The new approach of standardization of capillary electrophoresis

In this paper, we develope the new standardization methods to eliminate the influence in capillary electrophoresis (CE). The markers were used to determine the basis position and then correct the data of sample by the migration time of standard sample, and make the migration time of samples consistent with the standard sample by the criterion of the marker. The problem of time transition was corrected in this way. Then according to the peak height or peak area of the marker in the sample (peak height was used here) compared with the standard sample, the sample data was zoomed appropriately. The absorbance error was made to be correct. The wavelet de-noise method was also used to make the data smooth and get a good baseline.     

Effects of sample matrix and injection plug on dsDNA migration in capillary gel electrophoresis

Reproducible DNA migration times are required for accurate basepair assignment in restriction fragment mapping and polymerase chain reaction product identification. Our data shows DNA migration time shifts with changes in sample ionic strength. Secondly, loss of resolution with replaceable polyacrylamide gels was observed when increasing the length of the sample plug with pressure injection. An easy way to correct for the migration time shifts is to incorporate an internal DNA standard directly into the separation process by consecutively injecting the DNA sample and the DNA standard. This allows for compensation of any possible migration time variation caused by high ionic strength sample matrices. Also high-resolution separations can be maintained with large injection volumes (long injection plug) by using consecutive injections of 0.1 M Tris-acetate buffer and the DNA sample.     

Comparison of alpha-1-acid glycoprotein isoforms from healthy and cancer patients by capillary IEF

alpha-1-Acid glycoprotein (AGP) is a glycoprotein that presents different forms in the same individual, depending on the amino acid sequence and/or on the carbohydrate distribution of each form. Changes in these two types of heterogeneities are related to pathophysiological states. The aim of this work is to study the possibility of comparing AGP samples in terms of their CIEF profiles, what would facilitate in a future to perform studies about the role of AGP as a disease marker. In the present study, the CIEF profiles of AGP samples purified from sera of healthy donors and of ovary cancer and lymphoma patients are qualitatively and quantitatively compared. To make possible the comparison of those electrophoretical profiles, reliable assignment of AGP peaks is necessary. A computer program developed in our laboratory is used to select the migration parameters that make possible an accurate assignment of AGP peaks. Percentages of correct assignment of AGP peaks using the migration time of each peak relative to the migration time of an internal standard close to 95% are achieved. After peak assignment, a different distribution of the area percentage of AGP forms is observed when comparing samples from diseased and healthy individuals, the most acidic AGP forms being present in a higher proportion in the samples from cancer patients. Although the number of samples studied is too low to get any clinical significance from these results, this work provides a way to study the role of AGP as a biomarker.     

Reproducibility of sample separation using liquid or forced air convection thermostatted high performance capillary electrophoresis systems

Run-to-run sample separation reproducibility has been compared on two commercial high performance capillary electrophoresis units which differ in the mode by which the capillary temperature is thermostatted. Three standard analytes, differing dramatically in molecular character and size, were used for the analysis: benzoic acid, a 14 amino acid peptide from human chorionic gonadotropin, and ribonuclease A represent, respectively, small stable organic molecules, small peptides with little or no secondary structure, and proteins with secondary structure. These standards were evaluated with regard to reproducibility of migration time, peak area, and peak height. The analyses, performed in buffers of optimum pH for the separations, demonstrated that the liquid and forced air convection thermostatted systems both performed extremely well. The reproducibility, as judged by the percent coefficient of variance (% CV) of replicate analyses, was generally found to be less than 1 % (migration time); the reproducibility decreased in the order migration time > peak height > peak area. Whereas the absolute % CV values for MT_rel (migration relative to a standard) observed with the liquid thermostatted system were 2- to 4-fold lower than those observed with the forced air convection thermostatted system, there was little statistically significant difference between the two. As expected, the data indicated a reduction in reproducibility as the complexity of the analyte increased, perhaps as the result of an increased potential for wall interactions. Comparing separations in which low (≈?1 watt/meter [W/m] of capillary) and high (>5 W/m) Joule heat was generated by altering the sodium chloride content of the buffer revealed few statistically significant differences in the reproducibility obtained from the two systems. With these particular standard analytes and their respective buffer systems, there appears to be little difference between forced air convection and liquid thermostatting of the capillary.     

CZE of human alpha-1-acid glycoprotein for qualitative and quantitative comparison of samples from different pathological conditions

Alpha-1-acid glycoprotein (AGP) presents different forms, which may arise from differences in the amino acid sequence and/or in the glycosidic part of the protein. Changes in forms of AGP have been described in literature as a possible tumor marker. While most previous works have approached the study of glycopeptides and/or glycans obtained after fragmentation of the protein, in this work, a CZE method is developed to separate up to eleven peaks of intact forms of AGP. A computer program developed in our laboratory is used to select the migration parameters that make possible an accurate assignment of AGP peaks. Electropherograms of AGP samples purified from sera of cancer patients and healthy donors are qualitatively and quantitatively compared. Percentages of correct assignment of AGP peaks close to 100% are achieved by using either the migration time of each peak relative to that of the EOF marker or the effective electrophoretic mobility of the peaks. The computer program permits to select, among different hypotheses for peak allotment, that one providing the highest accuracy of assignment. In this way, some peaks with different charge-to-mass ratio and a different distribution of area percentage of AGP forms are observed when comparing samples from sick and healthy individuals. Thus, a method that permits to compare AGP forms existing in sera of individuals with different pathophysiological situations has been developed. A potential for using AGP forms analyzed by CZE as a disease marker and for using this technique for screening purposes is envisaged.     

Yoctomole analysis of ganglioside metabolism in PC12 cellular homogenates

We report an ultrasensitive method for the analysis of glycosphingolipid catabolism. The substrate G(M1) and the set of seven metabolites into which it can be degraded (G(A1), G(M2), G(A2), G(M3), LacCer, GlcCer, and Cer) were labeled with the highly fluorescent dye tetramethylrhodamine. CE with LIF detection was used to assay these compounds with 150 +/- 80 yoctomole mass (1 ymol = 10(-24) mol = 0.6 copies) detection limits and 5 +/- 3 pM concentration detection limits. An alignment algorithm based on migration of two components was employed to correct for drift in the separation. The within-day and between-day precision in peak height was 20%, in peak width 15%, and in adjusted migration time 0.03%. After normalization to total sample injected, the RSD in peak height reduced to 2-6%, which approaches the limit set by molecular shot noise in the number of molecules taken for analysis. PC12 cells were incubated with the labeled G(M1). Fluorescent microscopy demonstrated uptake by the cells. CE was used to separate a cellular homogenate prepared from these cells. A set of peaks was observed, which were tentatively identified based on comigration with the standards. Roughly 120 pL of homogenate was injected, which contained a total of 150 zmol of labeled substrate and products. Metabolite that preserves the fluorescent label can be detected at the yoctomole level, which should allow characterization of this metabolic pathway in single cells.     

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%.     

Comprehensive two-dimensional separations based on capillary high-performance liquid chromatography and microchip electrophoresis

A novel comprehensive two-dimensional (2-D) separation system coupling capillary high-performance liquid chromatography (cHPLC) with microchip electrophoresis (chip CE) is demonstrated. Reversed-phase cHPLC was used as the first dimension, and chip CE acted as the second dimension to perform fast sample transfers and separations. A valve-free gating interface was devised simply by inserting the outlet-end of LC column into the cross-channel on a specially designed chip. A home-made confocal laser-induced fluorescence detector was used to perform on-chip high-sensitive detection. The cHPLC effluents were continuously delivered to the chip and pinched injections of the effluents every 20 seconds were employed for chip CE separation. Gradient elution of cHPLC was carried out to obtain the high-efficiency separation. Free-zone electrophoresis was performed with triethylamine buffer to achieve high-speed separation and prevent sample adsorption. Such a simple-made comprehensive system was proved to be effective. The relative standard deviations for migration time and peak height of rhodamine B in 150 sample transfers were 3.2% and 9.8%, respectively. Peptides of the fluorescein isothiocyanate (FITC)-labeled tryptic digests of bovine serum albumin were fairly resolved and detected with this comprehensive 2-D system.     

多肽在PEG-400交联改性电泳毛细管柱中的缓冲溶液浓度效应

在PEG-400交联柱上,用多肽样品研究了毛细管区带电泳中缓冲溶液浓度对迁移时间、柱效、选择性、分离度及进样量等的影响.发现试样的峰高与缓冲液浓度存在近似线性的关系,并随着浓度的增加而增大.对于电动进样模式,应注意毛细管内和进样槽里试样浓度的不一致性.     

电迁移微离子色谱及其应用

本文提出一种新型的离子色谱方法,采用恒电流电场代替机械泵输送离子,研制出体积小、重量轻、成本低、易操作的电迁移微离子色谱仪,并检测了几种常见的阴离子,Cl~-、Br~-、NO_2~-的检测极限为10~(-11)～10~(-12)mol,峰高和保留时间重现性相对标准偏差均<3％,进样量与峰高的线性关系良好。     

Highly sensitive chemiluminescence detection of copper(II) in capillary electrophoresis with field-amplified sample injection

Field-amplified sample injection of copper(II) was investigated using capillary electrophoresis with chemiluminescence detection. The sensitivity of copper(II) has been improved markedly by the field-amplified sample injection technique and the detection limit reaches 2 x 10(-11) M. By injection of a short plug of water before sample introduction, the sensitivity can be further improved 5-fold and the detection limit reaches 4 x 10(-12) M. The relative standard deviations (n = 6) of the migration time and the peak height are 0.61% and 4.7% at 1.0 x 10(-9) M Cu(II), respectively. Parameters affecting the field-amplified sample injection, such as separation voltage and concentration of electrophoretic buffer, have been investigated.     

Precision of micellar electrokinetic capillary chromatography in the determination of seven antibiotics in pharmaceuticals and feedstuffs

Validation of analytical procedures is important for their efficient and reliable application. The International Conference on Harmonisation (ICH), Food and Drug Administration (FDA) and pharmacopoeia guidelines achieved a great deal in harmonising the definitions of the required validation characteristics. It is well known that poor reproducibility limits the practical implementation of capillary electrophoresis (CE). A precision study on four different MEKC methods was performed with 11 samples, containing seven antibiotics, by two analysts, in few days, on two capillary electrophoresis instruments. Five pharmaceutical preparations and three animal feeds were used. Precision was statistically analysed using migration time, peak area and height of each compound, as well as electroosmotic front (EOF). In 25 of 31 cases, the reproducibility of peak area, peak height and migration time was good (<5%). In most cases the reproducibility of peak area was much better than the reproducibility of peak height. The worst reproducibility that we observed was 12.7% for peak height and 7.6% for peak area.     

Microfluidic chip-capillary electrophoresis with dynamic multi-segment standard addition for rapidly identifying nephrolithiasis markers in urine

A microchip-CE device was fabricated for bed-side monitoring of nephrolithiasis biomarkers in urine by incorporating on-chip continuous passive mixing and standard addition to reduce sample matrix interference, increase sample throughput and eliminate accessories for active mixing. Under optimized conditions with buffer containing 20 mM borate and 0.5 mM CTAB at pH 10.3, sample and standards injected electrokinetically at -350 V for 10 s for online mixing in a Y-merging flow microchannel prior to CE separation and UV detection at 210 nm, both inhibitors (citrate, CA) and promoters (oxalate, OA and uric acid, UA) for nephrolithiasis can be separated and determined in human urine in a single run completed within 10 min after a simple 50-fold sample dilution and filtering. Satisfactory working ranges from 0.13-40, 0.25-40 and 0.025-40 mM, LOD 2.6, 6.1 and 0.7 μM, repeatability (%RSD, n=5) for migration time 1.40, 1.43, 0.47 and peak area 4.46, 6.10, 1.98, respectively, for CA, OA and UA are obtained for urine samples. The use of on-chip standard addition is shown to improve repeatability of the migration time, assist the identification of nephrolithiasis markers from difficult samples with noisy baseline and enlarge the working range for nephrolithiasis marker determination. The device developed can be used for both routine and emergency monitoring to deliver results on demand for bedside monitoring and public health protection. It provides an early detection of nephrolithiasis to enable timely treatments, ease anxiety of parents for neonates consuming suspected contaminated food, and quick results for patients in a critical condition.     

Determination of nitrate in seawater by capillary zone electrophoresis with chloride-induced sample self-stacking

A capillary zone electrophoresis (CZE) method was established to determine low concentration nitrate which was online preconcentrated with chloride-induced leading-type sample self-stacking for seawater samples. The sample self-stacking was based on transient isotachophoresis in which chloride served as leading ion, and dihydrogenphosphate in the background electrolyte (0.1 M phosphate) as the terminating one. Due to the small mobility difference between nitrate and chloride, the isotachophoresis time was so long that nitrate could not separate from the rear sharp boundary between chloride and the background electrolyte (BGE) when it migrated to the detection window. A zwitterionic surfactant, 3-(N,N-dimethyldodecylammonio)propane sulfonate was added to the BGE to enlarge the mobility difference for its selective interaction with anions. Thus, a highly conductive sample could be injected in a large volume with about fourfold sensitivity enhancement compared to that of field amplification sample stacking in which nitrate was dissolved in pure water. The relative standard deviations (n=5) of migration time, peak area, peak height were 0.1, 3.0, 1.5%, respectively. The limit of detection (S/N=3) for nitrate was 35 microg/l in seawater samples with relatively low concentration BGE (0.1 M sodium phosphate, pH 6.2). The overall procedure consisting of online preconcentration and separation was as simple as routine CZE except for a slightly longer sample injection time (3-4 min).     

Essential peak area normalisation for quantitative impurity content determination by capillary electrophoresis

Summary A number of papers have been published [1–5] which mention that normalisation of CE peak areas (ie division of peak areas by migration time) is necessary to ensure correct quantitation. However, there is a general unawareness of the impact of not performing this simple data manipulation upon impurity results when expressed as % area/area. An exercise has been conducted to exemplify the need for this normalisation using the separation of selected pharmaceuticals as illustrative examples. The impact of normalisation on % area/area results was demonstrated using the pharmaceutical ranitidine, and a synthetic precusor, as test solutes. The UV absorbance of each compound was determined and found to be equivalent. A solution of ranitidine hydrochloride was the spiked with a weighed amount of precursor. The % area/area CE results, when normalised, mateched the known weighed radio. Use of uncorrected peak area data in this instance would have resulted in a severe underestimation of impurity levels. A quantitative analysis of drug related impurities was conducted at three levels of operating voltage whilst keeping all other operating parameters constant. This produced electropherograms having identical peak profiles but each peak having a different retention time and peak area. However, when normalised, the results were identical at each level of operating voltage confirming the validity and necessity of normalisation. A chiral separation of a racemic pharmaceutical was conducted. The uncorrected peak area data indicated the test sample was not racemic whilst the corrected data correctly confirmed that the compound was racemic. The significance and impact upon reported purity data of not normalising peak area data has been clearly demonstrated in this paper.     

Principles for different modes of multiple-injection CZE

This paper introduces four different modes of multiple-injection CZE (MICZE). The validity of these MICZE models was evaluated by the experimental data. Prior to the application of MICZE, the electrophoretic conditions are developed in the single-injection mode by adjusting different experimental parameters such as pH, type and concentration of buffer additives and temperature. Based on the migration time difference (Deltatmig) between the analyte and the internal standard or injection marker, one or more MICZE modes can be employed. The injection marker is added to the sample to compensate for injection-volume fluctuations. The inter-plug distance is regulated by applying an electrical field over the capillary for a short period of time between each injection. After the final injection, the separation is completed by electrophoresis for a time period corresponding to that in the single-injection mode.     

Specific rotation measurements from peak height data, with a Gaussian peak model

A method is described whereby a sensitive laser-based polarimeter can be used to make very accurate and precise specific rotation measurements on microgram quantities of optically active materials. Flow-injection or liquid chromatography systems provide reproducible introduction of the sample into the polarimetric system. If a Gaussian distribution of the analyte concentration is assumed, the peak height can be used in the determination of specific rotation. This method provides a direct calibration with an absolute standard which yields more accurate and precise results than those obtainable by using peak area.     

On-line sample concentration via micelle to solvent stacking of cations prepared with aqueous organic solvents in capillary electrophoresis

In this paper, by injecting a SDS micellar plug before the sample prepared in aqueous organic solvents, we show the on-line sample preconcentration of cations via micelle to solvent stacking (MSS) using solvents of as low as 30%. This extends the choice of stacking techniques to include moderate amounts of organic solvent in the sample. The approach is akin to in-line solid phase extraction where the micellar plug acted as a transient micellar phase extractor. Basic studies were conducted (e.g. type and amount of organic solvent in the sample). The calculated sensitivity enhancement factors based on LOD obtained for the six test antipsychotic drugs were from 41 to 68. The peak signals were linear (R2 > 0.99) from 0.2 to 10.0 μg/mL. The intraday and interday reproducibility (n = 10) for migration time, peak height, and corrected peak area were from 0.2 to 13.6%. The technique was also tested on spiked wastewater sample with minimal sample treatment (i.e. dilution and centrifugation).     

Simultaneous analysis of polyhydroxylated alkaloids by capillary electrophoresis using borate complexation and evaluation of sweeping technique for sensitivity improvement

Summary Capillary zone electrophoresis coupled to UV detection was used for the simultaneous analysis of naturally occurring polyhydroxylated alkaloids. This separation was based on anin-situ complexation with borate ions. The effect of parameters such as borate concentration, capillary temperature and analyte molecular structure on migration times and selectivity were discussed. The best separation was obtained with a fused silica capillary (48.5 cm total length ×50 μm I.D., with a bubble factor of 3), 80 mM sodium tetraborate aqueous solution at pH 9.2 and temperature of 20°C. The method was validated and showed good data in terms of migration time and peak area reproducibility, selectivity, linearity and accuracy. The validated method was applied to determine miglitol in commercially available pharmaceutical tablets. To further improve method sensitivity, a sweeping technique involving borate ions was evaluated. This technique was found very sensitive to the analyte complexation with borate, borate concentration, and temperature as well as sample matrix. In the case of miglitol, a 35-fold improvement in peak height was achieved.     

Off-Line Concentration of Bisphenol A and Three Alkylphenols by SPE then On-Line Concentration and Rapid Separation by Reverse-Migration Micellar Electrokinetic Chromatography

A method for separation of four environmentally important compounds (three alkylphenols and bisphenol A) by cyclodextrin-modified reverse-migration micellar electrokinetic chromatography (CD-RM-MEKC) has been optimized. On-line concentration, stacking using reverse-migrating micelles and a water plug (SRW), was used to improve the limit of detection. Two main factors affecting the efficiency of enhancement by SRW, injection sample length and water-plug length, are discussed in detail. Compared with the normal 2-s injection, more than 121-fold enhancement of peak area and more than 71-fold enhancement of peak height were achieved with 120-s sample injection after 120-s water-plug injection. With this optimized separation method and SRW technology, limits of detection were found to be 0.089, 0.030, 0.0091, and 0.033 mg L^–1 for 4-nonylphenol, 4-tert-octylphenol, bisphenol A, and 4-tert-butylphenol, respectively. Calibration plots (R² 0.9968) and relative standard deviations (RSD, %) of corrected peak areas and migration time were also satisfactory. Combined with off-line concentration by solid-phase extraction (SPE) this method might be used to determine these contaminants at ppb levels in groundwater samples.