Complementary on-line preconcentration strategies for steroids by capillary electrophoresis

Complementary on-line preconcentration strategies are needed when analyzing different classes of solutes in real samples by capillary electrophoresis (CE) with UV detection. The performance of three different on-line preconcentration (focusing) techniques under alkaline conditions was examined in terms of their selectivity and sensitivity enhancement for a group of steroids, including classes of androgens, corticosteroids and estrogens. Electrokinetic focusing of large sample injection plugs (up to 28% of effective capillary length or 22.1 cm) directly on-capillary can be tuned for specific classes of steroids based on changes in their mobility (velocity) using a multi-section electrolyte system in CE. A dynamic pH junction was applied for the selective resolution and focusing of weakly acidic estrogens using borate, pH 11.0 and pH 8.0 in the background electrolyte and the sample, respectively. Sweeping, using an anionic bile acid surfactant and neutral gamma-cyclodextrin (gamma-CD) under alkaline conditions (pH 8), resulted in focusing and separation of the moderately hydrophobic (non-ionic) classes of steroids, such as androgen and corticosteroids. Optimal focusing and resolution of all test steroids under a single buffer condition was realized by a dynamic pH junction-sweeping format using borate, pH 11.0 and bile acid surfactant with gamma-CD in the BGE, whereas the sample is devoid of surfactant at pH 8.0. The design of selective on-line focusing strategies in CE is highlighted by the analysis of microgram amounts of ethynyl estradiol derived from a female contraceptive pill extract using the dynamic pH junction method, which resulted in over a 100-fold enhancement in concentration sensitivity.     

Dynamic pH junction-sweeping for on-line focusing of dipeptides in capillary electrophoresis with laser-induced fluorescence detection

In this paper, we developed a simple and effective on-line focusing technique combining dynamic pH junction and sweeping by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. Dynamic pH junction-sweeping is defined when the sample has a different buffer pH (dynamic pH junction condition) and is devoid of micelles (sweeping condition) relative to the background electrolyte (BGE). This hyphenated focusing mode was applied to the sensitive and selective focusing of four dipeptides: Tyr-Phe, Tyr-Leu, Trp-Gly, and Ala-Gln. Picomolar detectability of these dipeptides by CE-LIF detection was demonstrated through effective focusing of large sample volumes (up to 39% capillary length) using the dual pH junction-sweeping focusing mode. 25 mmol L(-1) sodium dihydrogen phosphate, pH 2.5 was used as the sample matrix, and 100 mmol L(-1) borate, 21 mmol L(-1) sodium dodecylsulfate (SDS), 16 mmol L(-1) Brij35, pH 9.0 as the background solution (BGS). The concentration detection limits (S/N = 3) of the four dipeptides were in the range of 1.0-5.0 pmol L(-1). The developed method has been successfully used for the determination of dipeptides in human serum samples.     

On-line sample preconcentration of cationic analytes by dynamic pH junction in capillary electrophoresis

To improve detection sensitivity of cationic analytes, a dynamic pH junction technique was examined. Dynamic pH junction is an on-line focusing method in capillary electrophoresis (CE) based on the difference in the analyte's mobility between the background electrolyte (BGE) and sample matrix. The effects of pH values and concentrations of the BGE and the sample matrix on dynamic pH junction were examined. Optimization of analyte focusing resulted in enhanced detection responses of about 100-160-fold in terms of peak heights for some anilines in comparison to conventional injections. In particular, the concentration limits of detection (LOD) (S/N = 3) for the test anilines obtained with dynamic pH junction were from 1.9 to 3.7 ppb with UV detection without any pretreatment procedure.     

Quantitative analysis of microcystin variants by capillary electrophoresis mass spectrometry with dynamic pH barrage junction focusing

Dynamic pH junction is an online focusing method in CE based on the electrophoretic mobility difference of analytes in the sample matrix and the background electrolyte. An advantage of this method over the conventional CE is that the sensitivity can be significantly improved. By injecting a long sample plug in the capillary and focusing the analytes at the pH boundary between the background electrolyte and sample matrix, the LOD can be improved by 10–100 folds. The dynamic pH junction method can be easily coupled with ESI‐MS. In this work, we used this method for the analysis of microcystins (MCs). The detection limits and dynamic ranges were studied. The separation was optimized by adjusting the injection time, and concentrations and pH values of the background electrolyte. The optimization of analyte focusing leads to enhanced detection response compared to conventional injections, achieving 200–400 fold higher averaged peak heights for four microcystin (MC) variants. More importantly, this method was successfully used for the quantitative analysis of microcystins (MCs) in crude algae samples from natural water bodies, making it promising for practical applications.     

Sensitive Method for Enantioseparation of Rivastigmine with Highly Sulfated Cyclodextrin as Chiral Selector by Capillary Electrophoresis

A sensitive method for enantioseparation of a basic drug rivastigmine and determination of its optical impurityby capillary electrophoresis with highly sulfated β-cyclodextrin(HS-β-CD)as the chiral selector is described.Ingeneral,enantioseparation of basic chiral compounds is carried out in acidic condition(pH 2.5)to prevent analytesfrom adsorption on the capillary wall.However,in the case of rivastigmine,the detection sensitivity was too limitedto determine the optical impurity of S-rivastigmine lower than 1% when buffer pH was 2.5.It was found that thedetection sensitivity was improved 1.6 times just by raising the buffer pH value from 2.5 to 5.8.The poor columnefficiency due to the adsorption of the analytes on the capillary wall was resolved by using dynamical coating of thecapillary wall with the linear polyacrylamide solution.The experirnental parameters such as the concentration ofHS-β-CD,buffer pH and buffer ionic strength were optimized,respectively.The method was validated in terms ofrepeatability,linearity,limit of detection(LOD)and limit of quantitation(LOQ).Using the present method,the op-tical purity of nonracemic rivastigmine with the enantiomeric excess(ee)value of 99.14% was determined.     

Online sample pre-concentration via dynamic pH junction in capillary and microchip electrophoresis

Various analytical techniques have been developed over the years to analyse a large diversity of biomolecules with a constant push towards ultra-sensitive detection. CE is at the forefront of the most powerful analytical tools available to date when considering its superior efficiency and resolution; however, the technique suffers from poor sensitivity as a result of the short path length at the detection site and small injection volumes (typically <1% capillary length). One of the approaches to abate the inherent problem is to employ clever chemistry using sample focusing techniques whereby a large sample plug can be injected, preconcentrated and separated, producing excellent sensitivity and efficiency at the detector. This particular review will focus on the use of dynamic pH junction as a means of improving sensitivity in CE and focuses on the use of a change in analyte ionisation due to different pHs between the sample and electrolyte. The review provides a fundamental discussion of the mechanisms, buffer and sample conditions required to concentrate various analytes and a comprehensive list of published works in tabular format for easy identification of suitable conditions for new applications. The review further encompasses the use of dynamic pH junction in CE and its involvement in combination with other preconcentrations techniques to produce high sensitivity enhancements recorded between the years 1990-2010.     

Extraction Properties of Modified Silica Gel for Metal Analysis by Energy Dispersive X-Ray Fluorescence

Abstract

The separation of water‐soluble vitamins by capillary zone electrophoresis was developed, in which on‐line concentration methods, namely field‐enhanced sample stacking and dynamic pH junction, were utilized to improve the detection sensitivity. The effects of some critical parameters, including pH and concentration of background electrolyte, sample matrix pH and concentration, and injection volume were examined. The effects of field‐enhanced sample stacking and dynamic pH junction on the separation resolution and concentration efficiency were compared. The limits of detection of the vitamins were from 6 to 119 ng ml^?1 (2.7×10^?8 to 53.4×10^?8 M) based on the signal‐to‐noise ratio of 3 and the relative standard deviations of migration time and peak area for each vitamin (1 µg ml^?1) were less than 3.5% using the field‐enhanced sample stacking as an on‐line concentration method. The developed method was applied to the analysis of water‐soluble vitamins in corns.     

Quantification of 5‐aminolevulinic acid by CE using dynamic pH junction technique

An online dynamic pH junction preconcentration method was developed for quantification of 5‐aminolevulinic acid (ALA) by CE with the separation time less than 6 min. The optimal dynamic pH junction of ALA was carried out between pH 9.3 borate buffer (BGE, 40 mM) and pH 2.5 phosphate buffer (sample matrix, 40 mM) when 4.1 cm of sample plug was hydrodynamically injected into an uncoated fused‐silica capillary (48.5 cm in length, id of 50 μm). If a 24 kV separation voltage was applied, the calibration curve of ALA peak area (200 nm) showed good linearity (R² = 0.9991) ranging from 0.01 to 6.5 mg/mL. The reproducibility of this system was excellent with RSDs (n = 10) of 2.5% for peak area response and 0.6% for migration time at ALA concentration of 0.5 mg/mL. The LOD was evaluated as 1.0 μg/mL (S/N > 3). Compared to conventional CE procedure, the sensitivity was successfully improved over 50‐fold. The analytical results of pharmaceutical formulations show a good agreement with those by HPLC (r = 0.94).     

Determination of urinary nucleosides via borate complexation capillary electrophoresis combined with dynamic pH junction-sweeping-large volume sample stacking as three sequential steps for their on-line enrichment

The combination of dynamic pH junction, sweeping (using borate complexation), and large volume sample stacking (LVSS) is investigated as three consecutive steps for on-line focusing in the sensitive quantitation of urinary nucleosides by CE-UVD. A low conductivity aqueous sample matrix free from borate and a high conductivity BGE (containing borate, pH 9.25) are needed to fulfill the required conditions for dynamic pH junction, LVSS, and sweeping. Parameters affecting the separation and the enrichment efficiency are studied such as buffer concentration, separation voltage, capillary temperature, sample composition, and sample injection volume. Prerequisite for the developed strategy is the extraction of the nucleosides from urine using a phenylboronate affinity gel, which is described to be a unique means for the selective enrichment of cis-diol metabolites under alkaline conditions. The impact of ionic constituents remaining in the eluate after extraction on focusing efficiency and resolution is investigated. The developed method is applied to the analysis of blank and spiked urine samples. Fundamental aspects underlying the proposed enrichment procedure are discussed. A detection limit as low as 10 ng mL^?1 is achieved. To the best of our knowledge, this LOD represents the lowest LOD reported so far for the analysis of nucleosides using CE with UV detection and provides a comparable sensitivity to CE/MS. Because of the high sensitivity, the proposed method shows a great potential for the analysis of nucleosides in human urine and other types of biological fluids. Schematic presentation of the developed three-step focusing mechanism (combining dynamic pHjunction, sweeping using borate complexation, and LVSS).      

Sensitive and high-throughput analyses of purine metabolites by dynamic pH junction multiplexed capillary electrophoresis: a new tool for metabolomic studies.

On-line sample preconcentration by a dynamic pH junction in conjunction with multiplexed capillary electrophoresis (CE) and UV detection represents a sensitive and high-throughput format for future metabolomic research, such as purine analysis. The optimization of purine focusing can be rapidly assessed by systematically altering the sample matrix properties, such as the buffer co-ion, pH and ionic strength using a 96-capillary array format. This method permits focusing of large sample injection volumes, resulting in over a 50-fold improvement in the concentration sensitivity. The limit of detection (S/N = 3) for purine metabolites was less than 8.0 x 10(-8) M under optimum conditions when using UV absorbance. Dynamic pH junction multiplexed CE demonstrated excellent linearity over a hundred-fold concentration range, as well as low inter-capillary precision in terms of normalized migration times and peak areas. The potential for clinically relevant high-throughput analyses of micromolar amounts of purine metabolites in urine was also demonstrated.     

Simultaneous determination of dihydroxybenzene and phenylenediamine positional isomers using capillary zone electrophoresis coupled with amperometric detection

In general capillary zone electrophoresis (CZE) separation models, o‐, m‐, and p‐phenylenediamine isomers can be separated in a weak acidic running buffer for their pK_a values being 4.52, 5.64, 6.04, respectively, while o‐, m‐, and p‐dihydroxybenzene isomers can be separated in a weak basic buffer for their pK_a values being 9.40, 9.40 and 10.04, respectively. So, it is hard to find a suitable running buffer at a fixed pH in normal CZE for simultaneous separation of these two groups of positional isomers. In this paper, a novel method based on alternately running two different pH buffers in CZE coupled with amperometric detection (CZE‐AD) was designed to simultaneously determine these two groups of positional isomers. It is found that when two different pH running buffers were employed alternately under appropriate order and time, the six analytes could be separated perfectly in less than 20 min and the detection limits were as low as 10^–7 mol/L. Furthermore, the effects of working electrode potential, pH and concentration of running buffer, separation voltage and injection time on CZE–AD were investigated. Experimental results demonstrated that the introduced method was practical to simultaneously determine two groups of positional isomers with different pK_a and had some advantages of high sensitivity, good repeatability and small sample requirement.     

Separation and Determination of Stereoisomeric Impurity of Folinic Acid Diastereomers by CE with Vancomycin as a Selector

A highly sensitive and rapid method was developed that involves capillary electrophoresis for separation and determination of the stereoisomeric impurity of folinic acid diastereomers. In this method, vancomycin was used as the chiral selector, and a solution of poly(dimethylacrylamide) (PDMA) was prepared for dynamic coating of the capillary wall to minimize the adsorption of vancomycin. This method was optimized for six factors including concentrations of the organic modifier and vancomycin, pH and concentration of the background electrolyte, column temperature, and separation voltage. The following conditions were established: 100 mM Tris-phosphate buffer (pH 6.0) containing 1.0 mM vancomycin and 5 % acetonitrile at 30 °C, and −15 kV applied voltage on the PDMA dynamically coated capillary. Preliminary validation was performed with the determination of limit of quantification and detection, accuracy, precision, and linearity. Under our optimized method, the folinic acid diastereomers were baseline-separated within 7.5 min, and a (6S,2′S)-calcium folinate sample with 0.08 % stereoisomeric impurity was determined.

     

Determination of Phenolic Acids in Herba Epilobi by ITP–CE in the Column-Coupling Configuration

The combination of capillary isotachophoresis (ITP) and capillary zone electrophoresis (CZE) in the column-coupling configuration has been optimized in a mode in which the background electrolyte employed in the CZE step was different from the leading and terminating electrolytes of the ITP step. The optimum composition of the electrolyte system was 0.01 M HCl, 0.02 M IMI, 0.2% HEC, pH 7.2 (leading electrolyte), 0.01 M HEPES, pH 8.2 (terminating electrolyte), and 25 mM MES, 50 mM TRIS, 30 mM boric acid, 0.2% HEC, pH 8.3 (background electrolyte). All solutions contained 20% methanol. The timing of the transfer of isotachophoretically stacked analyte zones into the CZE column was also optimized. An ITP–CZE method with UV detection at 270 nm was developed for separation of nine phenolic acids (protocatechuic, syringic, vanillic, cinnamic, ferulic, caffeic, ρ-coumaric, chlorogenic, and gentisic acids) in a model mixture and used for assay of some of these acids in a methanolic extract of herba epilobi. Application of ITP–CZE resulted in 100-fold better sensitivity than conventional CZE; limits of detection ranged between 10 and 60 ng mL⁻¹. When MES–TRIS–borate-based buffer, pH 8.3, was used in the CZE separation step the linearity of the ITP–CZE response was satisfactory (correlation coefficients were from 0.9937 to 0.9777). Repeatability was also satisfactory (RSD values ranged between 0.77% and 1.28% for migration times and between 1.65% and 13.69% for peak area). Revised: 23 March and 27 April 2006     

Rapid analysis of anthracycline antibiotics doxorubicin and daunorubicin by microchip capillary electrophoresis

A simple and rapid method has been developed for the analysis of anthracycline antibiotics doxorubicin (DOX) and daunorubicin (DAU) in human serum using mirochip-based capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. In this study, method development included studies of the effect of buffer pH, buffer concentration, organic solvents and separation voltage on sensitivity and separation efficiencies for the CE separation of DOX and DAU. Acetonitrile was found to have significantly improved the sensitivity and separation efficiency. The method was validated with regard to reproducibilities, linearity and limit of detection (LOD). The optimum electrophoretic separation conditions were 10 mM sodium tetraborate buffer at pH 9.5 with 40% acetonitrile (V/V) and a separation voltage of 2.1 kV. DOX and DAU were separated in 60 s under the optimum separation conditions. Linear relationships were obtained between the concentration and peak area (or peak height) in the 1–75 µg mL^{− 1} range and with the detection limits of 0.3 and 0.2 μg mL^{− 1} for DOX and DAU, respectively. The stability of both migration time and peak height of the analytes showed relative standard deviations of less than 5% (n = 9). The potential of this method was verified by spiking a human serum sample with the two drugs and analyzing the recovery ratios.     

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.     

Determination of Phosphoamino Acids by 6-Oxy-(N-succinimidyl acetate)-9-(2′-methoxycarbonyl)fluorescein and MEKC with LIF Detection

The fluorescent reagent 6-oxy-(N-succinimidyl acetate)-9-(2′-methoxycarbonyl)fluorescein (SAMF), has been newly synthesized for use as a label for characterization of phosphoamino acids by micellar electrokinetic capillary electrophoresis (MEKC) with laser-induced fluorescence (LIF) detection. The conditions for derivatization and separation of the phosphoamino acids, including pH, concentration of electrolyte, and Brij-35 concentration were optimized in detail. Derivatization was performed at 35 °C for 10min in borate buffer (pH 8.0). The derivatives were separated to baseline by use of running buffer containing 50 mM borate and 20 mM Brij-35 at pH 9.3. Detection limits ranged from 5 × 10^?11 to 1 × 10^?9 mol L^?1 (signal to noise ratio = 3). The method was used for characterization of the phosphoamino acids in a sample from hydrolysis of a novel protein kinase from tobacco cells.     

Online preconcentration of arsenic compounds by dynamic pH junction-capillary electrophoresis

An online preconcentration technique by dynamic pH junction was studied to improve the detection limit for anionic arsenic compounds by CE. The main target compound is roxarsone, or 3-nitro-4-hydroxyphenylarsonic acid, which is being used as an animal feed additive. The other inorganic and organoarsenic compounds studied are the possible biotransformation products of roxarsone. The arsenic species were separated by a dynamic pH junction in a fused-silica capillary using 15 mM phosphate buffer (pH 10.6) as the BGE and 15 mM acetic acid as the sample matrix. CE with UV detection was monitored at a wavelength of 192 nm. The influence of buffer pH and concentration on dynamic pH junction were investigated. The arsenic species focusing resulted in LOD improvement by a factor of 100-800. The combined use of C18 and anion exchange SPE and dynamic pH junction to CE analysis of chicken litter and soils helps to increase the detection sensitivity. Recoveries of spiked samples ranged between 70 and 72%.     

Development and validation of a capillary electrophoretic method for the determination of amiodarone and desethylamiodarone

Summary A simple, sensitive and rapid capillary electrophoretic method has been developed for the separation and quantification of amiodarone and its metabolite, desethylamiodarone. The compounds were separated in a capillary of 45 cm effective length and 75 μm i.d., by use of an applied voltage of 25 kV and an electrolyte containing 15mm ADA buffer (pH 7.5), 10mm SDS, and 70% (v/v) acetonitrile. The selectivity, precision, linearity, range, sensitivity, and robustness of the method were good. The applicability of the assay was demonstrated by analyzing these drugs in serum. Electrokinetic injection with field-amplified sample-stacking was used to increase sensitivity. The limit of detection of the serum assay was 6.46 ng mL⁻¹ and the precision 3.7%.     

Separation of alkylbenzyl quaternary ammonium compounds by capillary zone electrophoresis with sample stacking injection

Summary A systematic investigation of operational buffer systems, sample preparation and instrument parameters for achieving the best possible performance for determinating an homologous series of N-benzyl-N-alkyl-N,N-dimethylammonium chloride compounds by capillary zone electrophoresis with direct UV detection. The most effective separation was achieved within 3.5 min with the addition of acetonitrile (40%) in a phosphate buffer (20 mM pH 5.2) using a 40 cm fused-silica capillary operating at 25 KV and 20°C. Degassing of all electrolyte solutions and samples was very important. The linearity and repeatability for each compounds were satisfactory. To improve detection limits, on-column sample preconcentration, sample stacking, was investigated achieving a tenfold enrichment factor and quantitation limits about 10⁻⁷M.     

Selective and sensitive determination of protoberberines by capillary electrophoresis coupled with molecularly imprinted microextraction

In this work, we developed a novel molecularly imprinted solid‐phase microextraction with capillary electrophoresis method for the selective extraction and determination of protoberberines in complicated samples. The imprinted monolith was prepared in a micropipette tip‐based device by using acrylamide as the functional monomer, ethyleneglyoldimethacrylate as the cross‐linker and dimethylsulfoxide as the porogen, and exhibited an imprinting factor of 2.41 to berberine, 2.36 to palmatine and 2.38 to jatrorrhizine. Good capillary electrophoresis separation was achieved by using 20 mM phosphate buffer at pH 7 as running buffer with the addition of organic modifier of 10% methanol. Parameters such as sample pH value, sample flow rate and sample volume were investigated for imprinted monolith‐based solid‐phase microextraction. An imprinted solid‐phase microextraction with capillary electrophoresis method was developed, the method showed a wide linear range (0.3–50 μg/mL), good linearity (R² ≥ 0.9947) and good reproducibility (relative standard deviations ≤ 0.73%), the limit of detection was as low as 0.1 μg/mL, which was lower than some reported methods based on capillary electrophoresis for protoberberines. The method has been applied for determination of three common protoberberines in Cortex Phellodendri Chinensis, by using a molecularly imprinted monolith as the selective sorbent, most of the matrices in the Cortex Phellodendri Chinensis sample were removed and three protoberberines were selectively enriched and well determined.