[Highly sensitive detection of fluorescent whitening agents in flour using sheathless capillary electrophoresis-electrospray ionization-tandem mass spectrometry].

Fluorescent whitening agents (FWAs) are dyes that emit visible blue or blue-purple fluorescence upon ultraviolet-light absorption. Taking advantage of light complementarity, FWAs can compensate for the yellow color of many substances to achieve a whitening effect; thus, they are used extensively in various applications. FWAs are generally stable, but their presence in the environment can lead to pollution and accumulation in the body through the food chain. Recent studies have revealed that some types of FWAs, such as coumarin-based FWAs, may exhibit photo-induced mutagenic effects that can trigger allergic reactions in humans and even pose carcinogenic risks. Hence, the development of an accurate and highly sensitive method for detecting FWAs in food-related samples is a crucial endeavor. Owing to the high polarity and structural similarity of FWAs, the accurate determination of these substances in complex food samples requires an analytical method that offers both efficient separation and sensitive detection. Capillary electrophoresis (CE) exhibits essential features such as high separation efficiency, short analysis times, very small sample injection requirements, minimal use of organic solvents, and simple operation. Thus, it is often used as an effective alternative to liquid chromatographic techniques. Over the past few decades, electrospray ionization mass spectrometry (ESI-MS) has been utilized as a highly sensitive and accurate detection method in numerous chemical analytical fields because it enables the analysis of molecular structures. By combining the high separation efficiency of CE with the high sensitivity of ESI-MS, a powerful tool for identifying and quantifying trace amounts of FWAs in food samples may be obtained. In this study, we present a method based on sheathless CE coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS) for the simultaneous detection of six trace FWAs in flour. In the proposed method, the CE separation device is directly coupled to the mass spectrometer through a sheathless interface without the need for a sheath liquid for electric contact, thereby avoiding the dilution of the analytes and improving detection sensitivity. Various conditions that could affect extraction recovery, separation efficiency, and detection sensitivity were evaluated and optimized. The FWAs were effectively extracted from the sample matrix with reduced matrix effects by ultrasonic-assisted extraction at a temperature of 30 ℃ for 20 min using CHCl3-MeOH (3∶2, v/v) as the extraction solvent. The extract was centrifuged, dried under N2, and reconstituted in CHCl3-MeOH (1∶4, v/v) for subsequent analysis. During the detection process, the CE device was coupled to the ESI-MS/MS instrument via a highly sensitive porous spray needle, which served as the sheathless electrospray interface. The target FWAs were scanned in positive-ion mode (ESI+) to ensure the stability and intensity of the obtained signals. Additionally, multiple-reaction monitoring (MRM) mode and MS/MS analysis were used to simultaneously quantify the six targets with high selectivity. The developed sheathless CE-ESI-MS/MS method detected the FWAs with high sensitivity over wide linear ranges with low method limits of detection (0.04-0.67 ng/g). The recoveries of the six target FWAs at three spiked levels were between 77.5% and 97.2%, with good interday (RSD≤11.5%) and intraday (RSD≤10.2%) precision. Analyses of the six target FWAs in eight commercial flour samples were performed using this method, and four positive samples were identified. These results demonstrate that the proposed CE-ESI-MS/MS method is a promising strategy for the determination of trace FWAs in complex food sample matrices with efficient separation and high sensitivity.

Graves' Disease: Acquired Cause of a Moderate Increase in Hemoglobin A2 Level.

BACKGROUND: Hyperthyroidism can lead to diverse hematological disorders, such as microcytosis and a mild increase in hemoglobin A2 fraction. METHODS: This study reported a 31-year-old woman of Moroccan origin recently diagnosed with Graves' disease. Her blood tests revealed microcytosis, hypochromia, and a normal ferritin level. A phenotypic analysis of hemo-globin was performed using two techniques: capillary electrophoresis and reversed-phase high performance liquid chromatography. RESULTS: Both techniques indicated a slight increase in hemoglobin A2 level. These results initially suggested het-erozygous beta-thalassemia, eventually correlating with the concurrent presence of Graves' disease, as evidenced by the normalization of hemoglobin A2 level following treatment. CONCLUSIONS: This case highlights the importance of having clinical, biological, and therapeutic data for a relevant interpretation of a phenotypic hemoglobin study.

DNA Sizing with pg/µL Sensitivity for Cell-Free DNA Analysis with the µLAS Technology.

The µLAS technology enables in-line DNA concentration and separation in a microchannel. Here, we describe its operation to analyze the size profile of cell-free DNA (cfDNA) extracted from blood plasma. Operated on commercial systems for capillary electrophoresis, we provide the size distribution of healthy individuals or patients using an input of 10 µL.

Droplet Microfluidics with Capillary Electrophoresis for Glycan Biomarker Analysis.

Several glycoproteins are validated biomarkers of various diseases such as cancer, cardiovascular diseases, chronic alcohol abuse, or congenital disorders of glycosylation (CDG). In particular, CDG represent a group of more than 150 inherited diseases with varied symptoms affecting multiple organs. The distribution of glycans from target glycoprotein(s) can be used to extract information to help the diagnosis and possibly differentiate subtypes of CDG. Indeed, depending on the glycans and the proteins to which they are attached, glycans can play a very broad range of roles in both physical and biological properties of glycoproteins. For glycans in general, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) has become a staple. Analysis of glycans with CE-LIF requires several sample preparation steps, including release of glycans from the target glycoprotein, fluorescent labeling of glycans, and purification of labeled glycans. Here, we describe the protocol for glycan sample treatment in a microfluidic droplet system prior to CE-LIF of labeled glycans. The microfluidic droplet approach offers full automation, sample, and reagent volume reduction and elimination of contamination from external environment.

Native N-glycome profiling of single cells and ng-level blood isolates using label-free capillary electrophoresis-mass spectrometry.

The development of reliable single-cell dispensers and substantial sensitivity improvement in mass spectrometry made proteomic profiling of individual cells achievable. Yet, there are no established methods for single-cell glycome analysis due to the inability to amplify glycans and sample losses associated with sample processing and glycan labeling. In this work, we present an integrated platform coupling online in-capillary sample processing with high-sensitivity label-free capillary electrophoresis-mass spectrometry for N-glycan profiling of single mammalian cells. Direct and unbiased quantitative characterization of single-cell surface N-glycomes are demonstrated for HeLa and U87 cells, with the detection of up to 100 N-glycans per single cell. Interestingly, N-glycome alterations are unequivocally detected at the single-cell level in HeLa and U87 cells stimulated with lipopolysaccharide. The developed workflow is also applied to the profiling of ng-level amounts (5-500 ng) of blood-derived protein, extracellular vesicle, and total plasma isolates, resulting in over 170, 220, and 370 quantitated N-glycans, respectively.

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.

BACKGROUND: Capillary 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. RESULTS: An 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 1000 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. SIGNIFICANCE: Sodium 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.

Automated analyses of dried blood spots collected by volumetric microsampling devices.

BACKGROUND: Dried blood spot (DBS) sampling on cellulose cards suffers from varying blood haematocrit levels and from chromatographic effects, which have a direct impact on quantitative DBS analyses. Commercial volumetric microsampling devices were, therefore, introduced to mitigate these effects, however, these devices are not compatible with automated DBS processing systems and must be processed manually. RESULTS: Capillary electrophoresis (CE) instruments use fused-silica (FS) capillaries for precise and accurate liquid handling as well as for injection, separation, and quantitative analyses of liquid samples. These inherent features of an Agilent 7100 CE instrument were employed for the automated processing (elution and homogenization) of DBSs collected by hemaPEN® volumetric devices (2.74 µL of capillary blood per spot). The hemaPEN® samples were processed directly in CE vials by consecutive transfers of 56 µL of methanol and 14 µL of deionized water through the FS capillary in a sequence of 39 DBSs with repeatability of the liquid transfers better than 1.4 %. The resulting DBS eluates were homogenized by a quick air flush through the capillary and analyzed by the same capillary and CE instrument. Creatinine was selected as a clinically relevant model analyte and its endogenous concentrations in DBSs were determined by CE with capacitively coupled contactless conductivity detection (CE-C4D) in a background electrolyte solution consisting of 50 mM acetic acid and 0.1 % (v/v) Tween 20 (pH 3.0). The overall repeatability of the automated DBS processing and CE-C4D analyses of 39 DBSs was ≤7.1 % (peak areas) and ≤0.6 % (migration times), the calibration curve was linear in the 25-500 µM range (R2 = 0.9993) and covered all endogenous blood creatinine levels, the limit of detection was 5.0 µM, and sample throughput was >12 DBSs per hour. DBS ageing for 60 days and varying blood haematocrit levels (20-70 %) did not affect creatinine quantitative results (≤6.9 % for peak areas). Inter-capillary and inter-instrument repeatability was ≤7.7 % (peak areas) and ≤3.4 % (migration times) and demonstrated an excellent transferability of the proposed analytical concept among laboratories. SIGNIFICANCE AND NOVELTY: This contribution is the first-ever report on the use of a single off-the-shelf analytical instrument for fully automated analyses of DBSs collected by commercial volumetric microsampling devices and holds great promise for future unmanned quantitative DBS analyses.

Controllable concentric electric field line distribution for simultaneous separation of DNA.

An approach for the controllable separation and concentration of nucleic acid using a circular nonuniform electric field was proposed and developed. Using six different lengths of DNA molecules as standard samples, the distribution of the gradient electric field was increased from the outer circular electrode to the inner rod-shaped electrode, contributing to the migration of DNA molecules at a velocity gradient towards the region with the strongest inner electric field. The DNA molecules were arranged in a distribution of concentric circles that aligned with the distribution of concentric equipotential lines. The concentration of DNA multiplied with the alternation of radius. As a result, this platform allowed simultaneous DNA separation, achieving a resolution range of 1.17-3.03 through an extended electrophoresis time, resulting in enhanced concentration factors of 1.08-6.27. Moreover, the manipulation of the relative height of the inner and outer electrodes enabled precise control over the distribution and the deflection degree of electric field lines, leading to accurate control over DNA deflection.

[Annual review of capillary electrophoresis technology in 2023].

This paper serves as an annual review of capillary electrophoresis (CE) technology for 2023. The journals were selected based on their impact factor (IF), a universally recognized academic performance metric, combined with experimental work closely related to CE technology, to facilitate the rapid acquisition of significant research and application advancements in CE technology in 2023. A thematic search of the ISI Web of Science database yielded 669 research papers on CE technology published in 2023. This review highlights five experimental papers published in journals with IFs greater than 10.0, including Nature Communications, Nucleic Acids Research, Engineering, Journal of Medical Virology, and Carbohydrate Polymers, and 31 experimental papers from representative journals with IFs between 5.0 and 10.0, such as Analytical Chemistry, Analytica Chimica Acta, Talanta, and Food Chemistry. It also provides an overview of experimental research in journals with focused reporting on CE technology but with IFs less than 5.0, such as Journal of Chromatography A and Electrophoresis, as well as significant experimental research from key domestic Chinese core journals (Peking University). In 2023, all the latest scientific advancements reported in journals with an IF greater than 10.0 utilized previously reported CE methods, offering new breakthroughs for the promotion and application of CE technology. Additionally, new applications of CE in conjunction with mass spectrometry remained a hot topic. An increase in reports on the hardware aspects of CE, such as 3D printing and underwater systems, and significant breakthroughs in the analysis of non-solution samples, such as solid particles, cell vesicles, cells, viruses, and bacteria, was noted. CE is advantageous for the analysis of drugs and their components. In Chinese journals, the number of papers on CE applications exceeded that in previous years, with particular focus on the field of printing for new applications.

Top-Down Proteomics Analysis of Picogram-Level Complex Samples Using Spray-Capillary-Based Capillary Electrophoresis-Mass Spectrometry.

Proteomics analysis of mass-limited samples has become increasingly important for understanding biological systems in physiologically relevant contexts such as patient samples, multicellular organoids, spheroids, and single cells. However, relatively low sensitivity in top-down proteomics methods makes their application to mass-limited samples challenging. Capillary electrophoresis (CE) has emerged as an ideal separation method for mass-limited samples due to its high separation resolution, ultralow detection limit, and minimal sample volume requirements. Recently, we developed "spray-capillary", an electrospray ionization (ESI)-assisted device, that is capable of quantitative ultralow-volume sampling (e.g., pL-nL level). Here, we developed a spray-capillary-CE-MS platform for ultrasensitive top-down proteomics analysis of intact proteins in mass-limited complex biological samples. Specifically, to improve the sensitivity of the spray-capillary platform, we incorporated a polyethylenimine (PEI)-coated capillary and optimized the spray-capillary inner diameter. Under optimized conditions, we successfully detected over 200 proteoforms from 50 pg of E. coli lysate. To our knowledge, the spray-capillary CE-MS platform developed here represents one of the most sensitive detection methods for top-down proteomics. Furthermore, in a proof-of-principle experiment, we detected 261 ± 65 and 174 ± 45 intact proteoforms from fewer than 50 HeLa and OVCAR-8 cells, respectively, by coupling nanodroplet-based sample preparation with our optimized CE-MS platform. Overall, our results demonstrate the capability of the modified spray-capillary CE-MS platform to perform top-down proteomics analysis on picogram amounts of samples. This advancement presents the possibility of meaningful top-down proteomics analysis of mass-limited samples down to the level of single mammalian cells.

Automated Capillary Electrophoresis Immunoblot for the Detection of Alpha-Synuclein in Mouse Tissue.

Background: Alpha-synuclein (aSyn) is a key player in neurodegenerative diseases such as Parkinson's disease (PD), dementia with Lewy bodies, or multiple system atrophy. aSyn is expressed throughout the brain, and can also be detected in various peripheral tissues. In fact, initial symptoms of PD are non-motoric and include autonomic dysfunction, suggesting that the periphery might play an important role in early development of the disease. aSyn is expressed at relatively low levels in non-central tissues, which brings challenges for its detection and quantification in different tissues. Objective: Our goal was to assess the sensitivity of aSyn detection in central and peripheral mouse tissues through capillary electrophoresis (CE) immunoblot, considering the traditional SDS-PAGE immunoblot as the current standard. Methods: Tissues from central and non-central origin from wild type mice were extracted, and included midbrain, inner ear, and esophagus/stomach. aSyn detection was assessed through immunoblotting using Simple Western size-based CE and SDS-PAGE. Results: CE immunoblots show a consistent detection of aSyn in central and peripheral tissues. Through SDS-PAGE, immunoblots revealed a reliable signal corresponding to aSyn, particularly following membrane fixation. Conclusion: Our results suggest a reliable detection of aSyn in central and peripheral tissues using the CE Simple Western immunoblot system. These observations can serve as preliminary datasets when aiming to formally compare CE with SDS-PAGE, as well as for further characterization of aSyn using this technique.

[Deep eutectic solvents synergistic with carboxymethyl -ß-cyclodextrin on the improvement of chiral separation of metoprolol by capillary electrophoresis].

The physical and chemical properties of chiral drugs are very similar. However, their pharmacological and toxicological effects vary significantly. For example, one enantiomer may have favorable properties whereas the other may be ineffective or even have toxic side effects. Hence, exploring innovative strategies to improve enantiomeric resolution is of great importance. Metoprolol (MET) is a ß-receptor blocker used to treat hypertension, stable angina pectoris, and supraventricular tachyarrhythmia. Establishing chiral separation and analysis methods of MET enantiomers is important for enhancing the quality of chiral drugs. Capillary electrophoresis (CE) has the advantages of a small sample size, simple operation, high separation efficiency, and many alternative modes; therefore it is widely used in the field of chiral drug separation. The chiral selectors commonly used for CE-based chiral separation include cyclodextrin (CD) and its derivatives, polysaccharides, proteins, and macrocyclic antibiotics. CD is one of the most commonly used and effective chiral selectors for CE. The relatively hydrophobic structure inside the cavity and the relatively hydrophilic structure outside the cavity of CD enable it and chiral molecules to form inclusion compounds with different binding constants, thus achieving chiral separation. However, the use of CD alone as a chiral selector does not always yield satisfactory separation results. Hence, the addition of other additives, such as ionic liquids and deep eutectic solvents (DESs) to assist CD-based chiral separation systems has received extensive attention. Previous studies on the enantiomeric separation of MET by CE have focused on the addition of CD and its derivatives alone for separation. Few studies have been conducted on the synergistic addition of auxiliary additives to CD to improve the enantiomeric resolution of MET. In this study, three DESs, namely, choline chloride-D-glucose, choline chloride-D-fructose, and lactate-D-glucose, were used for the CE-based chiral separation of MET for the first time, and the synergistic effect of the DESs on the separation of MET enantiomers by CD-based capillary zone electrophoresis was speculated. For this purpose, an uncoated fused silica capillary with inner diameter of 50 µm, total length of 50 cm and effective length of 41.5 cm was used as the separation column. First, the effects of CD type, CD concentration, buffer pH, and buffer concentration on MET separation were investigated, and the optimal conditions (15 mmol/L carboxymethyl-ß-cyclodextrin (CM-ß-CD), pH=3.0, and 40 mmol/L phosphate buffer) were obtained. Other CE conditions were as follows: UV detection at 230 nm, applied voltage of 25 kV. All operations were carried out at 20 ℃. Next, three types of DESs were prepared as auxiliary additives via a mixed-heating method. The DESs were mixed in a 50 mL round-bottomed flask at a certain molar ratio and then heated in a water bath at 80 ℃ for 3 h until a clear and transparent liquid was obtained. The effects of different DESs and their mass fraction on chiral separation were subsequently studied. The optimal choline chloride-D-fructose mass fraction was ultimately determined to be 1.5%. The resolution of MET increased from 1.30 without DES to 2.61 with 1.5% choline chloride-D-fructose, thereby achieving baseline separation. Finally, the separation effect and mechanism were speculated. The MET chiral separation method established in this study is of great significance for improving the quality of chiral compounds and ensuring the safety and effectiveness of clinical drugs. Furthermore, it may be useful in the research and development of CE-based chiral separation techniques using CD derivatives with DESs.

Development of a capillary zone electrophoresis method to monitor magnesium ion consumption during in vitro transcription for mRNA production.

Messenger RNA (mRNA) vaccines represent a landmark in vaccinology, especially with their success in COVID-19 vaccines, which have shown great promise for future vaccine development and disease prevention. As a platform technology, synthetic mRNA can be produced with high fidelity using in vitro transcription (IVT). Magnesium plays a vital role in the IVT process, facilitating the phosphodiester bond formation between adjacent nucleotides and ensuring accurate transcription to produce high-quality mRNA. The development of the IVT process has prompted key inquiries about in-process characterization of magnesium ion (Mg++) consumption, relating to the RNA polymerase (RNAP) activation, fed-batch mode production yield, and mRNA quality. Hence, it becomes crucial to monitor the free Mg++ concentration throughout the IVT process. However, no free Mg++ analysis method has been reported for complex IVT reactions. Here we report a robust capillary zone electrophoresis (CZE) method with indirect UV detection. The assay allows accurate quantitation of free Mg++ for the complex IVT reaction where it is essential to preserve IVT samples in their native-like state during analysis to avoid dissociation of bound Mg complexes. By applying this CZE method, the relationships between free Mg++ concentration, the mRNA yield, and dsRNA impurity level were investigated. Such mechanistic understanding facilitates informed decisions regarding the quantity and timing of feeding starting materials to increase the yield. Furthermore, this approach can serve as a platform method for analyzing the free Mg++ in complex sample matrices where preserving the native-like state of Mg++ binding is key for accurate quantitation.

Dextrin-assisted gel electromembrane extraction of chiral drugs: Improving the extraction efficiency and investigation of enantioselectivity of extraction.

The present study investigates the use of dextrins (maltodextrin, ß-cyclodextrin, and hydroxypropyl-ß-cyclodextrin) to improve the efficiency of the agarose-based gel electromembrane extraction technique for extracting chiral basic drugs (citalopram, hydroxyzine, and cetirizine). Additionally, it examines the enantioselectivity of the extraction process for these drugs. To achieve these, dextrins were incorporated into either the sample solution, the membrane, or the acceptor solution, and then the extraction procedure was performed. Enantiomers were separated and analyzed using a capillary electrophoresis device equipped with a UV detector. The results obtained under the optimal extraction conditions (sample solution pH: 4.0, acceptor solution pH: 2.0, gel membrane pH: 3.0, agarose concentration: 3 % w/v, stirring rate: 1000 rpm, gel thickness: 4.4 mm, extraction voltage: 62.3 V, and extraction time: 32.1 min) indicated that incorporating dextrins into either the sample solution, membrane or the acceptor solution enhances extraction efficiency by 17.3-23.1 %. The most significant increase was observed when hydroxypropyl-ß-cyclodextrin was added to the acceptor solution. The findings indicated that the inclusion of hydroxypropyl-ß-cyclodextrin in the sample solution resulted in an enantioselective extraction, yielding an enantiomeric excess of 6.42-7.14 %. The proposed method showed a linear range of 5.0-2000 ng/mL for enantiomers of model drugs. The limit of detection and limit of quantification for all enantiomers were found to be < 4.5 ng/mL and <15.0 ng/mL, respectively. Intra- and inter-day RSDs (n = 4) were less than 10.8 %, and the relative errors were less than 3.2 % for all the enantiomers. Finally, the developed method was successfully applied to determine concentrations of enantiomers in a urine sample with relative recoveries of 96.8-99.2 %, indicating good reliability of the developed method.

Dynamic Stomach Model-Capillary Electrophoresis-ICPMS for Evaluation of Release and Transformation Behaviors of Arsenic Species from Microplastics during Digestion.

Microplastics (MPs) can act as carriers of environmental arsenic species into the stomach with food and release arsenic species during digestion, which threatens human health. Herein, an integrated dynamic stomach model (DSM)-capillary electrophoresis-inductively coupled plasma mass spectrometry (CE-ICPMS) is developed for online monitoring of the release and transformation behaviors of arsenic species loaded on MPs (As-MPs) in the simulated human stomach. The 3D-printed DSM with a soft stomach chamber enables the behaviors of gastric peristalsis, gastric and salivary fluid addition, pH adjustment, and gastric emptying (GE) to be controlled by a self-written program after oral ingestion of food with As-MPs. The gastric extract during digestion is introduced into the spiral channel to remove the large particulate impurity and online filtered to obtain the clarified arsenic-containing solution for subsequent speciation analysis of arsenic by CE-ICPMS. The digestion conditions and pretreatment processes of DSM are tracked and validated, and the release rates of As-MPs digested by DSM are compared with those digested by the static stomach model and DSM without GE. The release rate of inorganic arsenic on MPs is higher than that of organic arsenic throughout the gastric digestion process, and 8% of As(V) is reduced to As(III). The detection limits for As(III), DMA, MMA, and As(V) are 0.5-0.9 µg L-1 using DSM-CE-ICPMS, along with precisions of ≤8%. This present method provides an integrated and convenient tool for evaluating the release and transformation of As-MPs during human gastric digestion and provides a reference for exploring the interactions between MPs and metals/metalloids in the human body.

A novel method of multiplex SNP genotyping assay through variable fragment length allele-specific polymerase chain reaction: Multiplex VFLASP-ARMS.

Variable Fragment Length Allele-Specific Polymerase Chain Reaction (VFLASP) and Amplification Refractory Mutation System (ARMS) are reliable methods for detecting allelic variations resulting from single base changes within the genome. Due to their widespread application, allele variations caused by Single Nucleotide Polymorphisms (SNPs) can be readily detected using allele-specific primers. In the context of the current study, VFLASP was combined with ARMS method as a novel strategy to enhance the efficacy of both techniques. Clinically important base variations within SNP regions used in the study were detected by a fragment analysis method. To validate the accuracy of the developed VFLASP-ARMS method, specifically designed synthetic sequences were tested using a capillary electrophoresis system. Allele-specific primers exhibit differences solely at the 3' end based on the sequence of the SNP. Additionally, to increase the specificity of the primers, a base was intentionally added for incompatibility. Therefore, allele discrimination on fragment analysis has been made possible through the 3-6 bp differences in the amplicons. With the optimization of the system, designed synthetic sequences provided reliable and reproducible results in wild-type, heterozygous, and homozygous genotypes using the VFLASP-ARMS method. Hence, our results demonstrated that VFLASP-ARMS method, offers a novel design methodology that can be included in the content of SNP genotyping assays.

Determination of sulfonamide antibiotics by magnetic porous carbon solid-phase extraction coupled with capillary electrophoresis.

Sulfonamide antibiotics (SAs) have been widely used as antibacterial drugs for the prevention and treatment of livestock and poultry diseases, but they seriously threaten human health because they can accumulate in humans. Therefore, it is highly important to develop methods for monitoring sulfonamide residues in aquaculture and food. In this research, based on the generation of porous carbon (PC) by the pyrolysis of sodium citrate, magnetic porous carbon (PC@Fe3O4) was synthesized by a solvothermal method and used as an adsorbent for the magnetic solid-phase extraction of SAs. The effects of the proportion of PC in PC@Fe3O4, adsorbent dosage, adsorption time, eluent type, extraction pH, salt concentration and eluent dosage on the extraction efficiency were systematically studied. The adsorption performance and behavior of PC@Fe3O4 on SAs were evaluated using adsorption kinetics and adsorption isotherms, and the adsorption mechanism was preliminarily discussed. Under optimal conditions, combined with capillary electrophoresis diode array detection, a sensitive detection method for SAs was developed. The proposed method can be used for the determination of six SAs in fishpond water and milk samples, with a linear range of 0.5-200 ng mL-1, detection limits of 0.24-0.34 ng mL-1, and spiked recoveries of 85.9-109.0 %.

Influence of oligonucleotides structures for separation of diastereomers by capillary electrophoresis method using polyvinylpyrrolidone 1,300,000.

In the field of oligonucleotides drug discovery, phosphorothioate (PS) modification has been recognized as an effective tool to overcome the nuclease digestion, and generates 2n of possible diastereomers, where n equals the number of PS linkages. However, it is also well known that differences in drug efficacy and toxicity are caused by differences in stereochemistry of oligonucleotides. Therefore, the development of a high-resolution analytical method that enables stereo discrimination of oligonucleotides is desired. Under this circumstance, capillary electrophoresis (CE) using polyvinylpyrrolidone (PVP) is considered as one of the useful tools for the separation analysis of diastereomers. In this study, we evaluated the several oligonucleotides with the structural diversities in order to understand the separation mechanism of the diastereomers by CE. Especially, five kinds of 2'-moieties were deeply examined by CE with PVP 1,300,000 polymer solution. We found that different trend of the peak shapes and the peak resolution were observed among these oligonucleotides. For example, the better peak resolution was observed in 6 mer PS3-DNA compared to the rigid structure of 6 mer PS3-LNA. As for this reason, the computational simulation revealed that difference of accessible surface area caused by the steric structure of thiophosphate in each oligonucleotide is one of the key attributes to explain the separation of the diastereomers. In addition, we achieved the separation of sixteen peak tops of the diastereomers in 6 mer PS4-DNA, and the complete separation of fifteen diastereomers in 6 mer PS4-RNA. These knowledge for the separation of the diastereomers by CE will be expected to the quality control of the oligonucleotide drugs.

Microsatellite Instability Detection in Cancer: A Multiplex qPCR Approach that Obviates the Need for Matching Normal Samples.

BACKGROUND: Microsatellite instability (MSI) indicates DNA mismatch repair deficiency in certain types of cancer, such as colorectal cancer. The current gold standard technique, PCR-capillary electrophoresis (CE), requires matching normal samples and specialized instrumentation. We developed VarTrace, a rapid and low-cost quantitative PCR (qPCR) assay, to evaluate MSI using solely the tumor sample DNA, obviating the requirement for matching normal samples. METHODS: One hundred and one formalin-fixed paraffin-embedded (FFPE) tumor samples were tested using VarTrace and compared with the Promega OncoMate assay utilizing PCR-CE. Tumor percentage limit of detection was evaluated on contrived samples derived from clinical high MSI (MSI-H) samples. Analytical sensitivity, specificity, limit of detection, and input requirements were assessed using synthetic commercial reference standards. RESULTS: VarTrace successfully analyzed all 101 clinical FFPE samples, demonstrating 100% sensitivity and 98% specificity compared to OncoMate. It detected MSI-H with 97% accuracy down to 10% tumor. Analytical studies using synthetic samples showed a limit of detection of 5% variant allele frequency and a limit of input of 0.5 ng. CONCLUSIONS: This study validates VarTrace as a swift, accurate, and economical assay for MSI detection in samples with low tumor percentages without the need for matching normal DNA. VarTrace's capacity for highly sensitive MSI analysis holds potential for enhancing the efficiency of clinical work flows and broadening the availability of this test.

A new coaxial flow-through probe for electromembrane extraction of methadone from clinical samples on-line coupled to capillary electrophoresis.

BACKGROUND: A new design of a flow-through coaxial electromembrane extraction (EME) probe that can be on-line coupled with CE instrument is described and tested. The supporting base of the probe is a PDMS microchip with T-shaped channels into which two coaxially arranged capillaries for inlet and outlet solutions are inserted. The extraction part of the probe is a porous polypropylene hollow fiber, sealed at one end and modified with nitrophenyloctyl ether (NPOE) extraction fluid. The internal volume of the extraction probe is 1.1 µL. RESULTS: The EME probe was tested on laboratory samples and methadone was extracted into 3.0 M AcOH as acceptor. The concentration dependence was linear in the range of 0.1-1.0 µg mL-1 at EME 300 s/150 V and in the range of 0.5-10.0 µg mL-1 at EME 100 s/150 V. The enrichment factor was greater than 30 and the LOD was 0.21 µg mL-1. The EME of methadone in clinical samples showed a linear concentration dependence in human urine and a nonlinear concentration dependence in serum. The distribution of methadone in each phase of the extraction system and the effect of extraction membrane thickness on the enrichment factor were studied. The EME probe can be applied repeatedly. SIGNIFICANCE: The supporting base of EME probe and flow gating interface (FGI) are realized by a microfluidic PDMS microchips cast in the laboratory without the use of lithography. A supporting PDMS chip with coaxially arranged capillaries and extraction membrane forms a compact analytical instrument. The entire EME/CE analysis process is performed on a laboratory-made instrument and automated by LabView.