Analysis of melamine resins by capillary zone electrophoresis with electrospray ionization-mass spectrometric detection

A method for the determination of the major components of (methoxymethyl)melamine resins, with quantitative analysis of unreacted melamine by capillary zone electrophoresis (CZE) using electrospray ionization-mass spectrometry (ESI-MS) is presented. Using a low background electrolyte (BGE) pH, components are separated according to their charge/ionic radius ratio with a distinctly different separation selectivity compared to the HPLC methods commonly employed in melamine-resin analysis. The use of a time-of-flight mass spectrometer (TOF-MS) was concluded to be necessary, as the complex samples studied required maximum sensitivity and resolution, which is clearly superior for TOF-MS detectors over their quadrupole counterparts. A standard curve of free melamine was determined with an R(2) = 0.999 over a concentration range of an order of magnitude. This method offers the unique separation selectivity of CZE as well as a quicker analysis time, especially for dimers compared to the HPLC methods used to date.     

System zones in capillary zone electrophoresis

When working with capillary zone electrophoresis (CZE), the analyst has to be aware that the separation system is not homogeneous anymore as soon as a sample is brought into the background electrolyte (BGE). Upon injection, the analyte creates a disturbance in the concentration of the BGE, and the system retains a kind of memory for this inhomogeneity, which is propagated with time and leads to so-called system zones (or system eigenzones) migrating in an electric field with a certain eigenmobility. If recordable by the detector, they appear in the electropherogram as system peaks (or system eigenpeaks). However, although their appearance can not be forecasted and explained easily, they are inherent for the separation system. The progress in the theory of electromigration (accompanied by development of computer software) allows to treat the phenomenon of system zones and system peaks now also in very complex BGE systems, consisting of several multivalent weak electrolytes, and at all pH ranges. It also allows to predict the existence of BGEs having no stationary injection zone (or water zone, EO zone, gap, dip). Our paper reviews the theoretical background of the origin of the system zones (system peaks, system eigenpeaks), discusses the validity of the Kohlrausch regulating function, and gives practical hints for preparing BGEs with good separation ability not deteriorated by the occurrence of system peaks and by excessive peak-broadening.     

Development of capillary zone electrophoresis-electrospray ionization-mass spectrometry for the determination of lamotrigine in human plasma

A method of coupling capillary zone electrophoresis (CZE) with electrospray ionization-mass spectrometry (ESI-MS) detection has been developed for monitoring an antiepileptic drug, lamotrigine (LTG) in human plasma. The CZE-MS was developed in three stages: (i) CZE separation and ESI-MS detection of LTG and tyramine (TRM, internal standard) were simultaneously optimized by studying the influence of CZE background electrolyte (BGE) pH, BGE ionic strength, and nebulizer pressure of the MS sprayer; (ii) sheath liquid parameters, such as pH, ionic strength, organic modifier content, and flow rate of the sheath liquid, were systematically varied under optimum CZE-MS conditions developed in the first stage; (iii) MS sprayer chamber parameters (drying gas temperature and drying gas flow rate) were varied for the best MS detection of LTG. The developed assay was finally applied for the determination of LTG in plasma samples. The linear range of LTG in plasma sample assay was between 0.1-5.0 microg/mL with a limit of detection as low as 0.05 microg/mL and run time less than 6 min. Finally, the concentration-time profile of LTG in human plasma sample was found to correlate well when CZE-ESI-MS was compared to a more established method of high-performance liquid chromatography with ultraviolet detection.     

以L-白氨酸为手性选择剂用毛细管电泳法拆分12种手性药物

 建立了一种以L 白氨酸为手性选择剂用毛细管区带电泳法快速分离 12种手性药物的方法。实验结果表明 ,手性对映体的分离度受L 白氨酸浓度和缓冲液 pH的影响。在含有 70mmol/LL 白氨酸 ,5 0mmol/L硼砂 (pH9.0 )的溶液中 ,12种手性药物在 11min之内得到了基线分离。     

CE-MS of zein proteins from conventional and transgenic maize

In this work, an original CE-MS method has been developed to analyze the complex zein protein fractions from maize. A thorough optimization of: (i) zein protein extraction, (ii) CE separation, and (iii) electrospray-MS (ESI-MS) detection is carried out in order to obtain highly informative CE-MS profiles of this fraction. The developed CE-MS method provides good separation of multiple zein proteins based on their electrophoretic mobilities as well as adequate characterization of these proteins based on their M(r). Zein proteins with small M(r) differences (below 100 Da) were easily separated and successfully analyzed by CE-MS. Thus, apart of the so-called 15-kDa-beta-zein and 16-kDa-gamma-zein, which are demonstrated to be formed by a heterogeneous group of proteins, numerous alpha-zeins belonging to the 19- and 22-kDa fraction were also identified for the first time in this work. The usefulness of this CE-MS method was corroborated by comparing the zein-protein fingerprints of various maize lines including transgenic and their corresponding nontransgenic isogenic lines cultivated under the same conditions.     

Separation efficiency in protein zone electrophoresis performed in capillaries of different diameters

R-phycoerythrin (PHYCO, Mr 240 000), glucose-6-phosphate dehydrogenase (GPD, Mr 104 000) and two charge isomers of recombinant green fluorescent protein (GFP-1 and GFP-2, Mr 27 000) were subjected to capillary zone electrophoresis (CZE) in capillaries of 50, 100 and 150 microm inner diameter at various sample concentrations, electric field strengths, and lengths of the initial zone with the purpose of testing the hypothesis that protein - capillary wall interactions rather than thermal effects are predominantly responsible for the peak spreading of proteins in CZE. The efficiency of CZE was expressed in terms of the number of theoretical plates, N, or the plate height corrected by subtracting the contribution from initial zone length, H'. The latter has the advantage of solely reflecting contributions to the separation efficiency arising from intracolumn peak spreading in capillaries of different diameters. The separation efficiency measured varied widely, by two orders of magnitude, for these proteins under identical conditions, with GPD exhibiting the highest and PHYCO the lowest values of N. H' was found to be independent of sample concentrations within the concentration ranges studied, 1-100 microg/mL for PHYCO and 100-1000 microg/mL for GPD, while exhibiting a decrease with sample concentration for GFP, especially in 150 microm diameter capillaries, within the concentration range 1-100 microg/mL. H'was also found to be independent of electric field strength up to 300-400 V/cm for PHYCO and GFP. In all experiments, the CZE of proteins in 100 microm diameter capillaries provided a higher or, at least, equal efficiency, compared to that in 50 or 150 microm diameter capillaries. It may be concluded that the protein - capillary wall interactions and protein microheterogeneity are the dominant sources of peak spreading and their specific combinations are thought to be responsible for the wide variation in separation efficiency between proteins in CZE observed under identical conditions.     

Enantioseparation of warfarin and its metabolites by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) method with direct ultraviolet (UV)-absorbance detection is presented for the simultaneous enantiomeric separation of warfarin and its main metabolites, including warfarin alcohols, 4'-, 6-, and 7-hydroxywarfarin, using highly sulfated beta-cyclodextrin (HS-beta-CD) as the chiral selector. This chiral separation method was optimized in terms of the electrophoretic parameters, which included the concentration of HS-beta-CD used, the type and composition of organic modifier added to the background electrolyte (BGE) buffer, and the BGE buffer pH. Chiral separation of warfarin and its major metabolites was achieved with high resolution, selectivity, efficiency, repeatability, and reproducibility. This optimized chiral analysis of warfarin along with its metabolites was completed within a satisfactory electrophoresis time of 20 min.     

Characterization of the transmembrane serine receptor by capillary zone electrophoresis

Summary Capillary zone electrophoresis (CZE) was applied to the characterization of the transmembrane serine receptor in biosynthetic samples. The serine receptor, otherwise known as Tsr (taxis to serine and repellents), is a ∼ 60,000 Dalton intrinsic membrane protein whose periplasmic domain (ligand binding domain) reversibly binds the amino acid serine. In general, the electrophoresis of intrinsic membrane proteins is difficult due to severe solubility problems and adsorption which occurs during the electrophoretic run. This is due to the tendency of these types of proteins to undergo aggregation, self-aggregation and precipitation in aqueous environments. The addition of percentage levels of the surfactant, sodium dodecyl sulfate (SDS), to a tetraborate run buffer was shown to be effective both in enhancing the solubility of intact Tsr and in preventing the adsorption of intact Tsr to the fused-silica capillary wall during electrophoretic analysis. Critical separation parameters such as run buffer concentration, surfactant concentration and surfactant type were optimized to give the best separation profiles.     

Ampholytes as background electrolytes in capillary zone electrophoresis: sense or nonsense? Histidine as a model ampholyte

A lot of phenomena, occuring in capillary zone electrophoresis (CZE), are linked with the ionic concentration of the background electrolyte (BGE). If weak bases and acids are used as BGEs in CZE, at a pH where they are scarcely ionized, the ionic concentration of the BGE is very low and this brings a strong peak broadening, limited sample stacking and low sample load. Because the electromigration dispersion increases extremely, moreover, the existence of low-conductivity BGEs in CZE is a contradiction in terms. The behavior of ampholytes as BGE in CZE is examined, by means of histidine as a model ampholyte. For BGEs consisting of histidine, important parameters, including the ionic concentrations, buffer capacity, transfer ratio, and the indicator for electromigration dispersion E(1)m(1)/E(2)m(2), are calculated at various pH. Although the transfer ratio is fairly constant over the whole pH traject, the ionic concentration and buffer capacity decrease whereas the electromigration dispersion strongly increases near the pI of histidine. I.e., that ampholytes can be applied as BGEs in CZE, however, just not at pH near their pI value, except as the difference between the pK values of the basic and acidic group, the deltapK value, is very small. For ampholytes with a low deltapK value or at high concentrations, all the before-mentioned effects are less fatal, but in that case we can not speak of a real low-conductivity BGE. If ampholytes are used at pH near their pK values, the use of ampholytes as BGE is not advantageously compared with simple weak bases and acids. This has been confirmed by calculations and experiments.     

CZE and ESI-MS of Borate-Sugar Complexes

Capillary zone electrophoresis (CZE) with electrospray ionization (ESI) mass spectrometry (MS) was used to study borate (B^?1) and sugar (L) complexes (L _x B^?1). Boric acid was adjusted to pH 10 with ammonium hydroxide to create an ESI-MS compatible CZE background electrolyte. We show for the first time that the electrophoretic peaks for each injected sugar contained both the substrates (i.e., sugar and/or multimers) and products (i.e., L _x B^?1). The effects of sheath liquid, temperature, and borate concentration were studied. The molecular mass information obtained from the ESI-MS provided new evidence on the mechanisms of borate-sugar complexation. Direct infusion ESI-MS and CZE-ESI-MS experiments strongly suggest that the formation of L _x B^?1 was from the direct reaction of a sugar or sugar multimer (L _x ) and B^?1. Larger L _x B^?1, where x > 2 were observed. Separation in the CZE dimension allows for the simultaneous analysis of a sugar mixture and simplified the ESI-MS analysis of sugars of the same molecular mass. The increase in sugar electrophoretic mobility caused by the increase in borate concentration was discussed in terms of the formation of L _x B^?1 complexes. In addition, the separation of five nucleosides by CZE using a borate electrolyte and detection using ESI-MS is demonstrated.     

Determination of amino acids in tobacco samples by capillary electrophoresis/indirect absorbance detection with isolation of the electrolysis compartment and p-Aminobenzoic acid as a background electrolyte.

A fast, convenient and sensitive method of capillary zone electrophoresis (CZE) and indirect UV detection was proposed for the determination of 16 amino acids. p-Aminobenzoic acid (PAB) was selected as a background electrolyte (BGE). An isolated cell included a BGE buffer part and an electrode buffer one, which were jointed with a glass frit. The isolated cell can prevent PAB from the electrode reaction and improve the stability of the detection baseline. The separation conditions of amino acids were investigated, such as different BGEs, BGE concentration, buffer pH and electroosmotic flow (EOF) modifiers. Under the selected separation conditions, 14 amino acid peaks could be separated in 12 min. The detection limits of the amino acids were in the range of 1.7 - 4.5 micromol/L. The isolated cell is suitable for reagents reacting on the electrodes in capillary electrophoresis. The proposed method has been successfully applied to the determination of the amino acids in tobacco samples.     

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).     

Rapid separation of tetracycline derivatives and their main degradation products by capillary zone electrophoresis.

A mixture of five tetracycline (TC) derivatives: minocycline (MC), demeclocycline (DMCTC), doxycycline (DC), and sancycline (SC), as well as each TC derivative from its main degradation product were separated by capillary zone electrophoresis (CZE). The influence of the pH and the concentration and nature of the background electrolyte (BGE) on the separations was investigated. Ethylenediaminetetraacetic acid (EDTA; 1 mM) was used as additive in a 25 mM phosphate buffer (pH 2.3) because this BGE enabled the rapid separation of the TC derivatives and of each TC derivative from its respective degradation product in less than 6 min. After optimization of the separation conditions, the analytical characteristics of the method were investigated. The parameters involved were linearity, precision (repeatability and reproducibility), and limits of detection (LODs). LODs obtained for the five TC derivatives studied were about 3 microg/mL. Finally, the CZE method developed was applied to study the stability of TC derivatives and to analyze the TC derivative content in three different pharmaceutical preparations.     

New carrier electrolytes for the separation of chlorophenols by capillary electrophoresis

Optimum conditions for the separation of positional isomers of chlorophenols by capillary zone electrophoresis (CZE) were established. The behavior of five volatile electrolytes (L-cysteic acid, 3-amino-1-propanesulfonic acid, aminomethanesulfonic acid, diethylmalonic acid, and ammonium acetate) was compared. The best performance based on low electrophoretic current and high separation efficiency was obtained for diethylmalonic acid as working electrolyte. The influence of pH on the separation, using both uncoated fused-silica capillaries and modified capillaries (NaAMPS from EKT) with anionic coating, was discussed. Moreover, the effect of electrolyte concentration and applied voltage using fused-silica capillaries was studied. The optimum CZE conditions that allowed the separation of 16 chlorophenols were 20 kV, 30 mM diethylmaIonic acid, pH 7.25, and uncoated fused-silica capillary. Figures of merit such as run-to-run and day-to-day precision, linearity, and limits of detection were calculated.     

Characterization of transferrin glycoforms in human serum by CE-UV and CE-ESI-MS

Human transferrin (Tf) is a model glycoprotein for congenital disorders of glycosylation (CDG) diagnosis. In the last few years, new CE-UV methods for intact Tf glycoforms analysis have been developed using nonvolatile BGEs and organic modifiers. However, the use of these BGEs does not allow the coupling of these procedures with electrospray MS (ESI-MS). In this study, a new CE-UV separation method of Tf glycoforms is developed, using a double-layer stable coating and a volatile BGE based on ammonium acetate. The separation method is optimized using standard Tf and their potential is demonstrated applying the method to the analysis of sera Tf from healthy individuals and CDG patients. The CE-UV separation method has been coupled to ESI-MS detection. Main parameters such as sheath liquid composition are optimized in order to obtain a good sensitivity. The CE-ESI-MS method has also been used in serum samples obtaining the separation of the different proteins present in serum and partial separation of Tf glycoforms. Different mass spectra and deconvoluted molecular masses were obtained for each sialoform, allowing unequivocal glycoform identification.     

毛细管区带电泳法研究肾上腺素类药物的手性分离

使用β－环糊精（β－ＣＤ）及β－ＣＤ－羧甲基（ＣＭ－β－ＣＤ）作为手性选择剂,采用毛细管区带电泳法（ＣＺＥ）对去甲肾上腺素、肾上腺素和异丙肾上腺素的手性分离进行了研究。对影响这类药物手性分离的主要因素〔手性选择剂、背景电解质（ＢＧＥ）、分离体系的酸度和温度〕进行了讨论,并对手性识别机理进行了探讨。     

Influence of ignored and well-known zone distortions on the separation performance of proteins in capillary free zone electrophoresis with special reference to analysis in polyacrylamide-coated fused silica capillaries in various buffers. II. Experimental studies at acidic pH with on-line enrichment

The separation of acidic and basic model proteins was studied in capillary free zone electrophoresis in a polyacrylamide-coated, electroosmosis-free capillary at pH below their isoelectric points (pI) using various buffers at pH 2.7-4.8 with UV detection at 200 nm. The separation performance was significantly dependent on the coating quality, which may even differ within the same batch of capillaries. In addition, a washing step with 2 M HCl and the storage of the capillary in distilled water was essential for the performance. For high efficiency and resolution the choice of buffer constituents was extremely important which is discussed in quantitative terms in Part I. The most promising buffers were ammonium acetate and ammonium hydroxyacetate at pH 4 (ionic strengths: 0.12 and 0.15 M, respectively) with plate numbers up to 1,700,000 plates/m, corresponding to a zone width (2sigma) of only 1 mm in a capillary with 40 cm effective length, when the injected samples were dissolved in a 10-fold diluted background electrolyte (BGE), a zone even narrower than those obtained in polyacrylamide gel electrophoresis, the characteristic feature of which is remarkably thin zones. In the experiment giving this plate number, the calculated variance for longitudinal diffusion was larger than all the other calculated variances (those for the width of the starting zone, Joule heating, sedimentation and the curvature of the capillary). Interestingly, the effect of capillary curvature was significant. In addition, the sum of all other imaginable variances (corresponding to various types of slow on/off kinetics and hyper-sharp peaks) was in the most successful experiments only 28-50% of the variance for longitudinal diffusion. One hundred- to two hundred-fold dilution of the BGE improved the detection limits and provided high precision in both migration times and peak areas with ammonium hydroxyacetate and ammonium acetate as background electrolytes. However, that high dilution increased the variance 140-400% for these buffers, respectively, at least partly due to conductivity or pH differences between the sample and buffer zones (hyper-sharp peaks). Sedimentation of the enriched sample, a factor that has not previously been treated theoretically or experimentally, was probably another reason for our finding that peak heights did not increase when the sample was dissolved in a buffer diluted more than 200-fold, although pH changes and in some cases thermal expansion in the capillary also may contribute. Loss of protein may occur at the ionic strength 0.01 and lower due to precipitation. Limits of detection were in the range 4-17 pmol of proteins with ammonium acetate as BGE. No indication of denaturation of proteins at pH 4 was observed. However, the separation performance at pH 3 was not satisfactory and loss of proteins was observed, possibly indicating such problems. The protein mobilities decreased unexpectedly from pH 4 to 3--a further indication of conformation changes.     

In‐line preconcentration capillary zone electrophoresis for the analysis of haloacetic acids in water

Two in‐line enrichment procedures (large volume sample stacking (LVSS) and field amplified sample injection (FASI)) have been evaluated for the CZE analysis of haloacetic acids (HAAs) in drinking water. For LVSS, separation on normal polarity using 20 mM acetic acid–ammonium acetate (pH 5.5) containing 20% ACN as BGE was required. For FASI, the optimum conditions were 25 s hydrodynamic injection (3.5 kPa) of a water plug followed by 25 s electrokinetic injection (?10 kV) of the sample, and 200 mM formic acid–ammonium formate buffer at pH 3.0 as BGE. For both FASI and LVSS methods, linear calibration curves (r²>0.992), limit of detection on standards prepared in Milli‐Q water (49.1–200 μg/L for LVSS and 4.2–48 μg/L for FASI), and both run‐to‐run and day‐to‐day precisions (RSD values up to 15.8% for concentration) were established. Due to the higher sensitive enhancement (up to 310‐fold) achieved with FASI‐CZE, this method was selected for the analysis of HAAs in drinking water. However, for an optimal FASI application sample salinity was removed by SPE using Oasis WAX cartridges. With SPE‐FASI‐CZE, method detection limits in the range 0.05–0.8 μg/L were obtained, with recoveries, in general, higher than 90% (around 65% for monochloroacetic and monobromoacetic acids). The applicability of the SPE‐FASI‐CZE method was evaluated by analyzing drinking tap water from Barcelona where seven HAAs were found at concentration levels between 3 and 13 μg/L.     

Qualitative and quantitative analysis of active flavonoids in Flos Lonicerae by capillary zone electrophoresis coupled with solid-phase extraction

Flavonoids are an important bioactive group in the commonly used herbal medicine Flos Lonicerae. A new method of capillary zone electrophoresis (CZE) coupled with solid-phase extraction (SPE) was developed for simultaneous assay of flavonoid aglycones and glycosides in Flos Lonicerae. Optimum CZE separation was achieved with a background electrolyte (BGE) solution consisting of 80 mM boric acid and 20 mM phosphate acid, adjusted to pH 8.1, with 15% acetonitrile (v/v) added, and applying a separation voltage of 28 kV. The SPE method was used for pretreating the complex matrix of botanical materials and good reproducibility was obtained when avicularin was used as internal standard. Linearity of the method was excellent with correlation coefficients (r2) in the range of 0.9995-0.9999 and detection limits were lower than 0.6 microg/mL for the four flavonoids. The obtained recoveries varied between 93 to 104% while the relative standard deviations (RSDs) were below 4.4% (n=3). The developed CZE method was successfully used for the separation of eight flavonoids and the quantification of the four flavonoids in five species of Flos Lonicerae.     

Analysis of sub-ppb levels of Fe(II), Co(II), and Ni(II) by electrokinetic supercharging preconcentration, CZE separation, and in-capillary derivatization

The analysis of sub-ppb levels of Fe(II), Co(II), and Ni(II) in heat exchanger fluids of nuclear power plants is needed to monitor corrosion. A method involving preconcentration with electrokinetic supercharging (electrokinetic injection with transient ITP), CZE separation, and in-capillary derivatization with ortho-phenanthroline (o-Phe) for direct UV detection was thus developed. First, a multizone BGE was loaded into the capillary by successive hydrodynamic introduction of zones of (i) o-Phe-containing BGE, (ii) BGE for the zonal separation, and (iii) ammonium-based leading electrolyte. Metal cations were electrokinetically injected and stacked at the capillary inlet behind this last leading zone. Finally, a terminating electrolyte zone was hydrodynamically introduced. When a constant voltage was applied, metal ions kept on concentrating isotachophoretically, then separated in CZE mode, were complexed by migrating through an o-Phe zone, and finally detected by direct absorbance. To detect extremely thin peaks, it was attempted for the first time to focus the derivatization reagent by inducing a second transient ITP, before labeling analytes, already separated in CZE mode. With this arrangement, LODs were about 30 ppt in pure water. In heat exchanger fluid matrices containing 1000 ppm bore and 2 ppm lithium, only Fe(II) cation was detected among the three cations of interest at the 1 ppb level using the present method, and its LOD was about ten times higher, due to the lower loading rate during electrokinetic injection.