefficients for the 11 phenols were 0.982 to 0.999. Limits of detection (LoDs) were from 2.8 to 8.6 mg/L. These LoDs are well below European Union migration limits for these substances. It is concluded therefore that MCE offers a rapid and reliable analysis for the control of migration from plastics intended for food contact which employ these phenols as antioxidants.     

Determination of small phosphorus-containing compounds by capillary electrophoresis

The review focuses on the analysis of small phosphorus-containing compounds by capillary electrophoresis (CE) with different detection modes including UV absorption, laser-induced fluorescence, conductometry, amperometry, atomic spectrometry, and mass spectrometry. Determinations of phosphates, organophosphate, and chemical warfare agents in environmental samples such as water, soil and grains are emphasized. To achieve better sensitivity, high-resolving power, and reproducibility, the optimum separation conditions for various analytes (samples) are different. We compare the merits and demerits of the different detection modes for the detection of the analytes. Although the present methods are successful in many cases, there is still a need to develop high-throughput CE techniques for screening numerous environmental samples and sample stacking techniques in CE for the analysis of trace analytes.     

Electrophoretic mobility for peptides with post-translational modifications in capillary electrophoresis

Several semiempirical models for peptide electrophoretic mobility have been tested for capillary electrophoresis (CE) separation with a positively charged capillary using on-line CE combined with electrospray ionization-mass spectrometry (ESI-MS). In the current system with pH 2.7, the expression q/M(0.56) provided the best correlation with the electrophoretic mobility in the analysis of a set of 18 standard samples, where q is the calculated net charge and M is the molecular weight. The peptides resulting from various digests of horse heart myoglobin or bovine hemoglobin were used to demonstrate the validity of this correlation. Post-translationally modified peptides from tryptic digest of human myelin basic protein were also investigated and were found to provide excellent correlation with the linear plot when the total charge of the peptide was correctly calculated. If the total charge was not properly calculated then the post-translationally modified peptides fell off the linear plot. Using this method five arginine residues (residues 5, 49, 54, 97 and 130) were found to be partially citrullinated, four glutamine residues (residues 8, 103, 121 and 147) were found to be partially deamidated and both methionines at residues 21 and 167 were found to be partially oxidized. Three peptides were found with phosphorylation; TPPPSQGK (residue 98 to 105), FSWGAEGQR (residue 114 to 122), and SGSPMAR (residue 163 to 169) and Arginine at 107 was found to be partially monomethylated or dimethylated. The method may provide an excellent means of identifying the presence of peptides with post-translational modifications in conjunction with MS.     

Efficient and reproducible analysis of peptides by capillary electrophoresis using noncovalently bilayer-coated capillaries

The usefulness of a noncovalent capillary coating consisting of two layers of oppositely charged polymers for the separation of peptides with capillary electrophoresis (CE) was studied. Capillaries were coated simply by subsequently flushing with solutions of 1% m/v Polybrene and 1% v/v poly(vinylsulfonate) (PVS) forming a bilayer, which showed to produce a strong and highly reproducible electroosmotic flow (EOF) at low pH. Using this coating in combination with a background electrolyte (BGE) containing sodium phosphate (pH 2.5) and 0.01% v/v PVS, initially broadened and overlapping peaks were obtained for some test peptides. By omitting the PVS from the BGE, the peak width and shape of the peptides improved resulting in baseline separation. A systematic study of the influence of the BGE composition showed that considerable further enhancement of the separation efficiency was achieved by increasing the ionic strength of the BGE. Using a BGE of 200 mM tris(hydroxymethyl)aminomethane (Tris)-phosphate (pH 2.5) plate numbers for the peptides were in the 300 000-600 000 range and the relative standard deviation of the peptide migration times was less then 0.3% (n = 5). The use of Tris-phosphate instead of sodium phosphate allowed the current to stay within acceptable limits when 30 kV was used as separation voltage. Overall, the bilayer coating showed a remarkable EOF repeatability, as well as long-term stability. Compared to bare fused-silica capillaries the intraday and interday repeatability of migration times was very favorable and coated capillaries could be used for over a month performing analyses with low and high ionic strength BGEs without any performance deterioration. The usefulness of the bilayer-coated capillaries for the analysis of positively charged peptides was demonstrated by the fast and efficient separation of various closely related enkephalins and the baseline separation of an isomeric peptide/peptoid couple exhibiting efficiencies of over 550 000 plates.     

Investigation of unexpected migration behavior of adenosine in capillary zone electrophoresis

Summary The capillary zone electrophoresis of two common nucleosides, adenosine and inosine, was investigated. Both compounds were resolved in a 0.1 M sodium phosphate buffer, pH 7.5. Contrary to expectations, adenosine behaved at this pH— 5 pH units lower than the literature pK_a— as a negative ion, migrating behind mesityloxide (neutral marker) when working in normal polarity mode. To confirm the migration order, peaks were identified from absorption maxima, by high-speed scanning detector. The change in electrophoretic mobility with pH was investigated for the nucleosides, and 10 other background electrolytes were tried to match the separation capabilities of the sodium phosphate buffer. Most inorganic buffers showed comparable separation, while organic, Good-type buffers lacked selectivity.     

Characterization of poly(4-vinylpyridine 1-oxide) by free-solution capillary electrophoresis and micellar electrokinetic chromatography

The migration characteristics of poly(4-vinylpyridine 1-oxide) (PVP-NO) in phosphate buffers of acidic pH (20 mM H3PO4 or NaH2PO4) have been studied using both free-solution capillary electrophoresis (FSCE) and MEKC. To inhibit adsorption, 250 mM o-phosphoethanolamine (2-aminoethyl dihydrogen phosphate) was used. In FSCE, PVP-NO showed a narrow peak and a broader band, both having anionic behavior. These peak and band were attributed to the free and aggregated or micellized PVP-NO forms, respectively. According to surface tension measurements, the CMC of SDS in the BGE was 1.8 and 0.48 mM in the absence and in the presence of 1000 microg/mL PVP-NO, respectively, and the association of the polymer with SDS was completed at 9.7 mM SDS. Using MEKC, a narrow peak and a broader band also appeared at SDS concentrations of ca. 1 mM, and their intensity increased with the SDS concentration. These peak and band were attributed to the formation of mixed micelles constituted by both free PVP-NO/SDS and aggregated PVP-NO/SDS, respectively. The determination of PVP-NO by FSCE in commercial additives for laundry was demonstrated.     

Analysis of protamine peptides in insulin pharmaceutical formulations by capillary electrophoresis

Protamines are a group of highly basic peptides that are sometimes added to insulin formulations to prolong the pharmacological action. In this study, different methods were investigated to identify protamine in insulin formulations. Capillary electrophoresis in aqueous and non‐aqueous media was tested to separate these peptides with very close amino acid sequences. Different buffers (phosphate or formate, both acidified) and various additives (principally negatively charged and neutral surfactants) were investigated to optimize peptide separation. Finally, a micellar electrokinetic capillary chromatography method using a capillary of 120 cm effective length and an aqueous background electrolyte made up of 100 mM phosphate buffer (pH 2) and 50 mM Thesit® gave the best results, providing the separation of the four major protamine peptides within 25 min.     

A covalent modified hydrophilic capillary for enhanced capillary electrophoresis of biopolymers

δ-Gluconolactone was covalently coupled to aminopropyl derivatized capillary,which created hydrophilic brushes on the inner wall of the capillary.The coated capillary was shown to generate a stable electroosmotic flow(EOF) in the investigated pH range of 2.0-9.0 and to suppress effectively the adsorption of proteins.And it enabled separation of some biopolymer mixtures including basic proteins,DNA and tryptic digested bovine serum albumin(BSA) within 15 min with efficiencies up to 450,000 plates/m.The in...     

Determination of levodopa by capillary electrophoresis with chemiluminescence detection

A rapid and simple method using capillary electrophoresis (CE) with chemiluminescence (CL) detection was developed for the determination of levodopa. This method was based on enhance effect of levodopa on the CL reaction between luminol and potassium hexacyanoferrate(III) (K₃[Fe(CN)₆]) in alkaline aqueous solution. CL detection employed a lab-built reaction flow cell and a photon counter. The optimized conditions for the CL detection were 1.0 × 10⁻⁵ M luminol added to the CE running buffer and 5.0 × 10⁻⁵ M K₃[Fe(CN)₆] in 0.6 M NaOH solution introduced postcolumn. Under the optimal conditions, a linear range from 5.0 × 10⁻⁸ to 2.5 × 10⁻⁶ M (r = 9991), and a detection limit of 2.0 × 10⁻⁸ M (signal/noise = 3) for levodopa were achieved. The precision (R.S.D.) on peak area (at 5.0 × 10⁻⁷ M of levodopa, n = 11) was 4.1%. The applicability of the method for the analysis of pharmaceutical and human plasma samples was examined.