Capillary Isotachophoresis Research Articles

Investigation of on-line electrokinetic enrichment strategies for capillary electrophoresis of extracellular vesicles

This work explores strategies for electrokinetic preconcentration of extracellular vesicles (EVs) that are potential source of biomarkers for different diseases. The first approach that led to successful preconcentration of EVs is based on large volume sample stacking (LVSS), allowing an enrichment factor of 7 for CE of EVs with long-end injection (using a capillary with an effective length of 50 cm). Attempts were also made to perform multiple cycles of LVSS, field amplified sample stacking (FASS) and field amplified sample injection (FASI), to improve EVs preconcentration performance. The focus was then put on development of capillary isotachophoresis under high ionic strengths (IS) for electrokinetic enrichment of slow migrating EVs having heterogeneous mobilities. This approach relies on the use of extremely high concentrations of the terminating electrolyte (TE) to slow down the mobility of TE co-ions, rendering them slower than those of EVs. The limit of detection for intact EVs using the developed ITP-UV method reached 8.3 × 108 EVs/mL, allowing an enrichment of 25 folds and a linear calibration up to 4 × 1010 EVs/mL. The ITP-UV and ITP-LIF approaches were applied to provide the electrokinetic signature of EVs of bovine milk and human plasma as well as to visualize more specifically intravesicular fluorescently labelled EVs. The investigation of these strategies shredded light into the challenges still encountered with electrokinetic preconcentration and separation of heterogeneous EVs sub-populations which are discussed herein based on our results and other attempts reported in the literature.

Sorting and simultaneous quantitation of intact mixed-cell samples via capillary isotachophoresis.

A great need currently exists for rapid, inexpensive, and accurate methods for microbial analysis in the medical, food, industrial, and water quality fields. Here, a novel capillary isotachophoresis (CITP) method is presented for the focusing, sorting, and quantitation of intact cells in mixed samples based on their electrophoretic mobility ranges. Using a series of ion spacers dissolved in the sample, this technique results in several efficient cell peaks in the electropherogram corresponding to specific cell electrophoretic mobility ranges. The concentrations of different species in mixed-cell samples are determined from the cell peak areas and the known peak response factors for the cell species using a series of linear equations. Method design and optimization are discussed, including the choice of running buffer, pH, and ion spacers. Mixed-cell samples of up to four different species were focused and quantified as a proof-of-principle of the method. When sample cell concentrations were toward the middle of the linear response range, accuracies between 1% and 11% and relative standard deviations of 1%-14% were obtained, depending on the number of cell species in the mixture. This work provides a useful basis for future studies of cell quantitation using CITP, which could be potentially applied to a variety of fields including cell growth studies, microbial contamination testing, and sterility testing.

Determination of Organic Acids in Special Beers and Beer-Based Beverages Using Capillary Isotachophoresis

Organic acids in beer affect beer quality, including organoleptic properties and colloidal stability. A large proportion of the total organic acids in beer comes from the wort, while the rest is produced or transformed by yeast metabolism. Concentrations of five organic acids were determined in selected types of beer. Malic, succinic, citric, acetic and lactic acids were quantified. The concentration of acids varies according to the type of beer. The determination was made by the capillary isotachophoresis.

Chemical analysis of selected meads produced in Poland

The aim of this study was the analysis of 25 commercially available meads obtained from three leading producers in Poland. In the course of the analyses, the concentration of nine organic acids was determined using the capillary isotachophoresis technique, and the total polyphenol content (58–699 mg/L GAE) and the antioxidant activity were expressed as FRAP (234–6422 µmol/L Fe2+) using spectrophotometric methods. We were able to indicate the acids whose main source was honey—gluconic acid (561–2287 mg/L) and formic acid (35–176 mg/L), the one that was formed during alcoholic fermentation—succinic acid (280–845 mg/L), and also those originating from the additives in the form of fruit juices, or as a result of acidification—tartaric acid (< LOD–159 mg/L), malic acid (135–1611 mg/L) or citric acid (125–4576 mg/L). Our results provide a further contribution to the general knowledge of the chemical composition of meads, and, in particular, these are the first results of this kind for meads commercially available in Poland. The analysis of principal components showed the correlation structure of the examined parameters and the existence of two clusters containing specific meads.

Electrophoretic determination of chitin in insects

An electrophoretic method (on-line coupled capillary isotachophoresis with capillary zone electrophoresis with conductometric detection (cITP-CZE-COND)) for the determination of chitin in insects based on the analysis of glucosamine after acidic hydrolysis of the sample is described. Chitin is deacetylated and hydrolyzed to glucosamine by acidic hydrolysis (6 M sulfuric acid, 110 °C, 6 h). Under optimized electrophoresis conditions, glucosamine (GlcN) is separated from other sample components in cationic mode and detected with a conductometer within 15 min. The performance method characteristics of the GlcN assay, i.e., linearity (0.2–20 μmol), accuracy (103 ± 5%), repeatability (1.9%), reproducibility (3.4%), limits of detection (0.06 μmol/L) and quantification (0.2 μmol/L), were evaluated. On a series of 28 insect samples, it was proven that cITP-CZE-COND provides results of chitin content in insects comparable to the literature data. The important features of the developed cITP-CZE-COND method are easy sample treatment, high sensitivity and selectivity, and low running costs. It is clear from the above that the cITP-CZE-COND method is suitable for analysis of insect samples for chitin content.

Developed and Validated Capillary Isotachophoresis Method for the Rapid Determining Organic Acids in Children's Saliva.

One of the current challenges facing researchers is the search for alternative biological material, as opposed to routinely and invasively collected (such as blood), as the analysis of the former would provide information about the state of human health, allowing for the diagnosis of diseases in their early stages. With the search for disease biomarkers in alternative materials, the development of newer analytical solutions has been observed. This study aims to develop a reliable analytical method using the capillary isotachophoresis technique for the determination of organic acids in children's saliva, the presence/elevation of which can be used in the future for diagnostic purposes. Organic acids such as formic, lactic, acetic, propionic, and butyric acid, were determined in the saliva of healthy children without carious lesions. The limit of quantification determined in the validation process was found to vary from 0.05 to 1.56 mg/L, the recoveries at the two levels were determined to vary between 90% and 110% for level I, while for level II the corresponding values of 75% and 106% were found; the presentation, expressed as relative standard deviation values (RSD), did not exceed 5%. The parameters determined while validating the results method indicated that the obtained are reliable. The Red-Green-Blue (RGB) additive color model was used for the evaluation of the method. This comparative analysis allowed us to define the color of the method, which expresses whether it meets the given assumptions and requirements. According to the RGB model, the isotachophoresis method developed requires less reagent input, shorter sample preparation times, and results with lower energy consumption. Thus, the subject procedure may provide an alternative, routine tool for determining organic acids in human saliva, to be applied in the diagnosing of diseases of various etiological origins.

Recent advancement and development of capillary isotachophoresis: A review

Capillary Isotachophoresis (CITP) is an advanced technique that is used for the separation of substances with electrophoretic mobility and their charges. The isotachophoresis electrolyte system is presented, along with how the effective mobilities and molar amounts of the analytes influence the separation and time required to attain the steady state for a certain electrolyte condition. One approach called CITP uses a “moving border.” Between the leading and terminating electrolytes is a sample zone where all solutes travel at the same speed in distinct bands. In this article, we have described the different and latest developments of CITP, i.e., isotachophoresis in nanoparticle studies, in the hyphenation technique, in the bioanalytical sector, and in various food tracing elements. Although traditional Isotachophoresis is becoming less popular, isotachophoresis is becoming more used for a variety of samples as a preconcentration approach for CZE analysis.

Advances in the Applications of Capillary Electrophoresis to Tobacco Analysis

Background: Capillary electrophoresis (CE) has found a wide range of applications because of its high separation efficiency, low expense, short analysis time and minimal sample volume requirement. The tobacco quality depends on the nature and quantity of numerous substances. CE has been applied in the constituent analysis of tobacco and tobacco products for quality control and tobacco research. Methods: The advances in the applications of CE to tobacco analysis are reviewed. The main subjects cover the separation modes of CE, the detection techniques of CE, sample preparations and the applications of CE in the measurements of various constituents in tobacco samples. In addition, the CE-based metabonomic investigation of tobacco is also introduced. Results: Capillary zone electrophoresis, micellar electrokinetic chromatography, capillary isotachophoresis, capillary gel electrophoresis, capillary electrochromatography and non-aqueous CE have been applied in the determination of a variety of constituents in tobacco and tobacco products. The assayed substances include alkaloids, amines, saccharides, organic acids, inorganic ions, phenols, phenolic acids, flavonoids, amino acids, peptides, proteins, hormones, agricultural chemicals, etc. Conclusion: This review demonstrates that CE is a promising analytical technique in the field of tobacco analysis. It is anticipated that CE will find more and more applications in tobacco investigations.

Variation in the Content of Bioactive Compounds in Infusions Prepared from Different Parts of Wild Polish Stinging Nettle (Urtica dioica L.).

Nettle is a common plant that offers many health benefits and is grown all over the world. The content of active compounds in roots, stems, and leaves was determined based on the extraction procedure optimized using the Central Composite Design. Flavonols, phenolic acids, trigonelline, nicotinamide, nicotinic acids, and short-chain organic acids were determined with the use of LC–MS/MS and capillary isotachophoresis. Trigonelline, which was not previously reported in the roots and stems of nettle, was found in all parts of the plant and considerable variations in its content were observed (2.8–108 µg g−1). Furthermore, the Principal Component Analysis taking into account more variables demonstrated differences in the content of bioactive components between roots and aerial parts of nettle.

Bottom-Up Analysis of Proteins by Peptide Mass Fingerprinting with tCITP-CZE-ESI-TOF MS After Tryptic Digest.

The identification of proteins in samples of moderate to complex composition is primarily done by bottom-up approaches. Therefore, proteins are enzymatically digested, mostly by trypsin, and the resulting peptides are then separated prior to their transfer to a mass spectrometer. The following protocol portrays a bottom-up method, which was optimized for the application of CZE-ESI-TOF MS. Protein denaturation is achieved by addition of 2,2,2-trifluoroethanol (TFE) and heat treatment. Afterwards, disulfide bonds are reduced with tris-(2-carboxyethyl)phosphine (TCEP) and subsequently alkylated with iodoacetamide (IAA). The tryptic digest is performed in an ammonium bicarbonate buffer at pH8.0. The digested protein sample is then concentrated in-capillary by transient capillary isotachophoresis (tCITP) with subsequent CZE separation of tryptic peptides in an acidic background electrolyte. Hyphenation to a time-of-flight (TOF) mass spectrometer is carried out by a triple-tube coaxial sheath flow interface, which uses electrospray ionization (ESI). Peptide identification is done by peptide mass fingerprinting (PMF). The protocol is outlined exemplarily for a model protein, i.e., bovine β-lactoglobulin A.

At-line coupling of hollow fiber liquid-phase microextraction to capillary electrophoresis for trace determination of acidic drugs in complex samples

Direct analysis of complex samples is demonstrated by the at-line coupling of hollow fiber liquid-phase microextraction (HF-LPME) to capillary electrophoresis (CE). The hyphenation of the preparative and the analytical technique is achieved through a 3D-printed microextraction device with an HF located in a sample vial of a commercial CE instrument. The internal geometry of the device guides the CE separation capillary into the HF and the CE injection of the HF-LPME extract is performed directly from the HF lumen. The 3D-printing process ensures uniform dimensions of the devices, their constant position inside the sample vial, and excellent repeatability of the HF-LPME as well as the CE injection. The devices are cheap (∼0.01 €) and disposable, thus eliminating any possible sample-carryover, moreover, the at-line CE analysis of the extract is performed fully autonomously with no need for operator's intervention. The developed HF-LPME/CE-UV method is applied to the determination of acidic drugs in dried blood spot and wastewater samples and is characterized by excellent repeatability (RSD, 0.6–9.6%), linearity (r2, 0.9991–0.9999), enrichment (EF, 29–97), sensitivity (LOD, 0.2–3.4 μg/L), and sample throughput (7 samples/h). A further improvement of selected characteristics of the analytical method is achieved by the at-line coupling of HF-LPME to capillary isotachophoresis (ITP) with electrospray ionization-mass spectrometry (ESI-MS). The HF-LPME/ITP-ESI-MS system facilitates enhanced selectivity, matrix-free analytical signals, and up to 34-fold better sensitivity due to the use of ESI-MS detection and additional on-capillary ITP preconcentration of the HF-LPME extracts.

Simple extraction procedure for free amino acids determination in selected gluten-free flour samples

The simple extraction of flours samples followed by free amino acids determination procedures was studied and optimised. The conditions of amino acids derivatisation reaction with ninhydrin for chromatographic determination of free amino acids sum was discussed. The developed method was processed in terms of linearity, precision, accuracy, and limits of detection and quantification. Moreover, capillary isotachophoresis and HPLC methods were applied for individual free amino acids determination. The proposed extraction procedure is simple, fast and convenient for different flours samples. Studied procedures were used for free amino acids determination in twelve gluten-free flour samples (corn, oat, soy, rice, pumpkin, millet, peanut, hemp seed, buckwheat, amaranth, pea and chickpea) and the obtained results were compared with wheat flour.

Electrophoretic determination of taurine

An electrophoretic method (on-line coupled capillary isotachophoresis and capillary zone electrophoresis) with conductometric detection for the determination of free taurine in selected food and feed is described. Taurine is converted to isethionic acid by van Slyke method. Under optimized conditions (leading electrolyte: 5 mM HCl, 10 mM glycylglycine, and 0.05% 2-hydroxyethyl cellulose solution, pH 3.2; terminating electrolyte: 10 mM citric acid; background electrolyte: 50 mM acetic acid, 20 mM glycylglycine, and 0.1% 2-hydroxyethyl cellulose solution, pH 3.7), isethionic acid is separated from other sample components in anionic mode and detected using a conductimeter within 15 minutes. The performance method characteristics, such as linearity (25 – 1250 ng/mL), accuracy (99 ± 9%), repeatability (3.9%), reproducibility (4.3%), limits of detection (3 ng/mL) and quantification (10 ng/mL) were evaluated. By analysing 20 food and pet food samples the method was proved suitable for routine analysis. High sensitivity and selectivity, short analysis time and low costs are significant features of the presented method.

On a separation voltage polarity switching transient capillary isotachophoresis method for higher sample loading capacity and better separation performance.

Limited sample loading capacity is one of the major reasons that prevents the utility of capillary electrophoresis (CE) as a routine separation method as compared to liquid chromatography (LC). In our previous study, separation voltage polarity switching transient capillary isotachophoresis (PS-tCITP) was proposed. Both sample loading capacity and separation resolution could be improved using a single PS-tCITP instead of routine transient capillary isotachophoresis (tCITP). In this study, a detailed investigation on the optimization strategy of the PS-tCITP method was performed systematically. A possible mechanism of sample preconcentration in multiple PS-tCITP was first proposed to better understand the multiple PS-tCITP process. Several optimization experiments were then performed, including single PS-tCITP, paused PS-tCITP and multiple PS-tCITP, sequentially using a mixture of five peptides. By selecting an optimum polarity switching time, sample loading capacity of 100% capillary volume could be achieved in a single PS-tCITP. Introducing an additional pause between each polarity switching in a single PS-tCITP further improved the separation resolution. Experimental results showed a baseline separation of five selected peptide standards at 100% sample loading volume using a 100 min pause in a single PS-tCITP. To further improve separation efficiency while still maintaining 100% sample loading volume, a multiple PS-tCITP technique was developed through this study. Compared to the separation performance of the optimal single PS-tCITP at 100% sample loading volume with a 10 min pause, the separation window was improved by 54% and the peak capacity was improved by 48% in the optimal four PS-tCITP with the same sample loading volume and pause.

Development of an extraction procedure and analysis of electrostatically stabilized silanates from aqueous solutions

Abstract The analyzed organosilicon derivatives of electrostatically stabilized silanates belong to a group of pentacoordinated compounds. These derivatives are: 1-(N-morpholiniomethyl)spirobi(1-sila-2,5-dioxacyclopentan-3-on)at, 1-(N-morpholiniomethyl)spirobi(1-sila-2,5-dioxa-4-methylcyclopentan-3-on)at, 1-(N-morpholiniomethyl) spirobi(1-sila-2,5-dioxa-4-(i-propyl)cyclopentan-3-on)at. In the course of the work, the extraction process and optimization of conditions for separation and determination of a mixture of electrostatically stabilized silanates were carried out using capillary isotachophoresis. Proper leading electrolytes were elaborated developed and the terminating electrolyte: 4,4’-bis[(1-morpholiniomethyl)spirobi(1-sila-2,5-dioxacyclopentan-3-on)at] was proposed. The extraction process involved the use of three stationary phases: octadecyl, octyl and phenylpropyl. The highest recovery values, approx. 94%, were obtained on the phenylpropyl column. The optimum time of analysis by the isotachophoretic technique did not exceed 12 min. The developed method of separation and determination of electrostatically stabilized silanates expands the possibility of research on biological activity of this group of compounds in aqueous solutions and surface water vegetation.

Front cover: Purification of anionic fluorescent probes through precise fraction collection with a two‐point detection system using multiple‐stacking preparative capillary transient isotachophoresis

Front cover: Purification of anionic fluorescent probes through precise fraction collection with a two‐point detection system using multiple‐stacking preparative capillary transient isotachophoresis

SIGNIFICANTLY LOWER CONTENT OF ANTINUTRITIONAL SOLUBLE OXALATE IN AMARANTH MUTANT LINES DEVELOPED BY RADIATION MUTAGENESIS

Soluble oxalate with potentially dietary injurious implications for human health were determined in amaranth (Amaranthus spp.) gamma-irradiation induced mutant lines by capillary isotachophoresis and compared to their non-irradiated reference samples 'Ficha' (Amaranthus cruentus L.) and interspecific hybrid 'K-433' (Amaranthus hypochondriacus × Amaranthus hybridus) during the cultivation period 2011–2014. The canonical discriminant analysis demonstrated significant differences among mutants and their reference counterparts during multiyear evaluation. Two-way ANOVA approach identified the mutant line D282 as variant with the significantly and long-term lowest soluble oxalate concentration in comparison to respective reference samples as well as other mutant lines. Decrease in a content of this antinutritional factor could be a possible effect of radiation-induced mutation event(s) in the D282 line genome. Therefore, this mutant line might be a good matrix in the further breeding programme of this naturally gluten-free pseudocereal.

Capillary isotachophoresis with electrospray-ionization mass-spectrometric detection: Cationic electrolyte systems in the medium-alkaline range for selective analysis of medium strong bases

This work extends the present working range of isotachophoresis (ITP) with electrospray-ionization mass-spectrometric (ESI–MS) detection and describes for the first time a functional cationic electrolyte system for analyses at medium-alkaline pH. So far no ITP–MS application was published on the analysis of medium strong bases although there is a broad spectrum of potential analytes like biogenic amines, alkaloids or drugs, where this technique promises interesting gains in both sensitivity and specificity. The presented results include a selection of suitable sufficiently volatile ESI-compatible system components, discussion of factors affecting system properties, and recommendations for functional ITP electrolyte systems. Theoretical conclusions based on calculations and computer simulations are confirmed by experiments with a model mixture of beta-blockers. Practical applicability of the method is demonstrated on the example of analysis of sotalol in dried blood spots where direct injection of aqueous extract, ITP stacking and MS detection provide a fast, simple and sensitive technique with limits of quantitation on the sub-nM level.

Determination of Free Tryptophan in Beer Samples by Capillary Isotachophoretic Method

Tryptophan is essential amino acid and precursor for many neurotramsmiters that must be obtained from dietary proteins. However, its free form is easily absorbed and could increase the availability of this amino acid to the brain. Because of free tryptophan interaction with human health simple, eco-friendliness and low-cost method of determination are still needed. In this study, new and simple procedure for free tryptophan determination using capillary isotachophoresis is discussed. The method validation pointed good linearity, satisfactory selectivity, accuracy (recoveries varied from 98.4 to 100.1%), intra- and inter-day precision (coefficent of variation was < 5% for each standard solution and < 6% for real samples) and no matrix effect. The proposed procedure was successfully applied to analyse free tryptophan in beer samples and found contents varied from not detected to 40.74 ± 0.27 mg L−1. The obtained results were compared with chromatographic determination after derivatization with 2-chloro-1,3-dinitro-5-(trifluoromethyl)benzene and pointed better selectivity and accuracy of isotachophoretic procedure with similar precision. Due to the simplicity and flexibility, the proposed procedure is suitable for tryptophan analysis in complex matrices.

Antibody-free detection of amyloid beta peptides biomarkers in cerebrospinal fluid using capillary isotachophoresis coupled with mass spectrometry

This study reports a capillary isotachophoresis (ITP) – electrospray ionization mass spectrometry (ESI-MS) method for the determination of several amyloid β (Aβ) peptides, which are biomarkers of Alzheimer’s disease (AD) in cerebrospinal fluids (CSF). For the first time, these peptides have been detected directly from CSF by MS without recourse to an immunocapture-based sample pre-treatment. The antibody-free approach is based on the marriage between capillary ITP, a powerful on-line electrokinetic preconcentration technique, and MS for simultaneous detection of different Aβ peptides. To ensure a good performance, the ITP process of fluorescently labelled Aβ peptides was for the first time implemented and verified with laser induced fluorescent detection, prior to methodology transfer to MS detection. Better detection sensitivity was achieved with labelled Aβ peptides for both detection modes. Using hydroxyl ions as the terminating and acetate as the leading ions, our method allows efficient ITP preconcentration under alkaline conditions of the slowly migrating Aβ peptides to reach quantifiable concentration down to 50 pM. The developed ITP-MS approach allows reliable quantification of different fluorescently derivatized Aβ peptides, i.e. Aβ 1–42, Aβ 1–40 and Aβ 1–38 down to sub nM ranges in CSF samples from AD and non-demented (healthy control) patients. Good agreement (<20% deviation) for the determination of Aβ 1–42/Aβ 1–40 ratio in CSF was achieved between results obtained with this new ITP-MS and our recently developed method based on large volume sample stacking coupled to CE. Discrimination of one AD patient from two healthy controls was successfully made with the Aβ 1–42/Aβ 1–40 ratio obtained by the developed ITP-MS method for the tested cerebrospinal fluid samples.