Comparison of field‐enhanced and pressure‐assisted field‐enhanced sample injection techniques for the analysis of water‐soluble vitamins using CZE

A new online concentration method, namely pressure‐assisted field‐enhanced sample injection (PA‐FESI), was developed and compared with FESI for the analysis of water‐soluble vitamins by CZE with UV detection. In PA‐FESI, negative voltage and positive pressure were simultaneously applied to initialize PA‐FESI. PA‐FESI uses the hydrodynamic flow generated by the positive pressure to counterbalance the reverse EOF in the capillary column during electrokinetic sample injection, which allowed a longer injection time than usual FESI mode without compromising the separation efficiency. Using the PA‐FESI method, the LODs of the vitamins were at ng/mL level based on the S/N of 3 and the RSDs of migration time and peak area for each vitamin (1 μg/mL) were less than 5.1%. The developed method was applied to the analysis of water‐soluble vitamins in corns.     

A rapid and sensitive CE method with field-enhanced sample injection and in-capillary derivatization for selenomethionine metabolism catalyzed by flavin-containing monooxygenases

A rapid and sensitive electrophoretically mediated microanalysis method with field-enhanced sample injection (FESI) for in-capillary derivatization was developed to determine selenomethionine (SeMet) and selenomethionine selenoxide (SeOMet). Phthalic anhydride (PA) was selected as the derivatization reagent due to the fast reaction at room temperature and the stability of derivatives. The in-capillary derivatization was accomplished by electrophoretically mixing PA and sample plugs. PA reagent was introduced hydrodynamically into the capillary, whereas the sample solution was injected electrokinetically, thus allowing a selective preconcentration of the analytes by FESI. For FESI, the optimum sample solvent was 2 mM borate solution. The borate buffer was suitable for both in-capillary derivatization and separation of the derivatives. The combination of electrophoretically mediated microanalysis with FESI for in-capillary derivatization was successfully achieved with about 800-fold concentration sensitivity enhancement compared to direct CE-UV detection in the same setup. The present method is miniaturized and fully automated, which ensures the on-line derivatization, stacking, separation and detection in 10 min. Finally, the developed method was successfully applied to measure enzyme activities by analyzing the reaction mixtures of SeMet with human flavin-containing monooxygenases (FMO). The results showed that both FMO1 and FMO3, but not FMO5 could catalyze the Se-oxygenation of SeMet.     

Electrophoretic determination of albumin in urine using on-line concentration techniques

To improve the sensitivity of the UV-detection for the determination of trace amounts of albumin by capillary zone electrophoresis (CZE), five on-line preconcentration techniques, including field-amplified sample stacking (FASS), head-column field-amplified sample stacking (HC-FASS), stacking with a polymer solution, dynamic pH junction and large volume sample stacking (LVSS) with reversed polarity, were compared. Sensitivity enhancement factor and reproducibility were two factors that were used to assess the suitability of each method. To minimize protein adsorption on the capillary wall, capillaries were covalently modified with anionic polymer, poly(sulfopropylmethacrylate) coating. All used methods have good reproducibility. The maximum sensitivity enhancement factor (about 67-fold in terms of peak heights) was achieved with LVSS technique. The concentration limit of detection (LOD) (S/N=3) for the human serum albumin obtained with the optimized LVSS approach was 15 microg/ml with UV-detection. The method was further evaluated for the analysis of urine samples with gel-filtration-based sample-desalting procedure.     

Comparison of two capillary electrophoresis online stacking modes by analysis of polycyclic aromatic hydrocarbons in airborne particulates

Naphthalene, fluorene, pyrene, anthracene, phenanthrene, and chrysene were successfully separated by CD-modified MEKC (CD-MEKC) using 20 mM borate (pH 9.0) containing 90 mM SDS and 75 mM beta-CD. Two online stacking methods, i.e., sweeping and field-enhanced sample injection (FESI), were explored to enhance the detection sensitivity. The influences of some crucial parameters in sweeping and FESI procedures were investigated. For FESI method, a plug of water and low-conductivity sample matrix was used to increase the stacking efficiency. Compared with the sweeping method, FESI can increase the sensitivity in the range of 10-20-fold. The proposed method was used for the analysis of polycyclic aromatic hydrocarbons in airborne particulates.     

Rapid quantification of quinine by multi‐stacking in a portable microchip electrophoresis system

A new multi‐stacking pre‐concentration procedure based on field‐enhanced sample injection (FESI), field‐amplified sample stacking, and transient isotachophoresis was developed and implemented in a compact microchip electrophoresis (MCE) with a double T‐junction glass chip, coupled with an on‐chip capacitively coupled contactless conductivity detection (C⁴D) system. A mixture of the cationic target analyte and the terminating electrolyte (TE) from the two sample reservoirs was injected under FESI conditions within the two sample‐loading channels. At the double T‐junction, the stacked analyte zones were further concentrated under field‐amplified stacking conditions and then subsequently focused by transient‐isotachophoresis and separated along the separation channels. The proposed multi‐stacking strategy was verified under a Universal Serial Bus (USB) fluorescence microscope employing Rhodamine 6G as the model analyte. This developed approach was subsequently used to monitor the target quinine present in human plasma samples. The total analysis time for quinine was approximately 200 s with a sensitivity enhancement factor of approximately 61 when compared to the typical gated injection. The detection and quantification limits of the developed approach for quinine were 3.0 μg/mL and 10 μg/mL, respectively, with intraday and interday repeatability (%RSDs, n = 5) of 3.6 and 4.4%. Recoveries in spiked human plasma were 98.1–99.8%.     

On-line sample stacking of peptides in capillary electrophoresis for the study of prebiotic reactions between α,α-dialkylated amino acids and amino acid N-carboxyanhydrides

The reaction between α,α-dialkylated amino acids and amino acid N-carboxyanhydrides is slow leading to low concentrations of products (peptides). The detection by capillary electrophoresis of the analytes contained in such samples is therefore a challenging issue. In this work, on-line sample pre-concentration methods based on field-amplified sample stacking have been implemented and compared. Because of the high ionic strength present in the sample matrix, samples were diluted with an organic solvent prior to analysis to decrease the sample conductivity. Different modes of sample injection (field amplified sample injection (FASI), hydrodynamic normal sample stacking (NSS) or large volume sample stacking (LVSS)) were compared. Pre-concentration factors of 20 for FASI, about 30–40 for NSS and 60 for LVSS were obtained for the analysis of (l,l) dipeptide of valine in a large excess of isovaline and 0.2 M of ionic strength. For LVSS application and resolution optimisation, a new non-covalent coating based on the partial modification of the capillary surface was used to tune the electroosmotic flow magnitude and to pump the sample matrix out of the capillary. This on-line sample pre-concentration step allowed confirming that oligopeptides including α,α-dialkylated amino acids are formed during the reaction between α,α-dialkylated amino acids and N-carboxyanhydride amino acids.     

Influence of high‐conductivity buffer composition on field‐enhanced sample injection coupled to sweeping in CE

The aim of this work was to clarify the mechanism taking place in field‐enhanced sample injection coupled to sweeping and micellar EKC (FESI‐Sweep‐MEKC), with the utilization of two acidic high‐conductivity buffers (HCBs), phosphoric acid or sodium phosphate buffer, in view of maximizing sensitivity enhancements. Using cationic model compounds in acidic media, a chemometric approach and simulations with SIMUL5 were implemented. Experimental design first enabled to identify the significant factors and their potential interactions. Simulation demonstrates the formation of moving boundaries during sample injection, which originate at the initial sample/HCB and HCB/buffer discontinuities and gradually change the compositions of HCB and BGE. With sodium phosphate buffer, the HCB conductivity increased during the injection, leading to a more efficient preconcentration by staking (about 1.6 times) than with phosphoric acid alone, for which conductivity decreased during injection. For the same injection time at constant voltage, however, a lower amount of analytes was injected with sodium phosphate buffer than with phosphoric acid. Consequently sensitivity enhancements were lower for the whole FESI‐Sweep‐MEKC process. This is why, in order to maximize sensitivity enhancements, it is proposed to work with sodium phosphate buffer as HCB and to use constant current during sample injection.     

The microemulsion electrokinetic capillary chromatography combined with reversed‐electrode polarity stacking mode for enriching and quantifying lignanoids and ginsenosides in TCMs preparation Shengmai injection

An on‐line large volume sample stacking with polarity switching (LVSS) method was proposed for simultaneously determining lignanoids and ginsenosides in MEEKC. The parameters including the pH value and concentration of buffer solution, SDS, organic modifier, oil phase, running voltage, and temperature as well as injection time, sample matrix, stacking voltage, and time influencing separation and stacking were systematically optimized. The method was verified by performing precision, accuracy, stability, and recovery. Its reliability was proved by separating and quantifying two lignanoids and three ginsenosides in Shengmai injectionSMI. The sensitivity of these compounds was improved by MEEKC‐LVSS method for 6–11 times than conventional MEEKC. Thus, this developed on‐line MEEKC‐LVSS method was sensitive, practical, and reliable.     

Analysis of acrylamide in food products by in-line preconcentration capillary zone electrophoresis

Two in-line preconcentration capillary zone electrophoresis (CZE) methods (field amplified sample injection (FASI) and stacking with sample matrix removal (LVSS)) have been evaluated for the analysis of acrylamide (AA) in foodstuffs. To allow the determination of AA by CZE, it was derivatized using 2-mercaptobenzoic acid. For FASI, the optimum conditions were water at pH > or = 10 adjusted with NH3 as sample solvent, 35 s hydrodynamic injection (0.5 psi) of a water plug, 35 s of electrokinetic injection (-10 kV) of the sample, and 6s hydrodynamic injection (0.5 psi) of another water plug to prevent AA removal by EOF. In stacking with sample matrix removal, the reversal time was found to be around 3.3 min. A 40 mM phosphate buffer (pH 8.5) was used as carrier electrolyte for CZE separation in both cases. For both FASI and LVSS methods, linear calibration curves over the range studied (10-1000 microg L(-1) and 25-1000 microg L(-1), respectively), limit of detection (LOD) on standards (1 microg L(-1) for FASI and 7 microg L(-1) for LVSS), limit of detection on samples (3 ng g(-1) for FASI and 20 ng g(-1) for LVSS) and both run-to-run (up to 14% for concentration and 0.8% for time values) and day-to-day precisions (up to 16% and 5% for concentration and time values, respectively) were established. Due to the lower detection limits obtained with the FASI-CZE this method was applied to the analysis of AA in different foodstuffs such as biscuits, cereals, crisp bread, snacks and coffee, and the results were compared with those obtained by LC-MS/MS.     

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.     

Simultaneous enrichment and separation of neutral and anionic analytes through combining large volume sample stacking with sweeping in CE

In this work, we overcame the deficiencies of large volume sample stacking (LVSS) in separating low‐mobility and neutral analytes through combining LVSS with sweeping in CE, and employed this new approach to enrich and separate neutral and anionic analytes simultaneously. This technique was carried out with pressure injection of large‐volume sample followed by EOF as a pump pushing the bulk of low‐conductivity sample matrix out of the outlet of the capillary while analytes were swept by micelles and separated via MEKC without the electrode polarity switching. Careful optimization of the enrichment and separation conditions allowed the enrichment factors (EFs) of peak height and peak area of the analytes to be in the range of 9–33 and 21–35 comparing with the conventional injection mode, respectively. The five analytes were baseline separated in 15 min and the detection limits ranged from 26.5 to 55.8 ng/mL (S/N = 3). The developed method was successfully applied to determine adenine, caffeine, theophylline, reduced L‐glutathione (GSH) and oxidized L‐glutathione (GSSG) in two different teas with recoveries that ranged from 84.4 to 105.2%.     

Quantitation and on-line concentration of enantiomers in open-tubular capillary electrochromatography.

The feasibility of open-tubular capillary electrochromatography (OTCEC) with UV detector for quantitation of enantiomers is explored, and a simple on-line sample concentration method to improve detection sensitivity of negatively charged enantiomers more than 1000-fold is described. With a capillary of 25 microm ID, the limits of detection (LODs) for absolute concentration and for enantiomeric ratio are 10(-6) M and 0.6-0.8% (signal-to-noise ratio S/N = 10). Good linearity and reproducibility are observed. The detection sensitivity is enhanced by combination with field-enhanced sample injection (FESI). A water plug is introduced hydrodynamically into the capillary inlet end and then the sample solution prepared with water is introduced with electrokinetic injection. With this concentration technique, the LOD for absolute concentration is reduced to a 10(-9) M level. On the other hand, due to the peak-sharpening effect of FESI, the LOD for enantiomeric ratio for the first-eluted enantiomer is significantly improved, being 0.3%. Effects of the injection conditions, such as length of water plug, buffer concentration, injection voltage, and injection time on the enrichment efficiency are investigated. Online concentration of a racemic compound with two chiral centers is demonstrated.     

Effect of NaOH on large-volume sample stacking of haloacetic acids in capillary zone electrophoresis with a low-pH buffer

Large-volume sample stacking (LVSS) is an effective on-capillary sample concentration method in capillary zone electrophoresis, which can be applied to the sample in a low-conductivity matrix. NaOH solution is commonly used to back-extract acidic compounds from organic solvent in sample pretreatment. The effect of NaOH as sample matrix on LVSS of haloacetic acids was investigated in this study. It was found that the presence of NaOH in sample did not compromise, but rather help the sample stacking performance if a low pH background electrolyte (BGE) was used. The sensitivity enhancement factor was higher than the case when sample was dissolved in pure water or diluted BGE. Compared with conventional injection (0.4% capillary volume), 97-120-fold sensitivity enhancement in terms of peak height was obtained without deterioration of separation with an injection amount equal to 20% of the capillary volume. This method was applied to determine haloacetic acids in tap water by combination with liquid-liquid extraction and back-extraction into NaOH solution. Limits of detection at sub-ppb levels were obtained for real samples with direct UV detection.     

Solid-phase extraction and large-volume sample stacking with an electroosmotic flow pump in capillary electrophoresis for determination of methotrexate and its metabolites in human plasma

This paper describes approaches for large-volume sample stacking (LVSS) with an EOF pumpin CE for the determination of methotrexate (MTX) and its metabolites in human plasma. After pretreatment of plasma through a SPE cartridge, a large sample volume was loaded by hydrodynamic injection (3 psi, 70 s) into the capillary filled with phosphate buffer (70 mM, pH 6.0) containing 0.01% polyethylene oxide. Following removal of a large plug of sample matrix from the capillary using polarity switching (-25 kV), the separation of anionic analytes was subsequently performed without changing polarity again, achieving an improvement of sensitivity of around a 100-fold. The method was applied to therapeutic drug monitoring of MTX in one acute lymphoblastic leukemia patient. This study is one of very few applications showing the feasibility of LVSS in analysis of biological samples by CE.     

Improving the sensitivity of the determination of ceftiofur by capillary electrophoresis in environmental water samples: in-line solid phase extraction and sample stacking techniques

This paper describes two different approaches for increasing the sensitivity for the analysis of ceftiofur by capillary electrophoresis (CE). Two different techniques based on the introduction of an enlarged volume of sample, namely large volume sample stacking (LVSS) and in-line solid phase extraction (SPE) were studied and compared. LVSS allowed the on-column electrophoretic preconcentration of ceftiofur without modification of the separation capillary. In-line SPE-CE was developed by using a home-made microcartridge that was filled with a reversed-phase sorbent (C₁₈). The microcartridge was coupled in-line near the inlet of the separation capillary. LVSS and in-line SPE-CE allowed automated operation and improved sensitivity for the analysis of ceftiofur with respect to conventional CE. When environmental water samples were analyzed, an additional pretreatment step based on off-line SPE was necessary in both cases to further decrease the detection limits. In terms of sensitivity for the determination of ceftiofur in river water samples, the combination of off-line SPE with in-line SPE-CE was found the most sensitive with a detection limit of 10 ng L⁻¹, whereas the method based on the use of off-line SPE with LVSS presented a detection limit of 100 ng L⁻¹.     

高效毛细管电泳大体积样品堆积在线富集法测定蒲公英中阿魏酸、绿原酸和咖啡酸

采用大体积样品堆积(LVSS)在线富集模式,建立了高效毛细管电泳(HPCE)测定蒲公英中阿魏酸、绿原酸和咖啡酸含量的方法。主要考察了在毛细管区带电泳(CZE)分离模式下,缓冲液的pH和浓度对分离效果的影响,以及在LVSS在线富集模式下,进样时间对富集效果的影响。在最优条件下阿魏酸、绿原酸和咖啡酸可在12 min内得到分离,3个成分在0.5~25.0μg/mL浓度范围内均有较好的线性关系(r2=0.999),平均加样回收率分别为104.9%,98.0%和100.1%,RSD(n=6)分别为3.6%,2.6%和1.0%。定量限(S/N=10)分别为0.10,0.10和0.03μg/mL,检出限(S/N=3)分别为0.03,0.03和0.01μg/mL。相对于常规CZE模式,本方法的富集效果倍数为17~19倍。建立的方法可用于蒲公英的日常检测与质量控制。     

pH-independent large-volume sample stacking of positive or negative analytes in capillary electrophoresis

In capillary electrophoresis, the short optical path length associated with on-column UV detection imposes an inherent detection problem. Detection limits can be improved using sample stacking. Recently, large-volume sample stacking (LVSS) without polarity switching was demonstrated to improve detection limits of charged analytes by more than 100-fold. However, this technique requires suppression of the electroosmotic flow (EOF) during the run. This necessitates working at a low pH, which limits using pH to optimize selectivity. We demonstrate that LVSS can be performed at any buffer pH (4.0-10.0) if the zwitterionic surfactant Rewoteric AM CAS U is used to suppress the EOF. Sensitivity enhancements of up to 85-fold are achieved with migration time, corrected area, and peak height reproducibility of 0.8-1.6%, 1.3-3.7%, and 0.8-4.9%, respectively. Further, it is possible to stack either positively or negatively charged analytes using zwitterionic surfactants to suppress the EOF.     

Different strategies for the preconcentration and separation of parabens by capillary electrophoresis

Several strategies, namely, large volume sample stacking (LVSS), field‐amplified sample injection (FASI), sweeping, and in‐line SPE‐CE, were investigated for the simultaneous separation and preconcentration of a group of parabens. A BGE consisting of 20 mM sodium dihydrogenphosphate (pH 2.28) and 150 mM SDS with 15% ACN was used for the separation and preconcentration of the compounds by sweeping, and a BGE consisting of 30 mM sodium borate (pH 9.5) was used for the separation and preconcentration of the compounds by LVSS, FASI, and in‐line SPE‐CE. Several factors affecting the preconcentration process were investigated in order to obtain the maximum enhancement of sensitivity. The LODs obtained for parabens were in the range of 18–27, 3–4, 2, and 0.01–0.02 ng/mL, and the sensitivity evaluated in terms of LODs was improved up to 29‐, 77‐, 120‐, and 18 400‐fold for sweeping, LVSS, FASI, and in‐line SPE‐CE, respectively. These preconcentration techniques showed potential as good strategies for focusing parabens. The four methods were validated with standard samples to show the potential of these techniques for future applications in real samples, such as biological and environmental samples.     

Large Volume Sample Stacking (LVSS) in Capillary Electrophoresis (CE) with Response Surface Methodology (RSM) for the Determination of Phenolics in Food Samples

Abstract

A capillary electrophoresis method with large volume sample stacking (CE-LVSS) has been developed and validated for the simultaneous determination of seven phenolic compounds: naringin, rutin, carnosic acid, apigenin, quercetin, morin, and chichoric acid. Optimization was carried out by response surface methodology and a set of 20 experiments helped to optimize the parameters such as the concentration of buffer, buffer pH, and applied voltage. Analytes were separated using a 50?µm diameter capillary with 56?cm effective length and an extended light path using 20?mM borate buffer at pH 9.2. The LVSS method was optimized and a three- to fivefold improvement in detectability was achieved with injection at 100 mbar for 20?s followed by polarity switching at –20?kV for 6?s. The linearity values of all seven analytes were observed in the concentration ranges from 0.5 to 50?µg/mL for CE and 0.1 to 25?µg/mL for LVSS. The limits of detection were from 0.012 to 0.241 and 0.003 to 0.086?µg/mL for CE and LVSS. The obtained limits of quantitation were within 0.041 to 0.802 for CE and 0.012 to 0.286?µg/mL for LVSS. The recoveries were between 91.1 and 109.8% and 96.3 and 108.4% for CE and LVSS, respectively. The developed method has been successfully applied for the quantitative determination of analyzed components from food samples that are important sources of these compounds.     

Online concentration of aristolochic acid I and II in Chinese medicine preparations by micellar electrokinetic chromatography

In this study, an online concentration method in micellar electrokinetic chromatography (MEKC) applying field-enhanced sample injection (FESI) mode was developed for the detection of aristolochic acids (AAs) in Chinese medicine preparations. AA-I and AA-II were baseline separated with high separation efficiency, and 100-fold enhancement of the detection sensitivity was achieved compared with those obtained from normal capillary zone electrophoresis (CZE) or simple MEKC method. The proposed method was successfully applied for the determination of AAs in Chinese medicine preparations.