Field-amplified Sample Stacking Research Articles

A simple and green offline-online capillary electrophoresis stacking strategy for the simultaneous determination of hydrophobic compounds in complicated samples using sodium dodecyl sulfate as the solubilizer and pseudophase

BackgroundCapillary electrophoresis is a powerful analytical method featured with high separation efficiency, minimal sample requirements, and reduced organic solvents consumption. However, its low sensitivity hinders its wide application in determination of trace analytes especially for the weakly ionized hydrophobic compounds. Offline and Online capillary electrophoresis stacking methods are more favored to enhance detection sensitivity of analytes. The determination of two sesquiterpenes and an alkaloid from the dried root of Lindera aggregata merged as an example for developing a simple, sensitive and green method for the simultaneous determination of two hydrophobic compounds in complicated matrix samples. ResultsAn offline-online capillary electrophoresis stacking strategy by integrating micro matrix solid phase dispersion with field-amplified sample stacking and micelle to cyclodextrin stacking has been developed for the simultaneous determination of dehydrocostus lactone, linderane, norisoboldine in complex matrices. The optimized parameters were set at 65 mM sodium dihydrogen phosphate, 35% methanol, 180 s for sample injection and 210 s for cyclodextrin injection, 20 mM sodium dodecyl sulfate of sample matrix for online stacking; 1:1 sample to MCM-48, 180 s grinding time, and 1,000 μL of 20 mM sodium dodecyl sulfate elution for offline procedure. Under the optimum conditions, the method showed good linearity with correlation coefficients (R2≥0.9927), low limits of detection within the range of 25-50 ng mL-1, satisfactory repeatability and reproducibility below 3.98%, and acceptable recoveries between 94% and 97%. The developed method was successfully applied to two real samples, the root of L. aggregata and rat feces. SignificanceSodium dodecyl sulfate is firstly used as an eluent in micro matrix solid phase dispersion and plays a dual role throughout the analytical procedure, including extraction solvent in sample preparation and micelle pseudophase during online stacking. It brings great procedure convenience to the method. The sensitivity of this method can improve up to 1283-folds compared with the normal mode. Moreover, the overall strategy indicates satisfied green potential evaluated by greenness assessment tools

Capillary electrophoresis tandem mass spectrometry to determine multiclass cyanotoxins in reservoir water and spinach samples

Cyanotoxins constitute a group of toxic secondary metabolites, the presence of which in any water body poses a major health risk. Moreover, advanced organisms such as edible plants exposed to these toxins, are a possible pathway for human exposure. Green analytical chemistry is demanding environmentally friendly analytical techniques. In this sense, we propose the use of capillary electrophoresis coupled to tandem mass spectrometry (CE-MS/MS) to determine a mixture of eight cyanotoxins belonging to three different classes: cyclic peptides (microcystin-LR, microcystin-RR and nodularin), alkaloids (cylindrospermopsin and anatoxin-a) and three isomeric non-protein amino acids (β-methylamino-l-alanine, 2,4-diaminobutyric acid and N-(2-aminoethyl)glycine). Separation was achieved by using an acidic background electrolyte consisting of 2 M formic acid and 20% acetonitrile in water. Parameters affecting MS/MS detection and the sheath-liquid interface were also studied. Finally, a combination of pH-junction, field-amplified sample stacking (FASS) and acid barrage as online preconcentration strategies, was employed to improve sensitivity and efficiency. The online preconcentration applied, in combination with a dual cartridge solid-phase extraction (SPE) system, allows to obtain limits of detection in the very low range of µg·L−1 for these multiclass cyanotoxins in reservoir water samples (from 0.005 to 0.10 µg·L−1). Furthermore, for the first time cyanotoxins are analysed in spinach samples through CE-MS/MS using the same SPE procedure, following lyophilisation and solid-liquid extraction with 6 mL 80 % aqueous MeOH.

A pH-mediated field amplification sample stacking technique based on portable microchip electrophoresis heavy metal ion detection system.

We built a portable microchip electrophoresis heavy metal ion detection system and proposed a pH-mediated field amplified sample stacking (pH-mediated FASS) online preconcentration method. The pH-mediated FASS focuses and stacks heavy metal cations by controlling electrophoretic mobilities with a pH change between the analyte and the background electrolyte (BGE) in solution to improve the detection sensitivity of the system. We optimized and adjusted sample matrix solution (SMS) ratios and pH to create concentration and pH gradients for SMS and BGE. Furthermore, we optimize the microchannel width to improve the preconcentration effect further. The system and method analyzed soil leachates polluted with heavy metals and separated Pb2+ and Cd2+ within 90s, obtaining their levels at 58.01mg/L and 4.91mg/L with sensitivity enhancement factors (SEF) of 26.40 and 43.73. Compared with inductively coupled plasma atomic emission spectrometry (ICP-AES), the detection error of the system was less than 8.80%.

Liquid Plasticine-Based Electrokinetic Enrichment of Proteins.

Protein analysis is an important approach for disease diagnosis, in which sample pretreatment is an essential step since protein samples are often complex and many protein biomarkers are of low abundance. Here, given the good openness and light transmission of liquid plasticine (LP), which is a liquid entity formed by SiO2 nanoparticles and encapsulated aqueous solution, we developed a LP-based field-amplified sample stacking (FASS) system for protein enrichment. The system was composed of a LP container, a sample solution and a Tris-HCl solution containing hydroxyethyl cellulose (HEC). The system design, mechanism investigation, optimization of experimental parameters and characterization of LP-FASS performance for protein enrichment were well studied. Under the optimized experimental conditions of 1 % HEC, 100 mm Tris-HCl and 100 V in the LP-FASS system, a 40-80 times enrichment of proteins was obtained in 40 min using bovine hemoglobin (BHb) as the model protein using the constructed LP-FASS system. The simultaneous enrichment of multiple proteins (phycocyanin, BHb and cytochrome C) was also realized using the system. The LP-FASS system can serve as a new platform for protein enrichment which is easy to be combined with online and offline detections.

Highly Sensitive In-Capillary Derivatization and Field Amplified Sample Stacking to Analyze Narcotic Drugs in Human Serum by Capillary Zone Electrophoresis

An in-capillary derivatization (in-CAP-D) integrated with field amplified sample stacking (FASS) has been developed for the determination of morphine (MOR) and its metabolite, morphine-6-glucuronide (MOR-6-G) in human serum using capillary zone electrophoresis (CZE) and fluorescence detection (in-Cap-D-FASS-CZE). Acetonitrile was employed in removing proteins and extracting MOR and MOR-6-G into the clear supernatant containing codeine (COD) as an internal standard (IS). The derivatization was achieved in an in-capillary mode by introducing the acetonitrile-treated samples into a running electrolyte containing an oxidizing agent of potassium ferricyanide, whereas MOR, MOR-6-G and COD were oxidized into dimer derivatives with highly fluorescent intensity. The effectiveness and sensitivity of the in-Cap-D-FASS-CZE method were affected by many parameters, and the following conditions were found to be optimal: 70 m Mdisodium tetraboratedecahydrate (pH, 10.5), 0.30 mM ferrricyanide and a separation voltage of 10 kV. In order to perform the FASS, samples were electrokinetically injected for 20 s at 20 kV into the capillary that was pre-field with a 4 s water plug. Analysis was performed at ambient temperature (22 ± 1 °C). The method’s validation revealed good linearity with respect to peak area ratios of MOR and MOR-6-G with the IS and the corresponding concentrations over the ranges of 1–2000 and 1.2 to 2000 ng/mL, respectively. Following one oral dose of controlled-release MOR sulphate tablet, the validated in-Cap-D-FASS-CZE method successfully enabled the determination of MOR and MOR-6-G in clinical serum samples.

Paper-based analytical device for fast colorimetric detection of total hemoglobin and free hemoglobin from human blood sample

Total hemoglobin (THb) and free hemoglobin (FHb) are biomarkers of anemia and hemolysis, playing a critical role in human health status. This study reports a paper-based device by field amplified sample stacking (FASS) using colorimetric method to quantify both THb and FHb. The detection of THb was based on the color intensity of the Hb stacking band, a limit of detection (LOD) of 11 g∙L−1 and a linear working range of 16 ∼ 140 g∙L−1 were achieved. With selective chromatic reaction of the stacking band with o-tolidine, the sensitivity of Hb detection was further enhanced by 2000 times, a LOD of 25.8 mg∙L−1 and a linear range of 30 ∼ 1000 mg∙L−1 was obtained for FHb. This paper-based device was successfully used for detection of high concentration THb and low concentration FHb from the same blood sample, and both meeting the requirements of clinical diagnosis. In addition, the feasibility of on-site plasma preparation and online integration with the device was also preliminarily investigated with a blood separation membrane. This paper-based device could be used as a new platform for developing fast and low-cost Hb point-of-care testing devices.

Investigation of parameters and porous plug enhanced enrichment with field-amplified sample stacking in microchip

With the recent great interest in microfluidic devices, a better understanding of preconcentration technology has become increasingly important. Herein, concentration enrichment of charged samples is achieved by using field-amplified sample stacking (FASS) technology in the microchannel. This paper aims to develop a fundamental understanding of FASS and to propose a method to enhance the enrichment quality of FASS. First, numerical investigations are carried out to systematically study the effects of various parameters including the applied voltage, the charged properties of the sample, the buffer concentration ratio, the injection length, and the microchannel width on FASS enrichment performance. The results show that reducing the width of the microchannel is an effective way to improve the enrichment quality. The maximum enrichment ratio can be improved by 67.35% by reducing the width of the microchannel to less than 10 μm due to the inhibition of background buffer diffusion. Second, to improve traditional FASS performance, a high-conductance gradient boundary is established by photoinitiating fabrication of a porous plug at the enriched interface position. This structure provides a region that reduces the local size of the internal channel to less than 5 μm and has high flow resistance, but allows the electromigration of the charged analyte. Experimental results show that an electropherogram signal increases by a maximum factor of 329 in electrophoretic enrichment of fluorescein–Na with 5 × 10−7 M initial concentrations, and the enrichment quality of traditional FASS is greatly improved.

Recent Applications of Dynamic On-Line Sample Preconcentration Techniques in Capillary Electrophoresis

This review highlights recent applications of on-line sample preconcentration techniques in capillary electrophoresis (CE) from 2019 to 2021. Various preconcentration techniques based on the analyte velocity change in two or three discontinuous solutions system including field-amplified sample stacking, transient isotachophoresis, pH-mediated stacking, sweeping, and their modified and combined techniques have been employed to enrich and separate biological, environmental, food samples, and so on, in CE. More than 60 published research articles collected from Scopus databases described the dynamic on-line sample preconcentration techniques. This review provides comprehensive tables listing the applications of the on-line sample preconcentration techniques.

Sensitive detection of heart-failure biomarkers natriuretic peptides using multi-photon laser wave-mixing spectroscopy

Nonlinear laser wave-mixing spectroscopy is demonstrated as a fast and sensitive detection method for heart-failure biomarkers, pro-atrial natriuretic peptide (proANP) and brain natriuretic peptide (BNP). Wave mixing is an ultrasensitive optical absorption-based method and analytes can be detected in their native form or labeled with fluorophore and chromophore labels. In this study, we utilized Chromeo P540 dye to label the peptides for wave-mixing detection. The wave-mixing signal is created from the diffraction of incoming photons by the thermal grating at the capillary analyte cell. The signal beam is strong, collimated, and coherent (laser-like) and it is collected using a simple photodetector with an excellent signal-to-noise ratio. We demonstrated advantages of this technique over conventional assays including shorter analysis times, smaller sample requirements, and higher throughput. To enhance detection selectivity and sensitivity levels, wave mixing is effectively coupled to capillary zone electrophoresis (CZE) and field-amplified sample stacking (FASS) methods. We determined detection limits of 7.4 × 10−10 M or 55 zmol and 6.8 × 10−10 M or 51 zmol for proANP and BNP, respectively, and separated and detected both peptides within 2 min. Due to the challenges in the confirmatory diagnoses of heart failure, wave-mixing serves as a potentially beneficial screening tool in addition to the commonly used echocardiographic tests.

Green microemulsion electrokinetic chromatographic method for simultaneous determination of azelastine and budesonide

A facile, sensitive, and eco-friendly microemulsion electrokinetic chromatographic method coupled with field amplified sample stacking was developed for the simultaneous determination of azelastine (AZL) and budesonide (BUD) in their combined pharmaceutical preparations. The separation of both drugs was achieved using 13 mM borate buffer (pH 9.0) containing 1.2% sodium dodecyl sulphate (SDS), 2.4% n-butanol, and 1.0% ethyl acetate as the background electrolyte (BGE). A capillary tube (40 cm effective length; 75 μm internal diameter) of uncoated fused silica was used for separation of the studied analytes. A diode array detector was used to monitor the studied compounds at 195 nm. The applied voltage and temperature were adjusted at 28 kV and 25 °C, respectively. The method was linear over the concentration ranges of 0.5–50.0 μg/mL for azelastine and 1.0–20.0 μg/mL for budesonide. The sensitivity was improved by more than 10 folds by using field amplified sample stacking technique. Moreover, the method was applied for separation of budesonide epimers (22R-BUD and 22S-BUD) by increasing the concentration of SDS in the BGE to be 1.8%. Greenness evaluation was performed using analytical eco-scale, Green Analytical Procedure Index (GAPI) and Analytical GREEnness (AGREE) approaches. The excellent green profile of the developed method, confirmed by the three approaches, has supported its applicability in quality control laboratories.

Capillary electrophoresis-mass spectrometry as a tool for the noninvasive target metabolomic analysis of underivatized amino acids for evaluating embryo viability in assisted reproduction.

Monitoring metabolite uptake and excretion in the culture medium is a noninvasive technique that is used for the metabolic study of cleaving embryos after in vitro fertilization. Low sample consumption, the versatility of the detection, and optimal sensitivity and selectivity are essential elements for extracellular metabolome analyses, and can be conveniently achieved by combining CE with mass spectrometric detection. This paper reports a method for amino acid determination in a limited volume sample (8 μL)ofspent culture media collected after the cultivation of in vitro fertilized embryos. Special attention was focused on the sample preparation procedure. The sample was processed with acetonitrile, which facilitates online sample preconcentration via field-amplified sample stacking, and undesired sample evaporation was significantly reduced by the simultaneous addition of dimethyl sulfoxide. Key parameters that affected electrophoretic separation and mass spectrometric detection were investigated, including the type of buffers and organic solvent, optimization of their concentrations, and finally the settings for their ionization. The separation and quantification of 19 amino acids were achieved using 15% acetic acid as the background electrolyte with a sheath liquid consisting of an equimolar mixture of methanol and water. The applicability of the optimized system was demonstrated by determining the amino acid profile in 40 samples of spent cultivation medium in this pilot study. This developed method also has great potential for amino acid analyses in minute sample volumes of other biological matrices.

Ultrasensitive determination of underivatized adamantane analogs in biological fluids by capillary electrophoresis with contactless conductivity detection

Separation techniques are promising for the detection of adamantane analogs with various functional groups. The derivatization of adamantane analogs with chromophores or fluorophores should be conducted using a separation technique combined with a common ultraviolet–visible or fluorescence detector. However, limited research has been conducted on underivatized adamantane analogs. Therefore, we present a highly rapid and sensitive method for detecting underivatized adamantane analogs by integrating ultrasound-assisted dispersive liquid–liquid microextraction (UADLLME), field-amplified sample stacking (FASS)-related capillary electrophoresis (CE), and capacitively coupled contactless conductivity detection (C4D). In the proposed system, UADLLME is used for sample clean up and analyte enrichment, whereas FASS-related CE provides on-line concentration of underivatized adamantane analogs during CE separation. A mixture of memantine (MT), amantadine (AT), and rimantadine (RT) was separated at baseline within 8 min through the application of optimized UADLLME (mixing extraction solvent, 50 μL of 1,1,2,2-tetrachloroethane (C2H2Cl4) and dispersive solvent, 200 μL of acetonitrile; mixing solution was injected into 1 mL of the sample solution at pH 12.5), FASS (buffer, 1.5 M acetic acid; additive, 0.05 mM β-cyclodextrin; pH 2.5), and C4D (amplitude, 2 Vpp; frequency, 500 kHz). The calibration curve exhibited acceptable linearity, with a coefficient of determination higher than 0.99. The limits of detection (signal-to-noise ratio of 3) were estimated to be 0.9, 1.0, and 1.2 nM for MT, AT, and RT, respectively. The relative standard deviations of peak areas varied from 8.9% to 9.3% (n = 5), whereas the sensitivity improvement of three underivatized adamantane analogs ranged from 1342 to 1766. The feasibility and accuracy of the present method for the determination of MT, AT, and RT in human serum and urine was satisfactorily confirmed by the excellent recovery and relative error. The proposed method exhibited high enrichment factors and offers excellent precision, high accuracy, and a short analysis time.

Tween 20-capped gold nanoparticles for selective extraction of free low-molecular-weight thiols in saliva followed by capillary electrophoresis with contactless conductivity detection

Low-molecular-weight thiols are widely present in human fluids, and are regarded as a kind of potential broad-spectrum evaluation indicators for some clinical diseases. In this work, gold nanoparticles capped with Tween 20 were used for purification and microextraction of the main free thiols (cysteine, homocysteine, glutathione and methionine) in saliva based on Au-S bond formation. Ultrasound further sped up the releasing of the target analytes, and the releasing time needed was only 10 min, and the required sample volume was only 40 µL. The desorption solution could be directly injected for electrophoretic analysis without derivatization, and field-amplified sample stacking of electrophoretic online enrichment technology further improved the detection sensitivity. The synergistic enrichment effect made the enrichment factors of four analytes reach 1119–2067 times. This developed method was applied for the analyses of saliva samples of healthy volunteers. Acceptable sensitivity (LODs: 0.15–1.5 ng mL−1) and recoveries (97.6–116%) were obtained in the saliva sample matrix. This proposed method provides an alternative for the sensitive detection of low-molecular-weight thiols in noninvasive body fluids, which has potential application prospect in the preliminary noninvasive diagnosis of diabetes, cardiovascular diseases, etc.

Field amplified sample stacking and in-capillary derivatization for forensic analysis of morphine and morphine-6-glucuronide in human urine by capillary electrophoresis

A rapid, sensitive, selective and simple capillary zone electrophoresis (CZE) method, by integration of field amplified sample stacking (FASS) and in-capillary derivatization (in-CapD) coupled with fluorescence (FL) detection (CZE-FASS-in-CapD-FL), has been developed and validated for simultaneous determination of morphine and its metabolite, morphine-6-glucuronide (morphine-6-G)in human urine. The method was based on in-line derivatization by introducing the sample to running background electrolytes (BGE) of disodium tetraborate decahydrate and potassium ferricyanide, whereas analytes were oxidized into highly fluorescent dimer products. Various parameters influencing the sensitivity and efficiency of the proposed method were investigated, and the optimal conditions of the BGE were as follow: 60 mM disodium tetraborate decahydrate (pH 10.5), 0.25 mM potassium ferricyanide and separation voltage of 9 kV. For FASS, morphine and morphine-6-G were electrokinetically injected for 14 s at 16 kV into the capillary that was pre-injected with water plug for 4 s. The fluorescence signals were then detected at excitation and emission wavelengths of 340 and 450 nm, respectively. Analysis was performed at ambient temperature (22±1 °C). Validation of the method showed good linearity over morphine and morphine-6-G concentrations ranging from 2 to 2000 ng/mL and 2.5 to 2000 ng/mL, respectively. The mean recoveries were ranged from 97.72 to 87.26% and 97.14 to 85.43% for morphine and morphine-6-G, respectively. The validated CZE-FASS-in-CapD-FL method was effectively applied as quantitative forensic analysis of morphine and morphine-6-G in clinical urine samples.

High‐resolution numerical simulations of electrophoresis using the Fourier pseudo‐spectral method

We present the formulation, implementation, and performance evaluation of the Fourier pseudo-spectral method for performing fast and accurate simulations of electrophoresis. We demonstrate the applicability of this method for simulating a wide variety of electrophoretic processes such as capillary zone electrophoresis, transient-isotachophoresis, field amplified sample stacking, and oscillating electrolytes. Through these simulations, we show that the Fourier pseudo-spectral method yields accurate and stable solutions on coarser computational grids compared with other nondissipative spatial discretization schemes. Moreover, due to the use of coarser grids, the Fourier pseudo-spectral method requires lower computational time to achieve the same degree of accuracy. We have demonstrated the application of the Fourier pseudo-spectral method for simulating realistic electrophoresis problems with current densities as high as 5000 A/m2 with over tenfold speed-up compared to the commonly used second-order central difference scheme, to achieve a given degree of accuracy. The Fourier pseudo-spectral method is also suitable for simulating electrophoretic processes involving a large number of concentration gradients, which render the adaptive grid-refinement techniques ineffective. We have integrated the numerical scheme in a new electrophoresis simulator named SPYCE, which we offer to the community as open-source code.

Necessity and Challenges of Sample Preconcentration in Analysis of Multiple MicroRNAs by Capillary Electrophoresis.

Thousands of putative microRNA (miRNA)-based cancer biomarkers have been reported, but none has been validated for approval by the Food and Drug Administration. One of the reasons for this alarming discrepancy is the lack of a method that is sufficiently robust for carrying out validation studies, which may require analysis of samples from hundreds of patients across multiple institutions and pooling the results together. The capillary electrophoresis (CE)-based hybridization assay proved to be more robust than reversed transcription polymerase chain reaction (the current standard), but its limit of quantification (LOQ) exceeds 10 pM while miRNA concentrations in cell lysates are below 1 pM. Thus, CE-based separation must be preceded by on-column sample preconcentration. Here, we explain the challenges of sample preconcentration for CE-based miRNA analyses and introduce a preconcentration method that can suit CE-based miRNA analysis utilizing peptide nucleic acid (PNA) hybridization probes. The method combines field-amplified sample stacking (FASS) with isotachophoresis (ITP). We proved that FASS-ITP could retain and concentrate both near-neutral PNA with highly negatively charged PNA-miRNA hybrids. We demonstrated that preconcentration by FASS-ITP could be combined with the CE-based separation of the unreacted PNA probes from the PNA-miRNA hybrids and facilitate improvement in LOQ by a factor of 140, down to 0.1 pM. Finally, we applied FASS-ITP-CE for the simultaneous detection of two miRNAs in crude cell lysates and proved that the method was robust when used in complex biological matrices. The 140-fold improvement in LOQ and the robustness to biological matrices will significantly expand the applicability of CE-based miRNA analysis, bringing it closer to becoming a practical tool for validation of miRNA biomarkers.

Offline Selective Extraction Combined with Online Enrichment for Sensitive Analysis of Chondroitin Sulfate by Capillary Electrophoresis

A capillary electrophoresis (CE) method combined with online and offline enrichment for improving the detection sensitivity of chondroitin sulfate (CS) is established. The online enrichment method is based on the field-amplified sample stacking and large volume electrokinetic injection, and offline enrichment is based on the association between cetyltrimethylammonium chloride and CS. Experimental parameters affecting CE method such as the type and pH of background electrolyte, the injection mode and time and the steps of offline enrichment were optimized. Under optimum conditions, the calibration plot between CS concentration and peak area was linear in the range of 1~100μg/mL. The enrichment factor was 130 times and the limit of detection was 50ng/mL. The average recovery was 103.5% and the relative standard deviation of peak area was <2.0%. The method was successfully applied to the quantitative analysis of CS in drugs.

Quantification of imatinib and related compounds using capillary electrophoresis-tandem mass spectrometry with field-amplified sample stacking.

A quantification method for imatinib (IM), its major metabolite N-desmethyl imatinib (NDI), and a degradation by-product was developed using CE-MS combined with an online concentration technique. The use of multiple reaction monitoring (MRM)-MS/MS further improved the sensitivity of this technology. Liquid-liquid extraction (LLE) using tertiary butyl methyl ether yielded high recovery and reproducibility for the pretreatment of serum samples. The recovery rate exceeded 83% for all three analytes, and was 90% for IM. To improve quantification results, a conductivity-induced online analyte concentration technique, field-amplified sample stacking (FASS), was used. The S/N ratios were improved at least 10-fold when compared with conventional capillary zone electrophoresis. The detection limits were 0.2ng/mL for IM, 0.4ng/mL for NDI, and 4ng/mL for the degradation by-product. These results are superior to those previously obtained by other reported methods. The new method was validated in terms of its selectivity, intra- and interday repeatability and accuracy, and sample storage stability, following the guidelines issued by the European Medicines Agency. Considering the convenient pretreatment procedure (LLE), superior sensitivity, and fast analysis speed (<15min), this method can be useful in the determination of imatinib levels in blood.

Nanosized cation exchanger for the electrophoretic separation and preconcentration of catecholamines and amino acids.

The first example of application of nanosized polystyrene-based cation exchanger (NSCE) with sulfo groups as a dynamic coating of capillary walls was demonstrated. The conditions of dynamic coating formation were optimized and ensured the long-term stability of the coating. Capillary-to-capillary and day-to-day repeatabilities were 4% and 3%, correspondingly. The NSCE coating stability at various pH and influence of pH on the EOF mobility were investigated. The developed NSCE-modified coated capillaries provided improved resolution (Rs = 0.9-3.2 for catecholamines and Rs = 1.7-2.8 for amino acids) and efficiencies (330-520 ×103 t.p./m) of basic analytes, which are 1.5 times higher compared to untreated capillary. The optimized conditions were as follows: 50mM phosphate buffer solution at pH 2.2 with 5μM NSCE. The effect of the NSCE concentration in BGE on the electrophoretic mobilities of the analytes was investigated. The various online concentration techniques were tested in order to decrease the LODs. The simultaneous application of NSCE capillaries and field-amplified sample stacking provided the lowest LODs of catecholamines and amino acids and allowed to determine these analytes in human urine.

Ultrasound-assisted dispersive liquid-liquid microextraction coupled with field-amplified capillary electrophoresis for sensitive and quantitative determination of fluoxetine and norfluoxetine enantiomers in biological fluids.

A rapid, simple, and sensitive technique for the quantitative detection of fluoxetine and norfluoxetine enantiomers in biological fluids was developed based on the combination of field-amplified sample stacking (FASS)-related capillary electrophoresis (CE) with ultrasound-assisted dispersive liquid-liquid microextraction (UA-DLLME). The extraction efficiency of UA-DLLME was strongly related to extraction time, salt concentration, type of extraction and dispersion solvents, and volume of extraction and dispersion solvents. The extracted fluoxetine and norfluoxetine enantiomers in a mixture of 50% methanol and 50% deionized water were efficiently stacked using FASS and then separated using cyclodextrin-modified CE. Under optimal conditions of FASS (chiral selector, 3mM trimethyl-β-cyclodextrin; and background electrolyte, 100mM phosphate buffer) and UA-DLLME (extraction solvent, 200μL of acetone; and dispersed solvent, 50μL of C2H2Cl4 in 1mL of the sample solution), the obtained enrichment factors of fluoxetine and norfluoxetine enantiomers reached approximately 2000. The linear ranges for the quantification of fluoxetine and norfluoxetine enantiomers were 0.3-150 and 0.6-150nM, respectively. The relative standard deviations in peak areas and migration time for four analytes were less than 3.3% and 6.3%, respectively. The proposed system provided limits of detection (signal-to-noise ratio of 3) for four analytes corresponding to 0.1nM. The precision and accuracy for urine and serum samples were less than 6.8 and 8.3%, respectively. These findings suggested that the proposed system exhibited a high potential for the reliable determination of fluoxetine and norfluoxetine enantiomers in clinical samples. Graphical abstract.