Field-enhanced Sample Stacking Research Articles

Method Development for Determination of Doripenem in Human Plasma via Capillary Electrophoresis Coupled with Field-Enhanced Sample Stacking and Sweeping.

In this study, we established a novel capillary electrophoresis method for monitoring the concentration of doripenem in human plasma. As a time-dependent antibiotic, doripenem maximizes its antibacterial effects and minimizes the potential for antibiotic resistance through careful therapeutic drug monitoring. Two online preconcentration techniques, field-enhanced sample stacking (FESS) and sweeping, were coupled to enhance the detection sensitivity. Briefly, an uncoated fused silica capillary (40 cm × 50 μm i.d) was rinsed with a high conductivity buffer (HCB) composed of 150 mM phosphate buffer (NaH2PO4, pH 2.5) and 20% methanol. A large sample plug prepared in a low-conductivity phosphate buffer (50 mM NaH2PO4, pH 2.5) was then hydrodynamically injected (5 psi, 80 s) into the capillary. Under an applied voltage of -30 kV, the analyte was accumulated at the FESS boundary and swept by the negatively charged micelles toward the UV detector. Plasma samples were pretreated by solid-phase extraction (SPE) to eliminate endogenous interferences. The validation results demonstrated a high coefficient of determination (r2 > 0.9995) for the regression curve with impressive precision and accuracy: relative standard deviation (RSD) <5.86% and relative error <4.63%. The limit of detection (LOD, S/N = 3) for doripenem was determined to be 0.4 μg/mL. Compared to the conventional micellar electrokinetic chromatography method, our developed method achieved a sensitivity enhancement of up to 488-fold for doripenem. Furthermore, the newly developed method successfully quantified doripenem concentrations in plasma samples obtained from patients accepting doripenem regimens, proving its application potential in the clinical realm.

Determination of quetiapine and its metabolites in plasma by field-enhanced sample stacking.

Quetiapine is an atypical antipsychotic drug that can be used to treat mental disorders, including schizophrenia, bipolar disorder and Alzheimer's disease. Quetiapine is mainly converted into the active metabolites of norquetiapine and 7-hydroxyquetiapine by the liver enzymes CYP3A4 and CYP2D6. In this study, liquid-liquid extraction (LLE) was used as a sample pretreatment method to eliminate interferences in plasma. tert-Butyl methyl ether was chosen as the extraction solvent. Field-enhanced sample injection (FESS), an online preconcentration technique, was used to analyze quetiapine and its metabolites norquetiapine and 7-hydroxyquetiapine in plasma. The optimal separation condition was 120 mM phosphate (pH 4.0) containing 0.005% (w/v) polyvinyl pyrrolidone and 40% (v/v) methanol. The methanol plug was 0.3 psi for 6 s, the sample was electrokinetic injection by 10 kV for 60 s at positive polarity, and the separation voltage was set at 26 kV. In this experiment, quetiapine, norquetiapine and 7-hydroxyquetiapine were successfully extracted from plasma by the LLE method and stacking and separated by FESS within 15 min. The limits of detection (S/N = 3) of quetiapine, norquetiapine and 7-hydroxyquetiapine were 0.25 ng/mL, 0.50 ng/mL and 1.00 ng/mL, respectively. The linear ranges of quetiapine, norquetiapine and 7-hydroxyquetiapine were 3-120 ng/mL and the correlation coefficients were 0.999. Compared with that of the traditional capillary zone electrophoresis method, the sensitivity enrichment of analytes was 463-835-fold. The optimal experimental conditions were successfully applied to the analysis of plasma samples from patients taking quetiapine for the treatment of schizophrenia.

Capillary Electrophoresis-Mass Spectrometry with Multisegment Injection and In-Capillary Preconcentration for High-Throughput and Sensitive Determination of Therapeutic Decapeptide Triptorelin in Pharmaceutical and Biological Matrices.

A capillary electrophoresis–tandem mass spectrometry method with a multisegment injection and an in-capillary field-enhanced sample stacking for determination of therapeutic peptide triptorelin in pharmaceutical and biological matrices was developed. The CE separation conditions were optimized in order to obtain maximal separation efficiency, analytical signal intensity and stability, and minimal adsorption of the analyzed peptide onto the capillary wall (1 M formic acid—HFo, pH 1.88). The implementation of the field-enhanced sample injection into CE improved the value of limit of detection 50 times while the multisegment injection increased the sample throughput three times in comparison to a conventional CE approach. The proposed method was characterized by favorable performance parameters, such as linearity (r2 ≥ 0.99), limit of detection (5 ng mL−1 in water matrix, 25 ng mL−1 in plasma matrix), precision (relative standard deviation, 1.5–9.4% for intraday and 2.3–11.9% for interday reproducibility), or accuracy (relative errors in the range of 80–109%). The FDA-validated method was successfully applied to the analysis of triptorelin in the commercial drug Diphereline® 0.1 mg (powder for injection) and in spiked human plasma samples. Favorable performance parameters along with proven application potentialities indicate the usefulness of the proposed method for its routine use in drug quality control laboratories and for clinical analysis, such as determination of triptorelin levels in plasma (for pharmacokinetic study).

Electropreconcentration, gate injection, and capillary electrophoresis separation on a microchip

The nanochannel-based electropreconcentration is not compatible with successive capillary zone electrophoresis (CZE). In this study, the incompatibility is theoretically discussed and experimentally proven, and then, the development of a monolithic glass microfluidic chip for performing integrated electropreconcentration and CZE separation is described. The sample is electropreconcentrated at the interface of a micro- and nanochannel where electric double layer overlap conditions exist. Because an ion-depletion region develops at the leading front of the preconcentrated plug, a field-enhanced sample stacking effect occurs which limits the separation efficiency unless compensated for. The ion-depletion region was confirmed by monitoring the solution conductivity at discrete points in the microchannel during the preconcentration step. The solution conductivity decreased >20-fold during the preconcentration step. To overcome the effects of this region, a cross-intersection was used to shunt the ion-depleted buffer away from the analysis channel while reintroducing the running buffer. When the preconcentrated sample plug arrives at the cross-intersection, it is gate injected into the analysis channel so that fresh running buffer surrounds the plug. Under these conditions, three-peptide mixture was preconcentrated ∼200-fold in 60 s and the preconcentrated plug was successfully resolved with better than 1% relative standard deviations in migration times.

Determination of finasteride and its metabolite in urine by dispersive liquid–liquid microextraction combined with field-enhanced sample stacking and sweeping

The on-line preconcentration technique of field-enhanced sample stacking and sweeping (FESS-sweeping) are combined with dispersive liquid–liquid microextraction (DLLME) to monitor the concentrations of finasteride, which is used in the treatment of androgenetic alopecia, and its metabolite, finasteride carboxylic acid (M3), in urine samples. DLLME is used to concentrate and eliminate the interferences of urine samples and uses chloroform as an extracting solvent and acetonitrile as a disperser solvent. A high conductivity buffer (HCB) was introduced into capillary and then sample plug (90.7% capillary length) was injected into capillary. After applying voltage, the sodium dodecyl sulfate (SDS) swept the analytes from the low conductivity sample solution into HCB. The analytes were concentrated on the field-enhanced sample stacking boundary. The limit of detection for the analytes is 20 ng mL−1. The sensitivity enrichment of finasteride and M3 are 362-fold and 480-fold, respectively, compared with the conventional MEKC method. The on-line preconcentration technique of field-enhanced sample stacking and sweeping possess good selectivity because the endogenous steroid did not interfere the detection of finasteride and M3. The analytical technique is applied to investigate the concentrations in urine samples from patients who have been administered finasteride for the treatment of androgenetic alopecia; the amount of M3 detected in 12 h was 72.69 ± 4.18 μg.

Determination of baclofen and vigabatrin by microchip electrophoresis with fluorescence detection: application of field-enhanced sample stacking and dynamic pH junction

A novel microchip gel electrophoretic method for the assay of baclofen and vigabatrin for the first time.

Simultaneous quantification of energetically important metabolites in various cell types by CZE

A new CZE method was developed for the determination of 12 purine and pyrimidine nucleotides, two adenine coenzymes and their reduced forms, and acetyl coenzyme A in various cell extracts. As the concentration levels of these metabolites in living cells are low; CZE was combined with field-enhanced sample stacking. As a result, the separation conditions were optimised to achieve a suitable resolution at the relatively high sample volume provided by this on-line pre-concentration technique. The optimum BGE was 150 mM glycine buffer (pH 9.5). Samples were introduced hydrodynamically using a pressure of 35 mbar (3.5 kPa) for 25 s, and data were collected at a detection wavelength of 260 nm. An applied voltage of 30 kV (positive polarity) and capillary temperature of 25°C gave the best separation of these compounds. The optimised method was validated by determining the linearity, sensitivity and repeatability and it was successfully applied for the analysis of extracts from Paracoccus denitrificans bacteria and from stem cells.

Review of recent developments of on-line sample stacking techniques and their application in capillary electrophoresis

Abstract Capillary electrophoresis (CE) has become one of the most useful tools in separation science because of its high separation efficiency, low cost, versatility, ease of sample preparation and automation. However, some limitations of CE, such as poor concentration sensitivity due to its lower sample loading and shorter optical path length, limits its further applications in separation science. In order to solve this problem, various on-line sample preconcentration techniques such as transient isotachophoresis preconcentration, field-enhanced sample stacking, micelle to solvent stacking, micelle collapse, dynamic pH junction, sweeping, solid phase extraction, single drop microextraction and liquid phase microextraction have been combined with CE. Recent developments, applications and some variants together with different combinations of these techniques integrating in CE are reviewed here and our discussions will be confined to the past three years (2008–2011).

Fully electrochemical hyphenated flow system for preconcentration, cleanup, stripping, capillary electrophoresis with stacking and contactless conductivity detection of trace heavy metals

Successful coupling of electrochemical preconcentration (EPC) to capillary electrophoresis (CE) with contactless conductivity detection (C 4D) is reported for the first time. The EPC–CE interface comprises a dual glassy carbon electrode (GCE) block, a spacer and an upper block with flow inlet and outlet, pseudo-reference electrode and a fitting for the CE silica column, consisting of an orifice perpendicular to the surface of a glassy carbon electrode with a bushing inside to ensure a tight press fit. The end of the capillary in contact with the GCE is slant polished, thus defining a reproducible distance from the electrode surface to the column bore. First results with EPC–CE–C 4D are very promising, as revealed by enrichment factors of two orders of magnitude for Tl, Cu, Pb and Cd ion peak area signals. Detection limits for 10 min deposition time fall around 20 nmol L −1 with linear calibration curves over a wide range. Besides preconcentration, easy matrix exchange between accumulation and stripping/injection favors procedures like sample cleanup and optimization of pH, ionic strength and complexing power. This was demonstrated for highly saline samples by using a low conductivity buffer for stripping/injection to improve separation and promote field-enhanced sample stacking during electromigration along the capillary.

Diffusion as major source of band broadening in field-amplified sample stacking under negligible electroosmotic flow velocity conditions

Theory of field-amplified sample stacking (FASS) also called field-enhanced sample stacking is reevaluated considering the early work of Chien, Burgi and Helmer. The classical theory presented by Chien, Helmer and Burgi predicts the existence of maxima, which are ascribed to the counteracting principles of zone focusing and hydrodynamic dispersion. In contrast to their work, we here focus on cationic analytes separated in an acidic background electrolyte providing a very low electroosmotic flow velocity. Therefore, peak broadening due to differences in the local electroosmotic flow velocities in different compartments of the capillary can be regarded to be negligible. Consequently, peak broadening resulting from hydrodynamic dispersion will not be the dominant limitation of the accessible enrichment efficiency. In our experimental studies we, however, obtain an optimum value for the field enhancement factor (maximum of the enrichment efficiency, when varying the electric conductivity of the sample and the size of the sample injection plug) corresponding to a 10-fold dilution of the BGE in the sample solution. Comparing these experimental data with data modeled according to the revised theory, we show that this limitation of the loadability is caused by the unavoidable decrease of the analyte migration velocity in the BGE compartment of the capillary when injecting of a sample plug of lower electric conductivity (decrease in the local electric field strength). The additional diffusional band broadening limits the obtainable enrichment efficiency.

Capillary zone electrophoresis with field enhanced sample stacking as a tool for targeted metabolome analysis of adenine nucleotides and coenzymes in Paracoccus denitrificans

The main aim of this work was to demonstrate the applicability of capillary zone electrophoresis in combination with field enhanced sample stacking in targeted metabolome analyses of adenine nucleotides--AMP, ADP, ATP, coenzymes NAD(+), NADP(+) and their reduced forms in Paracoccus denitrificans. Sodium carbonate/hydrogencarbonate buffer (100 mM, pH 9.6) with the addition of beta-CD at a concentration of 10 mM was found to be an effective BGE for their separation within 20 min. Besides this, special attention was paid to the development of the procedure for the extraction of specific metabolites from the bacterium P. denitrificans. This procedure was not only optimised to achieve the highest metabolite yields but also to obtain a sample that was fully compatible with the online preconcetration strategy used. The developed methodology was finally applied in a study of the bacterium P. denitrificans at various stages of the active respiratory chain.

Analysis of aliphatic amines using head‐column field‐enhanced sample stacking in MEKC with LIF detection

This paper describes the use of head-column field-enhanced sample stacking for on-line sample concentration in MEKC; the method has been applied to the determination of C1-C9 aliphatic amines after their pre-capillary derivatization with 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA). The optimum derivatization, on-line concentration and separation conditions for these aliphatic amines were investigated in detail. The optimized molar ratio of CBQCA to amines was found to be 120:1, and 50 mmol/L borate buffer (pH 9.5) was used for derivatization in order to achieve high efficiency. Nine imidazole aliphatic amines were baseline separated within 12 min by using 50 mmol/L of sodium borate (pH 9.5) and 15 mmol/L of SDS as running buffer. Under the optimum conditions, the detection limits of these nine aliphatic amines ranged from 1.2 to 3.8 pmol/L. S/N=3, which was about 49-173-fold lower than those of conventional sample injections. After validation, the developed method was applied to the determination of C1-C9 aliphatic amines in river water with the average recoveries of 86.7-105%.

On‐line concentration and pressurized capillary electrochromatographic analysis of phytohormones in corn

A pressurized CEC (pCEC) method was developed for the separation of phytohormones, in which UV absorbance was used as the detector and a monolithic silica-ODS column as the separation column. The parameters (including the concentration of organic solvent in the mobile phase, pH of the electrolyte buffer, applied voltage) affecting the separation resolution were evaluated. Two on-line concentration techniques, namely, solvent gradient zone sharpening effect and field-enhanced sample stacking, were utilized to improve detection sensitivity. The combination of the two techniques proved to be beneficial to enhance the detection sensitivity by enabling the injection of large volumes of samples. Compared to the conventional injection mode, the enhancement in the detection sensitivities of phytohormones using the on-line concentration technique is in the range from 9- to 23-fold. The developed pCEC method was applied to evaluate phytohormones in corns.

Integration of various stacking processes in carrier ampholyte-based capillary electrophoresis

Field-enhanced sample stacking, field-enhanced sample injection as well as electrokinetic supercharging have been successfully integrated in carrier ampholyte-based capillary electrophoresis. Through the analysis of different test sample mixtures, it has been shown that the carrier ampholyte-based background electrolytes, in spite of their very low conductivity, allow efficient online preconcentration of analytes by field-amplified techniques. Sensitivity enhancement factors of the same magnitude as those usually encountered with classical conductive background electrolytes have been obtained in such carrier ampholyte-based buffers. Depending on the online preconcentration method that has been integrated, sensitivity enhancement factors between 50 and several thousands have been reached.

Analysis of tetracycline residues in bovine milk by CE‐MS with field‐amplified sample stacking

A rapid, sensitive, and robust CE-MS method has been developed for the determination of tetracycline, oxytetracycline, and chlortetracycline in milk. Field-amplified sample stacking with electromigration injection (FASS-EMI) was used for the online concentration of tetracyclines. The conditions of separation, MS detection, and stacking were systematically optimized. The optimum buffer composition was 35 mM Tris, 1.1% formic acid, 5% methanol, and 15% ACN. By using the online concentration method of field-enhanced sample stacking (FESI)-EMI stacking, the sensitivity was increased six- to seven-fold. The RSDs (n=6) of the relative migration time of tetracyclines were 1.1-1.4% for intraday and 2.4-2.9% for interday. The RSDs (n=6) of the relative peak area of tetracyclines were 3.2-4.6% for intraday and 4.7-6.1% for interday. The LODs (S/N=3) were 7.14 ng/mL for tetracycline, 11.4 ng/mL for oxytetracycline, and 14.9 ng/mL for chlortetracycline. The method has been successfully used to analyze tetracyclines residues in bovine milk.

Rapid determination of aristolochic acid I and II in medicinal plants with high sensitivity by cucurbit[7]uril-modifier capillary zone electrophoresis

Aristolochic acids (AAs) are commonly found in medicinal plants such as Radix aristolochiae and have been reported to cause acute hepatitis and end-stage renal failure. Hence, quantitative analysis and quality control for the plants containing AAs is of great importance. In this study, a novel macrocylcic molecule, cucurbit[7]uril (CB[7]) was employed as a modifier in capillary zone electrophoresis (CZE) for rapid determination of aristolochic acid I and II in medicinal plants. In similarity to other macrocyclic molecules, such as cyclodextrins (CDs), CB[7] can be used to manipulate selectivity in CE because it can form inclusion complexes with a variety of guest molecules. During the running process, CB[7] bears a positive charge in the pH range of 2.5-7.5 and can be adsorbed onto the inner wall of a fused-capillary, leading to a reversal of the electroosmotic flow (EOF). By applying a negative polarity, a rapid separation of AA-I and AA-II was achieved within 7min using 100mM phosphate buffer (pH 7.5) containing 3mM CB[7] and 10% acetonitrile (v/v) as modifiers, due to the same directions of the EOF and the electrophoretic mobilities of the analytes. By applying electrokinetic injection with field-enhanced sample stacking, two kinds of aristolochic acids in four medicinal plants were successfully determined with high sensitivity, high separation efficiency, repeatability and recovery. The proposed method was also used to determine AA-I and AA-II in two slimming pills with complex matrix.

Pressurized capillary electrochromatographic analysis of water-soluble vitamins by combining with on-line concentration technique

A pressurized capillary electrochromatography (pCEC) system was developed for the separation of water-soluble vitamins, in which UV absorbance was used as the detection method and a monolithic silica-ODS column as the separation column. The parameters (type and content of organic solvent in the mobile phase, type and concentration of electrolyte, pH of the electrolyte buffer, applied voltage and flow rate) affecting the separation resolution were evaluated. The combination of two on-line concentration techniques, namely, solvent gradient zone sharpening effect and field-enhanced sample stacking, was utilized to improve detection sensitivity, which proved to be beneficial to enhance the detection sensitivity by enabling the injection of large volumes of samples. Coupling electrokinetic injection with the on-line concentration techniques was much more beneficial for the concentration of positively charged vitamins. Comparing with the conventional injection mode, the enhancement in the detection sensitivities of water-soluble vitamins using the on-line concentration technique is in the range of 3 to 35-fold. The developed pCEC method was applied to evaluate water-soluble vitamins in corns.

Chitosan as Cationic Polyelectrolyte for the Modification of Electroosmotic Flow and Its Utilization for the Separation of Inorganic Anions by Capillary Zone Electrophoresis

Cationic polyelectrolyte of chitosan was used for the reversal of electroosmotic flow in capillary zone electrophoresis. The chitosan was dissolved in acetic acid solution, and stable electroosmotic flow was obtained at the chitosan concentrations between 50 and 300 microg/mL. Separation of inorganic anions was carried out using the dynamically coated capillary by capillary zone electrophoresis. Nine kinds of anions were separated and detected with the capillary. The electrophoretic mobility of the analyte anions decreased with increasing concentrations of chitosan in the migrating solution through ion-ion interaction, but the migration order of the analyte anions was not changed in the concentration range of the chitosan examined. The signal shape for the analyte anions was developed by using field-enhanced sample stacking with 10 mM sodium sulfate.

Application of poly(methacrylic acid-ethylene glycol dimethacrylate) monolith microextraction coupled with capillary zone electrophoresis to the determination of opiates in human urine

A novel poly(methacrylic acid-ethylene glycol dimethacrylate) (MAA-EGDMA) monolith microextraction method coupled with CZE was proposed for rapidly determining a mixture of opiates comprising heroin, 6-monoacetylmorphine, morphine, codeine, papaverine, and narcotine in human urine. The extraction device contained a regular plastic syringe, the poly(MAA-EGDMA) monolithic capillary tube (530 microm id x 3 cm) and a plastic pinhead, which connected the monolithic capillary tube and the syringe without leakage. In the polymer monolith microextraction, the sample solution was ejected via the monolithic capillary tube by a programmable syringe pump, followed by desorption with an aliquot of appropriate solution, which was collected into a vial for the subsequent analysis by CZE. The best separation was achieved using a buffer composed of 0.1 M disodium hydrogen phosphate (adjusted to pH 4.5 with 1 M hydrochloric acid) and 20% methanol v/v with temperature and voltage of 25 degrees C and 25 kV, respectively. By applying electrokinetic injection with field-enhanced sample stacking, detection limits of 6.6-19.5 ng/mL were achieved. Excellent method of reproducibility was found over a linear range of 80-2000 ng/mL.

Operational modes for transient isotachophoretic preconcentration-capillary zone electrophoresis and detectable concentration of rare earth ions.

Operational modes for transient isotachophoretic preconcentration capillary zone electrophoresis (tr-ITP-CZE) were studied by using 5 microM and 0.5 microM rare earth mixtures as analytes in comparison with field-enhanced sample stacking. After examination of several operational modes for tr-ITP, it was found that tr-ITP effectively occured even if both the leading electrolyte and the terminating electrolyte were injected after the sample plug. This was explained as the result of a field-enhanced stacking for both sample components and the leading and terminating ions. The observed theoretical plate numbers were 4-20 times higher than those obtained by normal stacking; and the estimated low limit of detectable concentration of rare-earth elements (REE) was ca. 0.1 microM which was 2.5 times lower compared to normal stacking. For the 0.5 microM sample, a concentration factor of 20 000 could be achieved after only tr-ITP.