Migration Time Reproducibility Research Articles

Electrophoretic Determination of L-Carnosine in Health Supplements Using an Integrated Lab-on-a-Chip Platform with Contactless Conductivity Detection.

The health supplement industry is one of the fastest growing industries in the world, but there is a lack of suitable analytical methods for the determination of active compounds in health supplements such as peptides. The present work describes an implementation of contactless conductivity detection on microchip technology as a new strategy for the electrophoretic determination of L-carnosine in complex health supplement formulations without pre-concentration and derivatization steps. The best results were obtained in the case of +1.00 kV applied for 20 s for injection and +2.75 kV applied for 260 s for the separation step. Under the selected conditions, a linear detector response of 5 × 10-6 to 5 × 10-5 M was achieved. L-carnosine retention time was 61 s. The excellent reproducibility of both migration time and detector response confirmed the high precision of the method. The applicability of the method was demonstrated by the determination of L-carnosine in three different samples of health supplements. The recoveries ranged from 91 to 105%. Subsequent analysis of the samples by CE-UV-VIS and HPLC-DAD confirmed the accuracy of the obtained results.

Plasma levels of creatine, 2-aminobutyric acid, acetyl-carnitine and amino acids during fasting measured by counter-current electrophoresis in PAMAPTAC capillary

Determination of the minor nitrogen metabolites, creatine, 2-aminobutyric acid (2-AB), and acetyl-carnitine (Ac-carn), in human plasma is performed by capillary electrophoresis (CE) in a 6 % PAMAPTAC coated fused silica capillary. 6 % PAMAPTAC generates a stable electroosmotic flow (EOF) of 6.31 × 10−9 m2V−1s−1; and the nitrogen metabolites are separated in a counter-current mode, which increases electrophoretic resolution. Baseline separation of creatine, 2-AB, Ac-carn, and most proteinogenic amino acids is achieved in 8.5 M acetic acid (AcOH) at pH 1.37 as background electrolyte (BGE) at an effective capillary length of 14.4 cm with migration times ranging from 4.95 min for creatine to 5.43 min for Ac-carn. 20 μL of plasma is deproteinized with the addition of 60 μL of acetonitrile (ACN) and a 21.8 mm sample zone is injected into the capillary, which corresponds to 6.9 % of total and 15 % of effective capillary length. The zone is focused using a large volume sample stacking technique with LODs of 0.2–0.4 μM and LOQs of 0.8–1.3 μM relative to the untreated plasma sample. Inter-day reproducibility of migration time is 0.7–1.1 % and for peak area 2.5–3.9 %. The CE method is used to monitor plasma levels of nitrogen metabolites during 60 h of fasting in 10 healthy lean volunteers. T-test showed statistically significant differences in plasma levels of creatine, 2-AB, Ac-carn and most proteinogenic amino acids, which return to their original concentrations after subsequent 48 h recovery.

Ultra-trace determination of oxaliplatin impurities by sweeping-MEKC-ICP-MS

Oxaliplatin represents a platinum-based cancerostatic drug that is widely used for the treatment of various types of cancer. There are two main platinum-containing impurities, impurity B and C, that can be formed as side products at very low concentrations. Their effect on biological systems can be like the oxaliplatin itself; however, this has not been fully investigated since there is a lack of methods for their determination in ultralow concentrations. In our work, we present a method for ultra-trace determination of oxaliplatin impurities B and C, and oxaliplatin itself, using online sweeping preconcentration micellar electrokinetic chromatography coupled with inductively coupled plasma mass spectrometry (MEKC-ICP-MS). This is the first application of online preconcentration with MEKC-ICP-MS to improve the detection limits of analytes. Under the optimal conditions, 25 mM sodium phosphate buffer at pH 2.15 with 175 mM SDS, and an injection time of 90 s at 50 mbar, baseline separation of all components within 6 min was achieved. The sweeping-MEKC-ICP-MS method was fully validated in terms of linearity, limits of detection and quantification, trueness, precision, and reproducibility of migration times. Limits of detection of 2, 1, and 3 ng mL−1 for impurity B, impurity C, and oxaliplatin, respectively, were obtained, which are 3,500-, 1,700-, and 2,100-fold lower than those for a MEKC-UV method also developed and validated as a part of this work. It also represents the detection of 98 femtograms of the impurity C (or 227 attomol of Pt) per injection. The sweeping-MEKC-ICP-MS method further benefits from a wide dynamic range up to six orders of magnitude (0.01–1,000 µg mL−1 for oxaliplatin) with coefficients of determination greater than 0.9989. ICP-MS provides characteristic element/isotope-specific and structure-independent detection. ICP-MS helped identify the co-separated impurity B counter-ion that can be wrongly assigned as the platinum-based impurity B using standard UV detection. Finally, the validated sweeping-MEKC-ICP-MS method was applied to the analysis of oxaliplatin samples with variable impurities concentrations (0.06–1.0%). The trueness and precision ranged between 76 and 115% and 2–19%, respectively. This method allows the accurate determination of oxaliplatin impurities from 0.0003% levels; therefore, it could be used in routine pharmaceutical laboratories for quality control purposes.

Fundamentals of Capillary Electrophoretic Migration and Separation of SDS Proteins in Borate Cross-Linked Dextran Gels.

Recent progress in the development and production of new, innovative protein therapeutics require rapid and adjustable high-resolution bioseparation techniques. Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using a borate (B) cross-linked dextran (D) separation matrix is widely employed today for rapid consistency analysis of therapeutic proteins in manufacturing and release testing. Transient borate cross-linking of the semirigid dextran polymer chains leads to a high-resolution separation gel for SDS-protein complexes. To understand the migration and separation basis of the D/B gel, the present work explores various gel formulations of dextran monomer (2, 5, 7.5, and 10%) and borate cross-linker (2 and 4%) concentrations. Ferguson plots were analyzed for a mixture of protein standards with molecular weights ranging from 20 to 225 kDa, and the resulting nonlinear concave curves pointed to nonclassical sieving behavior. While the 2% D/4% B gel resulted in the fastest analysis time, the 10% D/2% B gel was found to produce the greatest separation window, even higher than with the 10% D/4% B gel, due to a significant increase in the electroosmotic flow of the former composition in the direction opposite to SDS-protein complex migration. The study then focused on SDS-CGE separation of a therapeutic monoclonal antibody and its subunits. A combination of molecular weight and shape selectivity as well as, to a lesser extent, surface charge density differences (due to glycosylation on the heavy chain) influenced migration. Greater molecular weight selectivity occurred for the higher monomer concentration gels, while improved glycoselectivity was obtained using a more dilute gel, even as low as 2% D/2% B. This latter gel took advantage of the dextran-borate-glycoprotein complexation. The study revealed that by modulating the dextran (monomer) and borate (cross-linker) concentration ratios of the sieving matrix, one can optimize the separation for specific biopharmaceutical modalities with excellent column-to-column, run-to-run, and gel-to-gel migration time reproducibilities (<0.96% relative standard deviation (RSD)). The widely used 10% dextran/4% borate gel represents a good screening option, which can then be followed by a modified composition, optimized for a specific separation as necessary.

Rapid capillary gel electrophoresis analysis of human milk oligosaccharides for food additive manufacturing in-process control

Industrial production of human milk oligosaccharides (HMOs) represents a recently growing interest since they serve as key ingredients in baby formulas and are also utilized as dietary supplements for all age groups. Despite their short oligosaccharide chain lengths, HMO analysis is challenging due to extensive positional and linkage variations. Capillary gel electrophoresis primarily separates analyte molecules based on their hydrodynamic volume to charge ratios, thus, offers excellent resolution for most of such otherwise difficult-to-separate isomers. In this work, two commercially available gel compositions were evaluated on the analysis of a mixture of ten synthetic HMOs. The relevant respective separation matrices were then applied to selected analytical in-process control examples. The conventionally used carbohydrate separation matrix was applied for the in-process analysis of bacteria-mediated production of 3-fucosyllactose, lacto-N-tetraose, and lacto-N-neotetraose. The other example showed the suitability of the method for the in vivo in-process control of a shake flask and fermentation approach of 2′-fucosyllactose production. In this latter instance, borate complexation was utilized to efficiently separate the 2′- and 3-fucosylated lactose positional isomers. In all instances, the analysis of the HMOs of interest required only a couple of minutes with high resolution and excellent migration time and peak area reproducibility (average RSD 0.26% and 3.56%, respectively), features representing high importance in food additive manufacturing in-process control.Graphical abstract

A CE-ICP-MS/MS method for the determination of superparamagnetic iron oxide nanoparticles under simulated physiological conditions

Over the past few years, superparamagnetic iron oxide nanoparticles (SPIONs) have attracted much attention due to their medicinally attractive properties and their possible application in cancer diagnosis and therapy. However, there is still a lack of appropriate methods to enable quantitative monitoring of the particle changes in a physiological environment, which could be beneficial for evaluating their in vitro and in vivo behavior. For this reason, the main goal of this study was the development of a novel capillary electrophoresis-inductively coupled plasma mass spectrometry (CE-ICP-MS/MS) method for the determination of SPIONs suitable for the future examination of their changes upon incubation with proteins under simulated physiological conditions. The type and flow rate of the collision/reaction gas were chosen with the aim of simultaneous monitoring of Fe and S. The type and concentration of the background electrolyte, applied voltage, and sample loading were optimized to obtain SPION signals of the highest intensity and minimum half-width of the peak. Analytical parameters were at a satisfactory level: reproducibility (intra- and inter-day) of migration times and peak areas (presented as RSD) in the range of 0.23–4.98%, recovery: 96.7% and 93.3%, the limit of detection (for monitoring 56Fe16O+ by mass-shift approach) 54 ng mL−1 Fe (0.97 μM) and 101 ng mL−1 Fe (1.82 μM) for SPIONs with carboxyl and amino terminal groups, respectively. To the best of our knowledge, this is the first reported use of CE-ICP-MS/MS for the quantification of SPIONs and monitoring of interactions with proteins.

Consecutive and automatic detection of multi-gene mutations from colorectal cancer samples by coupling droplet array-based capillary electrophoresis and PCR-RFLP.

The efficacy of targeted therapy is associated with multi-gene mutation status. Carrying out effective multi-genotyping analysis in combination has been a challenge in clinical settings. We therefore developed a droplet-based capillary electrophoresis (CE) system coupled with PCR-restriction fragment length polymorphism (PCR-RFLP) technology to detect multi-gene mutations from a small volume of samples. A 16 × 16 200-nL droplet array for sample encapsulation was constructed on a glass chip. The electrophoresis system consisted of a tapered vertical capillary filled with polyvinylpyrrolidone, a laser-induced fluorescence detector, and a high voltage power supply. Notably, a droplet-based electrokinetic sample introduction method and a "∩" shape capillary were developed to facilitate consecutive droplet sampling using a home-made automatic control module. The DL2000 DNA marker was consecutively separated, achieving high migration time and plate number reproducibility. The system was further applied to detect PCR-RFLP products. For colorectal cancer (CRC) cell lines, KRAS, BRAF, and PIK3CA were genotyped with a sensitivity of 0.25%. For CRC patient specimens, 30 samples were consecutively and automatically multi-genotyped without inter-sample contamination, with a lowest mutation frequency of 0.37%. For the first time, we developed a droplet-based CE system for consecutive DNA analysis with low sample consumption. This automated CE system could be further developed to integrate the full process of gene mutation detection, serving as a more effective platform for individualised therapy.

Pyrolyzed cotton balls for protein removal: Analysis of pharmaceuticals in serum by capillary electrophoresis

Because of the inherent affinity of proteins for bare, fused silica capillaries, the analysis of protein-containing samples has proven a challenging task for capillary electrophoresis. The adsorption of proteins to the capillary walls effectively changes the zeta potential and thus affects the electro-osmotic flow leading to significant shifts in migration time, peak broadening, and poor reproducibility. While there are several well-known methods to remove proteins from samples prior to the analysis (including precipitation) or to prevent their adsorption to the capillary (semi-permanent coatings), those approaches are often expensive, time consuming, or simply unreliable. Aiming to address these needs, this manuscript reports on the use of pyrolyzed cotton balls, as a simple and widely accessible hydrophobic material to remove proteins from serum samples. The material retains enough flexibility so it can be placed directly into the sample vials and has enough capacity to capture more than 75% of the proteins in the sample (1% dilution of 1 mL of serum). The advantages of the material are demonstrated by performing the analysis of five representative drugs (in serum) by capillary electrophoresis obtaining a change in migration time of only 5 ± 1%, after 10 consecutive runs.

Approach for Profiling of Glycosphingolipid Glycosylation by Multiplexed Capillary Gel Electrophoresis Coupled to Laser-Induced Fluorescence Detection To Identify Cell-Surface Markers of Human Pluripotent Stem Cells and Derived Cardiomyocytes.

Application of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as tissue transplants in regenerative medicine depends on cell-surface marker-based characterization and/or purification. Glycosphingolipids (GSLs) are a family of highly diverse surface-exposed biomolecules that have been neglected as potential surface markers for hiPSC-CMs due to significant analytical challenges. Here, we describe the development of a novel and high-throughput-compatible workflow for the analysis of GSL-derived glycans based on ceramide glycanase digestion, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) labeling, and multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection (xCGE-LIF). GSL glycans were detected with highly reproducible migration times after repeated analysis by xCGE-LIF. We built up a migration time database comprising 38 different glycan species, and we showed exemplarily that as few as 10 pg of fucosyl lactotetra was detectable. GSL glycan profiling could be performed with 105 human induced pluripotent stem cells, and we quantitatively dissected global alterations of GSL glycosylation of human induced pluripotent stem cells (hiPSCs) and hiPSC-CMs by employing xCGE-LIF. In our study, we observed a general switch from complex GSLs with lacto- and globo-series core structures comprising the well-known human pluripotent stem cell marker stage-specific embryonic antigen 3 (SSEA3) and SSEA4 in hiPSCs toward the simple gangliosides GM3 and GD3 in hiPSC-CMs. This is the first description of GM3 and GD3 being highly abundant GSLs on the cell surface of stem cell-derived cardiomyocytes.

Chiral ligand exchange capillary electrochromatography with dual ligands for enantioseparation of D,L-amino acids

Utilizing block copolymers as coatings, a protocol of chiral ligand exchange capillary electrochromatography (CLE-CEC) protocol was designed and developed with dual ligands for D,L-amino acids enantioseparation. Four block copolymers including poly maleic anhydride-co-styrene-co-N-methacryloyl-L-histidine methyl ester [P(MAn-St-MAH)], poly maleic anhydride-co-styrene-co-N-methacryloyl-L-lysine methyl ester [P(MAn-St-MAL)], poly maleic anhydride-co-styrene-co-N-methacryloyl-L-phenylalanine methyl ester [P(MAn-St-MAP)] and poly maleic anhydride-co-styrene-co-N-methacryloyl-L-threonine methyl ester [P(MAn-St-MAT)] were synthesized by reversible addition fragmentation chain transfer polymerization reaction. Key factors affecting the enantioresolution were optimized, including the concentration of Zn (II) central ion, pH value of buffer solution and monomers of the block copolymers. The enantioresolution of the proposed CLE-CEC system could be enhanced dramatically by employing P(MAn-St-MAH) as the immobilized chiral ligand and by coordinating the synergistic effect of free ligand in buffer solution. The principle of improved enantioresolution of the CLE-CEC system with dual ligands was discussed. Well enantioseparation was successfully realized with 7 pairs of D,L-amino acids enantiomers baseline separation and 5 pairs part separation. For quantitative analysis of D,L-alanine, a good linearity was established in the range of 9.4 μM to 1.5 mM (r2 = 0.997) with the limits of detection (LODs) 3.7 μM of D-alanine, 2.0 μM for L-alanine, and limits of quantification (LOQs) 9.0 μM for D-alanine and 6.0 μM for L-alanine. The peak area and migration time reproducibility (n = 6) were 4.1% and 3.5% for D-alanine, 3.7% and 3.1% for L-alanine. Further, the enzyme kinetics study of alanine aminotransferase was investigated with the constructed CLE-CEC system.

SIMULTANEOUS DETERMINATION OF BENZOIC ACID AND SORBIC ACID IN FOOD PRODUCTS BY CAPILLARY ELECTROPHORESIS

In this work, a rapid and easy capillary electrophoretic method for simultaneous determination of benzoic acid and sorbic acid in food samples using direct UV detection was reported. The separation of acids was achieved in fused silica capillary at 28 kV. 20 mM borate buffer at pH 9.3 was used as running buffer; cinnamic acid was chosen as internal standard for quantification. Under the optimal separation conditions benzoic acid and sorbic acid were detected in 3.1 min. The linear ranges were between 0.005-0.4 mM. The correlation coefficients for each calibration curve were calculated as 0.999. The reproducibility of peak area and migration time for each acid were less than 3% (R.S.D.). The limits of detection were found 0.405 m g/mL for benzoic acid and 0.415 m g/mL for sorbic acid. The limits of quantification were 1.35 μg/mL and 1.38 μg/mL respectively.

Optimization and Validation of a New Capillary Electrophoresis Method with Conductivity Detection for Determination of Small Anions in Red Wines

A capillary electrophoresis (CE) method has been developed and validated for determination of organic acids (oxalate, tartrate, malate, malonate, pyruvate, succinate, acetate, citrate, and lactate) and inorganic anions (sulfate and phosphate) in red wines. The separations were carried out in an automated separation system equipped with wide-bore (300 μm i.d.) fluoroplastic capillary and contact conductivity detector used for monitoring the separation and quantification of the analytes. The fast method (analysis time less than 5 min.) provided a good linearity of calibration curves (R 2 > 0.9920) for the studied acids, as well as a good reproducibility of migration times (RSD < 1.5%). In total, 17 red wines were analyzed with the proposed method, including Vranec, Cabernet Sauvignon, and Merlot wines from various geographic areas (Demir Kapija, Kavadarci, Negotino, and Veles) in Macedonia. The used fully automated separation system (sample dilution not included) predetermined the developed CE method for routine analysis.

Surfactant-free microemulsion electrokinetic chromatography (SF-MEEKC) with UV and MS detection - a novel approach for the separation and ESI-MS detection of neutral compounds.

Microemulsion electrokinetic chromatography (MEEKC) is a powerful tool to separate neutral species based on differences in their hydrophobic and hydrophilic properties. However, as a major drawback the conventionally used SDS based microemulsions are not compatible with electrospray ionization mass spectrometry (ESI-MS). In this work, a surfactant-free microemulsion (SFME) consisting of water, ethanol, and 1-octanol is used for surfactant-free microemulsion electrokinetic chromatography (SF-MEEKC). Ammonium acetate was added to the SFME enabling electrophoretic separations. The stability of SFMEs containing ammonium acetate was investigated using small-angle X-ray scattering and dynamic light scattering. A method for the separation of a model system of hydrophobic and hydrophilic neutral vitamins, namely the vitamins B2 and D3, and the cationic vitamin B1 was developed using UV/VIS detection. The influence of the ammonium acetate concentration on the separation performance was studied in detail. The method was characterized concerning reproducibility of migration times and peak areas and concerning the linearity of the calibration data. Furthermore, SF-MEEKC was coupled to ESI-MS investigating the compatibility between SFMEs and the ESI process. The signal intensities of ESI-MS measurements of the model analytes were comparable for SFMEs and aqueous systems. Finally, the vitamin D3 content of a drug treating vitamin D3 deficiency was determined by SF-MEEKC coupled to ESI-MS using 25-hydroxycholecalciferol as an internal standard. Graphical abstract The concept of surfactant-free microemulsion electrokinetic chromatography coupled to electrospray ionization mass spectrometry.

Separation of small organic molecules using covalent organic frameworks-LZU1 as stationary phase by open-tubular capillary electrochromatography

Covalent organic frameworks (COFs) have attracted much attention because of their permanent nanoscale porosity and higher surface area compared to zeolites as well as robustness. COFs have great potential in several fields such as hydrogen storage, gas separation, and catalysis. However, COFs have not yet been applied in capillary electrochromatography. Herein, covalent organic frameworks-LZU1 (COF-LZU1) was used as the stationary phase in open-tubular capillary electrochromatography for the first time. Compared to the monoliths used in electrochromatography, the preparation technique of a COF-LZU1-coated capillary was simple and practical. The baseline separation of model analytes including alkylbenzenes, polyaromatic hydrocarbons, and anilines by the COF-LZU1-coated capillary was achieved based on the size selectivity of COF-LZU1 porous structure and hydrophobic interactions between the model analytes and organic ligands of COF-LZU1. The load capacity of the COF-LZU1-coated capillary for naphthalene was 0.6mg/mL. For three consecutive runs, the intraday relative standard deviations (RSDs) were 1.4–2.6% for the migration time and 2.7–8.7% for the peak area. The interday RSDs were 1.3–3.9% for the migration time and 3.7–9.7% for the peak area. The column-to-column reproducibility of migration time was in the range 1.0–3.9%. Moreover, the coated capillary was used for >300 runs with no changes in the separation efficiency. Thus, COFs have great potential in capillary electrochromatography and may provide a new method for chromatographic separation.

Microchip electrophoresis with electrochemical detection for the determination of analytes in the dopamine metabolic pathway.

A method for the separation and detection of analytes in the dopamine metabolic pathway was developed using microchip electrophoresis with electrochemical detection. The microchip consisted of a 5 cm PDMS separation channel in a simple-t configuration. Analytes in the dopamine metabolic pathway were separated using a background electrolyte composed of 15 mM phosphate at pH 7.4, 15 mM SDS, and 2.5 mM boric acid. Two different microchip substrates using different electrode materials were compared for the analysis: a PDMS/PDMS device with a carbon fiber electrode and a PDMS/glass hybrid device with a pyrolyzed photoresist film carbon electrode. While the PDMS/PDMS device generated high separation efficiencies and good resolution, more reproducible migration times were obtained with the PDMS/glass hybrid device, making it a better choice for biological applications. Lastly, the optimized method was used to monitor l-DOPA metabolism in a rat brain slice.

New pressure-assisted sweeping on-line preconcentration for highly polar environmentally relevant nitrosamines: Part 2. Cationic and anionic surfactants with zero-flow capillaries.

We recently introduced a pressure-assisted sweeping-reversed migration-EKC (RM-EKC) method for preconcentration of neutral polar N-nitrosamines with low affinity for the micellar phase. The type of surfactant and phase ratio are dominant factors in dictating the magnitude of interactions between analyte and micellar phase, thus four surfactants (anionic and cationic) with a range of functionalities (SDS, ammonium perfluorooctanoate (APFO), bile salts, and cetyltrimethylammonium chloride (CTAC)) were evaluated for sweeping-RM-EKC of highly polar N-nitrosamines. All gave acceptable results for sweeping-RM-EKC when used in high concentrations (≥200 mM) with low EOF. While no single surfactant was superior by all measures, all but the bile salts had useful performance characteristics. APFO showed the narrowest peak widths and highest number of theoretical plates, though two species co-migrated at all concentrations (25-300 mM); SDS and the cationic surfactant CTAC also showed good separation characteristics and could resolve all peaks, but CTAC had wider separation window. Various types of capillaries coated for EOF control were compared for use with anionic and cationic surfactants. A commercial zero-EOF capillary coated with a polymer bearing sulfonic acid functional groups showed superior EOF suppression and reproducibility of migration time with all surfactants.

Study of immobilized metal affinity chromatography sorbents for the analysis of peptides by on-line solid-phase extraction capillary electrophoresis-mass spectrometry.

Several commercial immobilized metal affinity chromatography sorbents were evaluated in this study for the analysis of two small peptide fragments of the amyloid β-protein (Aβ) (Aβ(1-15) and Aβ(10-20) peptides) by on-line immobilized metal affinity SPE-CE (IMA-SPE-CE). The performance of a nickel metal ion (Ni(II)) sorbent based on nitrilotriacetic acid as a chelating agent was significantly better than two copper metal ion (Cu(II)) sorbents based on iminodiacetic acid. A BGE of 25 mM phosphate (pH 7.4) and an eluent of 50 mM imidazole (in BGE) yielded a 25-fold and 5-fold decrease in the LODs by IMA-SPE-CE-UV for Aβ(1-15) and Aβ(10-20) peptides (0.1 and 0.5 μg/mL, respectively) with regard to CE-UV (2.5 μg/mL for both peptides). The phosphate BGE was also used in IMA-SPE-CE-MS, but the eluent needed to be substituted by a 0.5% HAc v/v solution. Under optimum preconcentration and detection conditions, reproducibility of peak areas and migration times was acceptable (23.2 and 12.0%RSD, respectively). The method was more sensitive for Aβ(10-20) peptide, which could be detected until 0.25 μg/mL. Linearity for Aβ(10-20) peptide was good in a narrow concentration range (0.25-2.5 μg/mL, R(2) = 0.93). Lastly, the potential of the optimized Ni(II)-IMA-SPE-CE-MS method for the analysis of amyloid peptides in biological fluids was evaluated by analyzing spiked plasma and serum samples.

Capillary electrophoresis to determine entrapment efficiency of a nanostructured lipid carrier loaded with piroxicam

A simple and fast capillary electrophoresis method has been developed to determine the amount of piroxicam loaded in a drug delivery system based on nanostructured lipid carriers (NLCs). The entrapment efficiency of the nanostructured lipid carrier was estimated by measuring the concentration of drug not entrapped in a suspension of NLC. The influence of different parameters on migration times, peak symmetry, efficiency and resolution was studied; these parameters included the pH of the electrophoretic buffer solution and the applied voltage. The piroxicam peak was obtained with a satisfactory resolution. The separation was carried out using a running buffer composed of 50mM ammonium acetate and 13.75mM ammonia at pH 9. The optimal voltage was 20kV and the cartridge temperature was 20°C. The corresponding calibration curve was linear over the range of 2.7–5.4µg/mL of NLC suspension. The reproducibility of migration time and peak area were investigated, and the obtained RSD% values (n=5) were 0.99 and 2.13, respectively.

Large‐volume sample stacking with polarity switching for monitoring of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) reactions by capillary electrophoresis

Nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is a membrane glycoprotein involved in the hydrolysis of extracellular nucleotides. Its main substrate is ATP yielding AMP and pyrophosphate. NPP1 has been proposed as a novel drug target, for diabetes type 2 and the treatment of calcium pyrophosphate dihydrate deposition disease leading to inflammatory arthritis. The monitoring of NPP1 reactions is difficult because its velocity is very slow requiring highly sensitive analytical procedures. In this study, a method of large-volume sample stacking with polarity switching was developed, and separations were optimized. Large sample volumes were loaded by hydrodynamic injection (5 psi, 13 s) followed by removal of a large plug of sample matrix from the capillary using polarity switching (-10 kV). The stacked analytes were subsequently separated in phosphate buffer (100mM, pH 9.2) at 20 kV. The validated method was found to be linear (R(2) = 0.9927) in the concentration range of 0.05-50μM of AMP, with high accuracy and precision. The determined LOD and LOQ of AMP were 18 nM and 60 nM, respectively. Compared to a previously reported CE procedure using sweeping technique, a fivefold improvement of sensitivity was achieved. Moreover, the new technique was faster, and reproducibility of migration times was improved (RSD value = 1.2%). Importantly, adenine nucleotide analogs and derivatives tested as NPP1 inhibitors could be completely separated from the substrate ATP and the enzymatic product AMP. The method was applied to NPP1 inhibition assays investigating nucleotide-derived inhibitors in the presence of ATP.

Preparation of poly(methyl acrylate) microfluidic chips with surface-modified by hyperbranched polyamide ester and their application in the separation of biomolecules

The surface of poly (methyl acrylate) (PMMA) microfluidic chips were modified using hyperbranched polyamide ester via chemical bonding. The contact angles of the modified chips were measured. The surface morphology was observed by scanning electron microscope (SEM) and stereo microscope. The results showed that the surface of the modified chips was coated by a dense, uniform, continuous, hydrophilic layer of hyperbranched polyamide ester. The hydrophilic of the chip surface was markedly improved. The contact angle of the chips modified decreased from 89.9 degrees to 29.5 degrees. The electro osmotic flow (EOF) in the modified microchannel was lower than that in the unmodified microchannel. Adenosine and L-lysine were detected and separated via the modified PMMA microfluidic chips. Compared with unmodified chips, the modified chips successfully separated the two biomolecules. The detection peaks were clear and sharp. The separation efficiencies of adenosine and L-lysine were 8.44 x 10(4) plates/m and 9.82 x 10(4) plates/m respectively, and the resolutions (Rs) was 5.31. The column efficiencies and resolutions of the modified chips were much higher than those of the unmodified chips. It was also observed that the modified chips possessed good reproducibility of migration time. This research may provide a new and effective method to improve the hydrophilicity of the PMMA surface and the application of PMMA microfluidic chips in the determination of trace biomolecules.