A novel cross-H-channel interface for flow injection-capillary electrophoresis to reduce sample requirement and improve sensitivity

In this study, a cross-H-channel interface was constructed for coupling flow injection with capillary electrophoresis (FI-CE) to reduce sample requirement and sensitivity loss in the typical FI-CE. Based on this cross-H-channel interface, a new FI-CE system was established, in which sample introduction was performed by directly injecting sample solution along a thin capillary (50 μm, i.d.) to the interface from an injection syringe. The sample requirement was reduced distinctly and usual sample dilution in the sample transport process was obviously decreased, thereby spontaneously enhancing the sensitivity. Moreover, because of the unique construction of the cross-H-channel interface, field amplified sample stacking (FASS) and high-speed CE were skillfully combined to further improve the sensitivity and to shorten separation time. The versatility of this new FI-CE was demonstrated by determination of ephedrine (E) and pseudoephedrine (PE) in human urine. Up to 45 repeated injections per hour and clearly baseline separation of E and PE in less than 1 min were achieved, giving limits of detection (LODs) of 0.23 and 0.21 μg mL(-1) for E and PE, respectively, and yielding relative standard deviation (RSD) values of the migration time and the peak height (n=5) of 2.6% and 3.1% for E, 2.3% and 3.3% for PE, respectively. In contrast to typical FI-CE, approximately 8-250-fold decreases in sample volume requirement, 7-fold shortening in separation time and 50-fold improvements in sensitivity were obtained.     

Capillary electrophoresis mass spectrometry with sheathless electrospray ionization for high sensitivity analysis of underivatized amino acids

A high durability sheathless electrospray ionization interface of CE-MS is applied for the sensitive analysis of underivatized amino acids. The sheathless interface was realized using an ionophore membrane-packed electro-conduction channel. The interface functioned well with a volatile alkaline background electrolyte (BGE) and uncoated fused-silica capillaries for CE-MS analysis of underivatized amino acids. High electroosmotic flow with alkaline BGE facilitated high separation efficiency (>100,000 theoretical plates) and short analysis time (<15 min). Both the short-term stability and long-term durability are particularly suited for routine applications. Using electrokinetic injection and the multiple reaction monitoring (MRM) mode with a triple-quadrupole analyzer, high sensitivity was achieved, which yielded detection limits of 0.05-0.81 μM. For the quantitation of underivatized amino acids, quantification precisions (RSDs) for intra- and inter-day analyses were less than 3%. Recoveries from serum were 96.3-101.8% for isotope dilution mass spectrometry (IDMS). When compared with HPLC-IDMS for human serum samples, highly agreeable (96.9-102.0%) results were obtained with the proposed CE-IDMS method.     

Determination of myo-inositol phosphates in food samples by flow injection-capillary zone electrophoresis

A capillary electrophoresis (CE) method with indirect photometric detection was developed to identify and quantify myo-inositol phosphates in food samples. A flow-injection (FI) system including a micro-column containing anionic exchange resin was used for the solid-phase extraction of the myo-inositol phosphates with a view to their preconcentration. The FI system was automatically coupled to CE equipment via a mechanical interface. The overall analysis time was shortened by incorporating an FI system for myo-inositol hexakisphosphate monitoring. The limit of detection for myo-inositol phosphates as determined by FI-CE ranged from 11 to 26 micromol/L and the coefficient of variation from 3.9 to 5.0%. On the other hand, the limit of detection and coefficient of variation for myo-inositol hexakisphosphate as monitored by the FI system were 75 micromol/L and 2.9%, respectively. The proposed method was successfully applied to a variety of food samples with recoveries ranging from 96.0 to 107.7% and the precision from 3.9 to 7.9%. Based on the results, the content of myo-inositol hexakisphosphate in nuts was two or three times higher than that in legumes.     

Simultaneous Determination of Organic Acids in Wine Samples by Capillary Electrophoresis and UV Detection: Optimization with Five Different Background Electrolytes

A simple technique is described for the routine capillary electrophoretic determination of organic acids in wine samples. Several aromatic and non‐aromatic compounds, including phthalic acid, benzoic acid, sorbic acid, boric acid, and phosphate, were evaluated as background electrolytes in order to obtain the highest resolution and detection sensivity. Factors that affect capillary electrophoretic separation such as the concentration and pH of the background electrolyte (BGE), the concentration of the electroosmotic flow modifier (EOF), and methanol addition to the electrolyte were investigated systematically. Tartaric, malic, succinic, acetic, and lactic acids were determined simultaneously in approximately six minutes using an electrolyte containing 3 mM phosphate and 0.5 mM myristyltrimethylammonium bromide (MTAB) as electroosmotic flow modifier at pH 6.5. This method is quantitative, with recoveries in the 90–102% range and linear up to 50 mg L^–1. The precision is better than 1% and the procedure shows the appropriate sensibility, with detection limits between 0.015 and 0.054 mg L^–1. The proposed method was successfully employed for the determination of organic acids in wine samples by direct sample injection after appropriate dilution and filtration.     

Online Concentration by Head-column Field-amplified with Large-volume Sample Stacking Using Flow Injection-Capillary Electrophoresis for the Analysis of Four Active Components in Cold Medicines.

A simple, effective, and sensitive online concentration method for the detection of dextromethorphan hydrobromide (Dex), chlorphenamine hydrogen maleate (Chl), pseudoephedrine hydrochloride (Pse) and paracetamol (Par) based on flow injection-capillary electrophoresis (FI-CE) analysis with head-column field-amplified sample stacking and large-volume sample stacking was developed. The background electrolyte (BGE) was a solution composed of 55 mM borate-15% (v/v) acetonitrile (ACN) (pH 9.3). The sample was injected electrokinetically between plugs of water. Under the optimum conditions, about a 30-fold improvement in the concentration sensitivity relative to normal CE methods was achieved, giving low limits of detection (LOD) of 1.94 x 10(-5), 0.64 x 10(-5), 1.16 x 10(-5) and 2.84 x 10(-5) mg/mL for Dex, Chl, Pse and Par, respectively. The repeatability (defined as RSD) was 1.01, 1.91, 0.89 and 0.92% with the peak-area evaluation and 1.94, 3.98, 2.66 and 3.27% with the peak-height evaluation for Dex, Chl, Pse and Par, respectively. This method has been successfully applied to the analysis of commercial pharmaceutical preparations containing Dex, Chl, Pse and Par.     

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.     

Analysis of glycosaminoglycan monosaccharides by capillary electrophoresis using indirect laser-induced fluorescence detection

Two methods for monosaccharide analysis by capillary electrophoresis (CE) using counterelectroosmotic and coelectroosmotic modes with indirect laser-induced fluorescence detection were optimised and compared. A mixture of seven glycosaminoglycan-derived hexoses was separated in alkaline fluorescein-based electrolytes and detected in both counterelectroosmotic and coelectroosmotic conditions. The fluorescein concentration and pH of the background electrolyte, and the influence of the reversal of electroosmotic flow by addition of hexadimethrine bromide on the separation were studied. Coelectroosmotic CE conditions provided better resolution and limits of detection. A 10(-6) M fluorescein solution at pH 12.25 containing 0.0005% (w/v) hexadimethrine bromide was used as background electrolyte. Quality parameters such as run-to-run, day-to-day precision and limits of detection were calculated, and better figures of merit were obtained for the coelectrooosmotic conditions than for the counterelectroosmotic mode. The coelectroosmotic method was applied to the quantitation of the hexosamine contents in glycosaminoglycans after acid hydrolysis. The method proved to be suitable for the determination of dermatan sulfate in heparin down to 2% (w/w).     

流动注射-毛细管电泳分流式电动进样歧视效应特征的研究

以碱性药物盐酸伪麻黄碱和酸性药物布洛芬为对象研究了分流式电动进样(一种用于流动注射-毛细管电泳(FI-CE)联用系统的新进样方法)歧视效应的特性。结果发现:在样品介质与运行缓冲液一致的条件下,FI-CE分流式电动进样产生的歧视效应与电动进样相似,但获得的校正曲线的线性明显优于电动进样,而与没有歧视效应的压力进样所获得的线性相似。利用这些特征提高了同时测定复方布洛芬片中少量组分盐酸伪麻黄碱和主要组分布洛芬的分析性能。在24次/h的采样频率下,盐酸伪麻黄碱的检测限为0.7 mg/L,比采用压力进样的毛细管电泳法所得的检测限低30%。连续进样11次分析含有13.1 mg/L盐酸伪麻黄碱和81.4 mg/L布洛芬的试样溶液,峰面积的相对标准偏差分别为2.8%(盐酸伪麻黄碱)和1.2%(布洛芬),明显优于采用压力进样的毛细管电泳法。用该法测定了两批复方布洛芬片中两种组分的含量,所得结果与高效液相色谱法的测定结果一致。     

Analysis of drying oils used as binding media for objects of art by capillary electrophoresis with indirect UV and conductivity detection

Capillary electrophoresis (CE) was applied to analyse the long-chain fatty acid composition of vegetable oils, and their degradation products formed upon ageing when drying oils are used as binding media. The analytes were detected with contactless conductivity detection (CCD) and indirect UV absorption, both detectors positioned on-line at the separation capillary. The long-chain fatty acids were resolved in a background electrolyte (BGE) consisting of phosphate buffer (pH = 6.86, 15 mM) containing 4 mM sodium dodecylbenzensulfonate, 10 mM Brij 35, 2% (v/v) 1-octanol and 45% (v/v) acetonitrile. As in this system dicarboxylic analytes, the products of oxidative degradation of unsaturated fatty acids, cannot be determined, a suitable background electrolyte was developed by the aid of computer simulation program PeakMaster. It makes use of a 10 mM salicylic acid, 20 mM histidine buffer, pH 5.85, which combines buffering ability with the optical properties obligatory for indirect UV detection. This buffer avoids system eigenpeaks, which are often impairing the separation efficiency of the system. Separation of the dicarboxylic analytes was further improved by a counter-directed electroosmotic flow (EOF), obtained by dynamically coating the capillary wall with 0.2 mM cetyltrimethylammonium bromide. Long-chain fatty acids and their decomposition products could be determined in recent and aged samples of drying oils, respectively, and in samples taken from two paintings of the 19th century.     

Study on characteristics of bias caused by FI-CE split flow electrokinetic injection

The characteristics of bias caused by split-flow electrokinetic injection (SEKI), a new type of sample injection method used in coupled flow injection-capillary electrophoresis system (FI-CE), was investigated using pseudoephedrine hydrochloride, a basic drug, and ibuprofen, an acidic drug, as model analytes. It was found that bias imposed by SEKI under the condition of continuous sample matrix/running buffer was similar to that done by electrokinetic injection (EKI). The linearity of calibration curve provided by SEKI was similar to that offered by non-bias hydrodynamic injection (HDI) but significantly better than that obtained by EKI. These features were exploited to improve analytical performances in simultaneous determination of the minor ingredient of pseudoephedrine hydrochloride and the major ingredient of ibuprofen in a pharmaceutical preparation. Detectability of 0.7 mg/l for pseudoephedrine hydrochloride was achieved at a sample throughput rate of 24 times per hour, which is 30% lower than that obtained by HDI-based conventional CE. Relative standard deviations (RSDs) of 2.8% for the minor ingredient and 1.2% for the major ingredient were produced in 11 runs of a test solution containing 13.1 mg/l pseudoephedrine hydrochloride and 81.4 mg/l ibuprofen. This is an improvement compared to that obtained by HDI-based conventional CE. Analytical results for two batches of compound ibuprofen tablets by the SEKI-based FI-CE approach were in good agreement with that obtained by a conventional high performance liquid chromatographic method. __________ Translated from Chinese Journal of Chromatography, 2005, 23(2) (in Chinese)     

Simple in‐house flow‐injection capillary electrophoresis with capacitively coupled contactless conductivity method for the determination of colistin

An in‐house flow‐injection capillary electrophoresis with capacitively coupled contactless conductivity detection method was developed for the direct measurement of colistin in pharmaceutical samples. The flow injection and capillary electrophoresis systems are connected by an acrylic interface. Capillary electrophoresis separation is achieved within 2 min using a background electrolyte solution of 5 mM 2‐morpholinoethanesulfonic acid and 5 mM histidine (pH 6). The flow‐injection section allows for convenient filling of the capillary and sample introduction without the use of a pressure/vacuum manifold. Capacitively coupled contactless conductivity detection is employed since colistin has no chromophore but is cationic at pH 6. Calibration curve is linear from 20 to 150 mg/L, with a correlation coefficient (r²) of 0.997. The limit of quantitation is 20 mg/L. The developed method provides precision, simplicity, and short analysis time.     

Capillary electrophoretic method for the determination of inorganic and organic anions in real samples. Strategies for improving repeatability and reproducibility

Selectivity and robustness of the pyromellitic acid (PMA) based background electrolyte was improved in order to increase its applicability for routine analysis of inorganic and organic anions in real samples. An electrolyte composed of 6.75 mM PMA, 0.5 mM hexamethonium hydroxide as electroosmotic flow (EOF) modifier, Ca(2+) 0.05 mM as complexation agent and pH adjusted to 7.6 with TEA 1M allows for the separation of 22 inorganic and organic anions in less than 17 min. Good RSDs for within-day migration time reproducibility (0.03-0.9%) and day-to-day analyses (0.04-1.4%) were obtained by the use of two internal standards, allowing for an accurate compound identification. The detection limits ranged from 0.1 to 0.4 mgL(-1) (S/N=3) for hydrodynamic injection (1250 mbars). The applicability of the proposed method was demonstrated by the analysis of inorganic and organic anions in diverse real samples. The recoveries obtained ranged from 93 to 106%.     

On-line preconcentration and quantitative analysis of peptide hormone of brain and intestine using on-column transient isotachophoresis coupled with capillary electrophoresis/electrospray ionization mass spectrometry

A new approach was described to achieve very sensitive analysis of peptide hormone of brain and intestine by capillary electrophoresis coupled with a transient isotachophoresis (tITP) preconcentration method. The system used was electrospray ionization mass spectrometry (ESI-MS) as detector and equipped with a sheath flow configuration. The effects of sample matrix, pH and concentration of leading electrolyte (LE), sample injection time, and ESI-MS instrumental parameters on the efficiency of the sample stacking were investigated in detail. Under the optimized conditions, lower than micromole (0.01 microM) concentrations of the peptides were easily detected. Compared to traditional hydrodynamic injection methods, about 40-230-fold increase in detection sensitivity was obtained by this technique. A distinguishing feature of the described approach is that the background electrolyte can serve as terminating electrolyte (TE), which simplifies the process of the experiments. The method was further evaluated by the analysis of low concentration active peptide mixtures spiked in hypothalamus tissue of the rat.     

Spectrophotometric determination of chloride in mineral and drinking waters using sequential injection analysis

An on-line sequential injection system has been developed for spectrophotometric determination of chloride in drinking mineral, natural, and ground waters. Samples containing different concentrations of chloride were analyzed. The analysis is based on detection of the red iron(III) thiocyanate complex. The complex was monitored spectrophotometrically at 480 nm using de-ionized water as the carrier stream at a flow rate of 3.21 mL min(-1). The method was found to be linear within the range 0-50 mg L(-1) chloride; the detection limit was 3.01 mg L(-1). The fully automated method can be used to analyze 37 samples per hour with a relative standard deviation (RSD) better than 2.50%.     

Online preconcentration by electrokinetic supercharging for sensitive determination of berberine and jatrorrhizine in biological samples

Electrokinetic supercharging, a convenient and powerful online preconcentration technique in capillary electrophoresis, was introduced and evaluated for the determination of two alkaloids, berberine and jatrorrhizine, in mice fecal samples for the first time. The method depended on using a bare fused silica capillary (50 cm × 50 μm i.d.) and applying the voltage of 25 kV with UV detection at 205 nm. Parameters that affect the separation and preconcentration efficiency have been optimized. The optimum conditions used were as follows: background electrolyte consisting of 40mM sodium dihydrogenphosphate containing 30% methanol (v/v); hydrodynamic injection of 20mM KCl (50 mbar × 150 s) as the leading electrolyte; electrokinetic injection of the sample (+15 kV, 120 s) followed by the hydrodynamic injection of 30mM dodecyl trimethyl ammonium chloride (50 mbar × 12 s) as the terminating electrolyte. The results showed that the detection sensitivity of berberine and jatrorrhizine was, respectively, improved up 2740- and 2928-fold compared with normal injection, providing limits of detection lower than 3 ng/mL with good repeatability in areas (relative standard deviation < 3%). In summary, the developed method proved its ability in analyzing trace alkaloids in complicated biological samples.     

Application of a contactless conductometric detector for the simultaneous determination of small anions and cations by capillary electrophoresis with dual-opposite end injection

A contactless conductometric detection (CCD) system for capillary electrophoresis (CE) with a flexible detection cell was applied for the simultaneous determination of small anions and/or cations in rain, surface and drainage water samples. The applied frequency, the amplitude of the input signal, the electrolyte conductivity and electrode distance were found to be the most significant factors affecting the detection sensitivity. 2-(N-Morpholino)ethanesulfonic acid/histidine-based (MES/His) electrolytes were used for direct conductivity detection of anions and cations, while ammonium acetate was selected for indirect conductivity determination of alkylammonium salts. For the simultaneous separation procedure, involving dual-opposite end injection, an electrolyte consisting of 20 mM MES/His, 1.5 mM 18-crown-6 and 20 microM cetyltrimethylammonium bromide provided baseline separation of 13 anions and cations in less than 6 min. The detection limits achieved were 7-30 micrograms/l for direct conductometric detection of various common inorganic cations and anions, excluding F- (62 micrograms/l) and H2PO4- (250 micrograms/l), and 35-178 micrograms/l for indirect conductometric detection of alkyl ammonium cations. The developed electrophoretic method with conductometric detection was compared to ion chromatography.     

Rapid speciation of Se(IV) and Se(VI) by flow injection–capillary electrophoresis system with contactless conductivity detection

A flow injection–capillary electrophoresis system with contactless conductivity detection and hydrostatic-pressure-generated flow was used for the fast and sensitive speciation of Se(IV) and Se(VI). The sample throughput was 25 samples per hour using a background electrolyte solution containing 8.75 mM l-histidine (His) adjusted to pH 4.00 with acetic acid. The repeatability of peak areas (n=8) was better than 1.41% and the limits of detection were 190 g L^–1 and 7.5 g L^–1 for Se(IV) and Se(VI), respectively. The interference from carbonate, typically present in water samples, was eliminated by using a low-pH electrolyte in which carbonate is uncharged and migrates at the EOF front. The method was applied to the analysis of Se(IV) and Se(VI) in soil samples that were spiked with both selenium species and the results for recovery of both selenium species were in good agreement with their introduced concentrations.     

Solid-phase extraction-capillary electrophoresis-mass spectrometry for the determination of tetracyclines residues in surface water by using carbon nanotubes as sorbent material

Last years chemical properties of carbon nanotubes (CNTs) have attracted high interest. One of the most important issues is the capability of CNTs to adsorb analytes on its surface. In this work, such property has been used to preconcentrate trace tetracyclines from environmental water samples at the trace level. Multi-walled carbon nanotubes (MWNTs) have showed higher capacity than other two single-walled carbon nanotubes (SWNTs). Preconcentration of the samples was performed in a flow system at-line coupled to the CE-MS equipment. The preconcentration of tetracyclines on MWNTs followed by capillary electrophoresis-mass spectrometry allows the detection of 0.30-0.69 microg/L of tetracyclines for the analysis of 10 mL of samples. Recoveries for the analysis of spiked samples ranged from 98.6 to 103.2% and the precision from 5.4 to 8.2%. Separation of tetracylines in the electrophoretic system was achieved using 50 mM formic acid at pH 2.0 as a background electrolyte. Atmospheric pressure electrospray ionization mass spectrometry detection was accomplished using 50:50 (v/v) methanol/water containing 0.5% (v/v) formic acid as a sheath liquid.     

Sensitive indirect photometric detection of inorganic and small organic anions by capillary electrophoresis using Orange G as a probe ion

This study addresses the two major problems in the use of dyes as highly absorbing probes for indirect photometric detection in capillary electrophoresis (CE). First, effective electroosmotic flow (EOF) modification or suppression to allow separation and detection of a wide mobility range of analytes is not straightforward when electrolytes containing increased dye concentrations are used. The suppression of EOF to less than + 5x10(-9) m(2)V(-1)s(-1) was achieved with a combination of a poly(ethylenimine) (PEI)-coated capillary and the addition of the neutral polymer hydroxypropylmethylcellulose (HPMC) to the background electrolyte. Second, the deterioration of baselines due to adsorption of the dye probe to the capillary wall is generally a problem. In this work, baseline quality at higher probe concentrations was significantly improved by a rather unusual but highly effective combination of a simultaneous application of a slight overpressure (25 mbar) at the injection end during the separation, and the use of a relatively narrow capillary of 50 microm inner diameter. Both measures would appear to be counterproductive. Optimisation of the probe concentration with regard to signal-to-noise ratio resulted in an electrolyte of 4 mM Orange G, 0.05% HPMC buffered at pH 7.7 by the addition of 10.0 mM histidine isoelectric buffer. Very high separation efficiencies of 128 000-297 000 plates were made possible by the relatively high probe concentration. Combined with excellent detection sensitivity, even with the introduction of hydrodynamic flow and a reduced optical path length, these measures resulted in limits of detection ranging from 0.216 to 0.912 microM with a deuterium lamp light source (248 nm) and from 0.147 to 0.834 microM with a 476 nm blue light-emitting diode (LED) light source. Reproducibility over 30 consecutive runs without changing the electrolyte was excellent, with relative standard deviation (RSD) values of 0.14-0.80% for migration time, 1.27-3.36% for peak area and 0.88-5.12% for peak heights. The optimised electrolyte was used for the analysis of inorganic anions in air filter samples, providing good agreement with results obtained by ion chromatography.     

Chemometrical examination of active parameters and interactions in flow injection-capillary electrophoresis

The first detailed examination of flow injection-capillary electrophoresis (FI-CE) active parameters and their interactions via response surface methodology (RSM) is presented. Specifically, RSM in the form of a Box-Behnken design was implemented to effectively predict the significance of capillary length, voltage and injection volume on the optimization of an in-house built FI-CE analyzer. Initial studies were performed assessing peak height and peak shape of the model compound N,N-dimethylformamide. Optimum model conditions were then derived and used in the model separation of two small molecules, nicotinamide adenine dinucleotide, reduced form (NADH) and benzenesulfonamide. By implementing the RSM approach, detailed examination of active FI-CE parameters was possible, including the ability to reveal a significant interactive effect. This work is not only highly significant for advancing FI-CE developments, but instructive for investigators actively exploring other coupled analytical techniques and associated experimental parameters.