Single bubble in-tube microextraction coupled with capillary electrophoresis.

Headspace (HS) extraction is a sample pretreatment technique for volatile and semivolatile organic compounds in a complex matrix. Recently, in-tube microextraction (ITME) coupled with CE using an acceptor plug placed in the capillary inlet was developed as a simple but powerful HS extraction method. Here, we present single bubble (SB) ITME using a bubble hanging to the capillary inlet immersed in a sample donor solution as a HS of submicroliter volume (∼200 nL). The analytes evaporated to the bubble were extracted into the acceptor phase through the capillary opening, then electrophoresis of the enriched extract was carried out. Since the bubble volume was much smaller than a conventional HS volume (∼1 mL), it was filled with the evaporated analytes rapidly and the analytes could be enriched much faster compared to conventional HS-ITME. Owing to the high surface-to-volume ratio of the SB, 5 min SB-ITME yielded the enrichment factor values similar to those of 10 min HS-ITME. When 5 min SB-ITME at room temperature was applied to a tap water sample, the enrichment factors of 2,4,6-trichlorophenol (TCP), 2,3,6-TCP, and 2,6-dichlorophenol were 53, 41, and 60, respectively, and the LOQs obtained by monitoring the absorbance at 214 nm were 5.6-8.3 ppb, much lower than 200 ppb, the World Health Organization guideline for the maximum permissible concentration of 2,4,6-TCP in drinking water.

Application of capillary electrophoresis-nano-electrospray ionization-mass spectrometry for the determination of N-nitrosodimethylamine in pharmaceuticals.

After a presence of highly hepatotoxic and potentially carcinogenic N-nitrosodimethylamine was detected in certain lots of sartan, ranitidine, metformin, and other pharmaceuticals, local regulatory authorities issued recalls of suspected products, and concerns of the pharmacotherapy safety were widely discussed. Since then, testing of a representative sample of each produced lot of these pharmaceuticals is required as a part of quality control processes. Hence, an interface-free CE-nanoESI system coupled with MS detection was employed for the development of a simple and economical method for quantitative detection of this contaminant in the valsartan drug substances and finished formulations used as model matrices. In this arrangement, a fused-silica capillary was used as both a separation column and a nanoESI emitter providing high ionization efficiency and sensitivity. The optimized procedure was found to have sufficient selectivity, linearity, accuracy, and precision. The established LOD and LOQ values were 0.3 and 1.0 ng/mL, respectively. The practical applicability of the method was tested by analyses of commercially available Valsacor® tablets. The results obtained prove that the developed procedure represents a promising alternative to currently available GC- and LC-based methods. Furthermore, after an adjustment of the separation conditions, the CE-nanoESI/MS system can be conceptually used for the determination of NDMA in other suspected pharmaceuticals.

A molecular basis behind heterophylly in an amphibious plant, Ranunculus trichophyllus.

Ranunculus trichophyllus is an amphibious plant that produces thin and cylindrical leaves if grown under water but thick and broad leaves if grown on land. We found that such heterophylly is widely controlled by two plant hormones, abscisic acid (ABA) and ethylene, which control terrestrial and aquatic leaf development respectively. Aquatic leaves produced higher levels of ethylene but lower levels of ABA than terrestrial leaves. In aquatic leaves, their distinct traits with narrow shape, lack of stomata, and reduced vessel development were caused by EIN3-mediated overactivation of abaxial genes, RtKANADIs, and accompanying with reductions of STOMAGEN and VASCULAR-RELATED NAC-DOMAIN7 (VDN7). In contrast, in terrestrial leaves, ABI3-mediated activation of the adaxial genes, RtHD-ZIPIIIs, and STOMAGEN and VDN7 established leaf polarity, and stomata and vessel developments. Heterophylly of R.trichophyllus could be also induced by external cues such as cold and hypoxia, which is accompanied with the changes in the expression of leaf polarity genes similar to aquatic response. A closely-related land plant R. sceleratus did not show such heterophyllic responses, suggesting that the changes in the ABA/ethylene signaling and leaf polarity are one of key evolutionary steps for aquatic adaptation.

Miniaturized LC in Molecular Omics.

Miniaturized LC has evolved at an exponential rate over the last 50 years. In the past decade, it has received considerable attention in the field of bioanalytical separation science and technology due to the need to measure different classes of biomolecules present in a variety of matrixes on a global scale to gain a deeper understanding of complex biological processes. This field has become a dominant area underpinning the molecular omics research (e.g., proteomics, metabolomics, lipidomics, and foodomics), allowing key insights into the function and mechanism of small to very large biomolecules on a molecular level. This Feature highlights the recent advances in molecular omics focusing on miniaturized LC technology combined with mass spectrometry-based platforms, with a particular emphasis on the strategies adopted and applications using new and sensitive nanoscale analytical methodologies.

Analyte focusing by micelle collapse for liquid extraction surface analysis coupled with capillary electrophoresis of neutral analytes on a solid surface.

Liquid extraction surface analysis (LESA) has an advantage of directly sampling analytes on a surface, thus avoiding unnecessary dilution by homogenization of the bulk sample commonly practiced in solid sample analysis. By combining LESA with CE, the additional advantage of separating analytes before detection can be accomplished. For neutral molecules, MEKC needs to be used. Since the detection sensitivity of CE in general suffers from the small capillary dimension, analyte focusing by micelle collapse was employed for enhanced extraction in LESA and sample preconcentration for MEKC. In addition, using a commercial CE instrument, the LESA process was performed much faster and more reliably compared to our first demonstration of LESA-CE using a homemade CE setup. Three neutral water-insoluble pesticides sprayed on an apple skin were directly extracted, preconcentrated, and analyzed by the automated LESA-analyte focusing by micelle collapse-MEKC with high sensitivity in 10 min. The relative standard deviations of the migration times and peak heights were 0.8-2.1 and 1.2-3.0%, respectively when ametryn was used as an internal standard. The limits of detection obtained with UV absorbance at 200 nm were 1.8-6.4 ppb.

Synergistic coupling of in-line single-drop microextraction and on-line large-volume sample stacking for capillary electrophoresis/mass spectrometry.

Single-drop microextraction (SDME) and large-volume sample stacking using an electroosmotic flow pump (LVSEP) were coupled with capillary electrophoresis/mass spectrometry (CE/MS) for sample cleanup and preconcentration. Without filtration or centrifugation of a soil sample containing debris, SDME using a pentanol acceptor drop was directly applied to the sample. After SDME, a large volume of the enriched pentanol extract was injected and further concentrated by LVSEP. For the drop formation in SDME and the sample matrix removal in LVSEP, a run buffer vial was temporarily placed to the electrospray tip, without any physical modification of the CE/MS interface. This method enabled the double preconcentration by SDME and LVSEP, achieving 600~1300-fold enrichments of anionic analytes including pesticide and herbicide compounds to provide limits of detection in the range of 0.4~0.8 ppb in soil. Graphical abstract ᅟ.

Rotational-State-Dependent Dispersion of Molecules by Pulsed Optical Standing Waves.

We report on the rotational-state-dependent, transverse acceleration of CS_{2} molecules affected by pulsed optical standing waves. The steep gradient of the standing wave potential imparts far stronger dipole forces on the molecules than propagating pulses do. Moreover, large changes in the transverse velocities (i.e., up to 80 m/s) obtained with the standing waves are well reproduced in numerical simulations using the effective polarizability that depends on the molecular rotational states. Our analysis based on the rotational-state-dependent effective polarizability can therefore serve as a basis for developing a new technique of state selection for both polar and nonpolar molecules.

In-line coupling of single-drop microextraction with capillary electrophoresis-mass spectrometry.

Single-drop microextraction (SDME) was in-line coupled with capillary electrophoresis-mass spectrometry to provide sample cleanup and enrichment simultaneously. Since there is no outlet vial in a conventional capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) configuration, it is not easy to hang a single drop in the capillary inlet for extraction. We overcame the difficulty of coupling SDME and CE-MS by using a temporary outlet reservoir. Basic drugs such as methamphetamine, amphetamine, phenethylamine, methoxyphenamine, and mephentermine were extracted from a basic sample solution to an acidic acceptor drop covered with a thin octanol layer formed at the capillary inlet tip. Compared to the CE-MS method in the multiple reaction monitoring (MRM) mode, the in-line SDME-CE-MS/MS technique showed 130∼150-fold enrichment in 10 min. The relative standard deviations (RSDs) of peak height ranged from 9 to 13 %. RSDs can be reduced from 4 to 6 % using mephentermine as an internal standard. We examined the pretreatment of sample with and without SDME from human urine under the full-scan mode, which confirmed that many metabolites were cleaned up by the selective extraction method of SDME. Even if the analytes from human urine were analyzed under the MRM mode used as a mass filter, there was an isobaric compound causing a disturbance to the analysis. However, in-line SDME-CE-MS/MS made it possible to perform a sample cleanup as well as sample enrichment. The research is extremely advantageous in that it is rapid, convenient, and highly sensitive for the analysis of biological samples using a commercially available instrument.

Highly sensitive analysis of catecholamines by counter-flow electrokinetic supercharging in the constant voltage mode.

Electrokinetic supercharging is one of the most powerful sample-stacking methods that combines field amplified sample injection and transient ITP. In counter-flow electrokinetic supercharging, a constant counter pressure is applied during sample injection in order to counterbalance the movement of the injected sample zone. As a result, there will be a pronounced increase in the amount of sample injected and the portion of the capillary available for electrophoresis. In this report, counter-flow electrokinetic supercharging optimization factors such as the electric field application in the constant voltage and constant current modes, the magnitude of counter pressure, and the terminating electrolyte concentrations were investigated. The enrichments obtained with a 30 min injection of 10 nM catecholamines in 5 mM terminating electrolyte solution in the constant voltage mode applying a counter pressure of 1.3 psi were 41,000-fold for dopamine, 50,000-fold for norepinephrine, and 32,000-fold for epinephrine, yielding detection limits of 1.3, 1.4, and 1.2 nM, respectively, with absorbance detection at 200 nm.

Headspace-single drop microextraction with a commercial capillary electrophoresis instrument.

Automated coupling of headspace-single drop microextraction (HS-SDME) and CE has been demonstrated using a commercial CE instrument. When a drop hanging at the inlet tip of a capillary for CE is used as the acceptor phase, HS-SDME becomes a simple but powerful sample pretreatment technique for CE before injection to facilitate sample cleanup and enrichment. By combining HS-SDME with an on-line sample preconcentration technique, large volume sample stacking using an electroosmotic flow pump, the sensitivity can be improved further. The overall enrichment factors for phenolic compounds were from 1900 to 3400. HS-SDME large volume sample stacking using an electroosmotic flow pump was successfully applied to a red wine sample to obtain an LOD of 4 nM (0.8 ppb) for 2,4,6-trichlorophenol which is a precursor for 2,4,6-trichloroanisole causing the foul odor in wine called cork taint.

Novel colorimetric assay of LSD1 activity using gold nanoparticles.

We have developed a simple and sensitive strategy for colorimetric LSD1 enzyme activity assay using avidin modified gold nanoparticles. The strategy is based on the vivid color change of a gold nanoparticle solution from red to violet upon addition of a test solution of peptide-antibody treated with LSD1. Thus, the presence of LSD1 in a sample can be determined by simple visual inspection with the naked eye. In addition, a wide range of LSD1 concentrations (13 pM to 0.13 µM) were quantitatively determined by spectrophotometry, which shows the possibility of quantitative analysis of the over expression levels of LSD1 in cancer tissue samples.

Introduction of a Capillary Gel Electrophoresis-Based Workflow for Biotherapeutics Characterization: Size, Charge, and N-Glycosylation Variant Analysis of Bamlanivimab, an Anti-SARS-CoV-2 Product.

Coronavirus Disease 2019 (COVID-19) is a major public health problem worldwide with 5-10% hospitalization and 2-3% global mortality rates at the time of this publication. The disease is caused by a betacoronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The receptor-binding domain (RBD) of the Spike protein expressed on the surface of the virus plays a key role in the viral entry into the host cell via the angiotensin-converting enzyme 2 receptor. Neutralizing monoclonal antibodies having the RBD as a target have the ability to inhibit angiotensin-converting enzyme 2 (ACE2) receptor binding, therefore, prevent SARS-CoV-2 infection, represent a promising pharmacological strategy. Bamlanivimab is the first anti-spike neutralizing monoclonal antibody, which got an emergency use authorization from the FDA for COVID-19 treatment. Albeit, bamlanivimab is primarily a neutralizing mAb, some of its effector function related activity was also emphasized. The effector function of antibody therapeutics is greatly affected by their N-linked carbohydrates at the conserved Fc region, possibly influenced by the manufacturing process. Various capillary gel electrophoresis methods are widely accepted in the biopharmaceutical industry for the characterization of therapeutic antibodies. In this paper we introduce a capillary gel electrophoresis based workflow for 1) size heterogeneity analysis to determine the presence/absence of the non-glycosylated heavy chain (NGHC) fragment (SDS-CGE); 2) capillary gel isoelectric focusing for possible N-glycosylation mediated charge heterogeneity determination, e.g., for excess sialylation and finally, 3) capillary gel electrophoresis for N-glycosylation profiling and sequencing. Our results have shown the presence of negligible amount of non-glycosylated heavy chain (NGHC) while 25% acidic charge variants were detected. Comprehensive N-glycosylation characterization revealed the occurrence of approximately 8.2% core-afucosylated complex and 17% galactosylated N-linked oligosaccharides, suggesting the possible existence of antibody dependent cell mediated cytotoxicity (ADCC) effector function in addition to the generally considered neutralizing effect of this particular therapeutic antibody molecule.

Capillary electrophoretic mobility shift assay for binding of DNA with NFAT3, a transcription factor from H9c2 cardiac myoblast cells.

Nuclear factor of activated T-cells (NFAT) is a transcription factor involved in the development of cardiac and skeletal muscle and the nervous system. NFAT is activated by calcium signal pathway and translocated into the nucleus. The quantification of binding between NFAT and NFAT-specific DNA gives important information about cardiac hypertrophy. We investigated the binding of NFAT3 in nuclear extracts from H9c2 cells to its specific DNA by capillary electrophoretic mobility shift assay. The binding reaction time required for stable formation of the DNA-NFAT3 complex was 3 h and the separation of the complex and free DNA was achieved within 10 min by CE. The formation of NFAT3-DNA complex was confirmed by the competitive reaction. Comparison of the ratios of complex/free DNA peak area for 1 µM endothelin-1 (ET-1)-treated cells and control cells showed the NFAT3 translocation into the nucleus promoted by ET-1. The binding constant between NFAT3 and DNA was estimated to be 7.7×10(9) M(-1) at 4°C.

High-sensitivity capillary and microchip electrophoresis using electrokinetic supercharging.

Electrokinetic supercharging (EKS) is considered as one of the most powerful online preconcentration techniques in electrophoresis. It combines the efficient preconcentration power of field-amplified sample injection and the exceptional selective nature of transient isotachophoresis. It has a wide range of applications to different types of analytes ranging from small ions to large proteins and DNA fragments. This comprehensive review--up to date--provides listing for all the works, developments, and advances in EKS. The review will pay particular attention to innovations, new methodologies for manipulation, challenges for improving the detection sensitivity, and various applications of EKS in capillaries and microchips.

Facile and highly efficient three-phase single drop microextraction in-line coupled with capillary electrophoresis.

A high-performance version of in-line, three-phase direct immersion-single drop microextraction (DI-SDME) coupled with capillary electrophoresis (CE) was demonstrated using a commercial CE instrument, and all the major and minor details were described to provide an easy-to-follow and user-friendly protocol. The excellent sample cleanup and enrichment power of this method was demonstrated with nonsteroidal anti-inflammatory drugs (NSAIDs) in human urine. The only preparation of urine samples was the addition of HCl to acidify the urine sample to pH 2. The acidic NSAIDs in the acidified urine sample were extracted into a basic acceptor drop covered with a thin organic layer attached to the inlet tip of a capillary immersed in the sample. A simple but powerful DI-SDME-CE method could be carried out automatically without any modification of the existing CE instrument. For improved performance, sample agitation and heating were employed by installing a microstirrer and a thermostating jacket in the sample tray. With 10 min of DI-SDME at 35°C with stirring, NSAIDs such as ketoprofen, ibuprofen, and naproxen in urine were enriched 340-970-fold with intraday and interday RSDs of 0.8-2.4% and 1.1-3.6%, respectively. The LODs obtained with in-line coupled CE/UV were 10-50 nM (2-10 µg/L). The performance of DI-SDME-CE/UV was also demonstrated by determining the naproxen level in human urine collected 24 h after taking a single oral dose of the drug. The spike recovery of naproxen from a single-point standard addition to the urine sample was 80%. Our high-performance three-phase DI-SDME-CE method is quite promising for the analysis of ionizable trace analytes in a complex sample matrix.

A doubly signal-amplified DNA detection method based on pre-complexed [Ru(bpy)3]2+-doped silica nanoparticles.

Easy detection: The target DNA in a 10-100 aM range can be detected by pre-complexed nanoparticles without additional amplification or target labeling. The [Ru(bpy)(3)](2+)-doped silica nanoparticles are hybridized to form a complex with highly enhanced sensitivity (see scheme). This method will be a significant improvement over conventional microarray/fluorescence readout systems.

Applications of deep eutectic solvents to quantitative analyses of pharmaceuticals and pesticides in various matrices: a brief review.

Pharmaceuticals and pesticides are important analytes of interest in clinical, environmental, and food analyses for ensuring public health. Sample pretreatment steps are often prerequisites for the quantitative analysis of these compounds, which are generally present in low concentrations in samples with complex matrices. In compliance with the current trend towards green analytical chemistry, the replacement of conventional toxic organic solvents with ecofriendly and safe solvents has been pursued in developing sample pretreatment methods. Subsequent to several reports in 2017, deep eutectic solvents (DESs) have been increasingly applied as desirable alternative solvents in numerous types of sample pretreatment methods for the analysis of pharmaceuticals and pesticides. The present review summarizes analytical methods involving DESs as extraction solvents and as the reaction media or functional materials for preparing adsorbents to quantify pharmaceuticals and pesticides in various matrices.

A bifunctional molecule as an artificial flavin mononucleotide cyclase and a chemosensor for selective fluorescent detection of flavins.

Flavins, comprising flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and riboflavin (RF, vitamin B(2)), play important roles in numerous redox reactions such as those taking place in the electron-transfer chains of mitochondria in all eukaryotes and of plastids in plants. A selective chemosensor for flavins would be useful not only in the investigation of metabolic processes but also in the diagnosis of diseases related to flavins; such a sensor is presently unavailable. Herein, we report the first bifunctional chemosensor (PTZ-DPA) for flavins. PTZ-DPA consists of bis(Zn(2+)-dipicolylamine) and phenothiazine. Bis(Zn(2+)-dipicolylamine) (referred to here as XyDPA) was found to be an excellent catalyst in the conversion of FAD into cyclic FMN (riboflavin 4',5'-cyclic phosphate, cFMN) under physiological conditions, even at pH 7.4 and 27 degrees C, with less than 1 mol % of substrate. Utilizing XyDPA's superior function as an artificial FMN cyclase and phenothiazine as an electron donor able to quench the fluorescence of an isoalloxazine ring, PTZ-DPA enabled selective fluorescent discrimination of flavins (FMN, FAD, and RF): FAD shows ON(+), FMN shows OFF(-), and RF shows NO(0) fluorescence changes upon the addition of PTZ-DPA. With this selective sensing property, PTZ-DPA is applicable to real-time fluorescent monitoring of riboflavin kinase (RF to FMN), alkaline phosphatase (FMN to RF), and FAD synthetase (FMN to FAD).

Single drop microextraction using commercial capillary electrophoresis instruments.

Single drop microextraction (SDME), a simple and efficient sample preconcentration method for capillary electrophoresis (CE), was performed using two different commercial CE instruments. With a CE instrument providing adjustable forward and backward pressures, a single drop of an aqueous acceptor phase covered with a thin layer of an organic phase was formed at the capillary tip by programming the sample-handling functions of the instrument to perform 3-phase SDME where the organic film is essentially a membrane. Analytes from an acidic donor phase were concentrated into a basic acceptor phase yielding 190-fold enrichment in 10 min. When the donor phase was agitated using a microstirrer, a 2000-fold enrichment was obtained in 10 min. For a less flexible CE instrument, 2-phase SDME was carried out with a large volume pentanol drop held by a Teflon sleeve, yielding 110-fold enrichments in 30 min. We demonstrate a practical and automated SDME-CE technique with accuracy and reproducibility that is easy to practice to attain matrix isolation and high concentration sensitivity.

Double sample preconcentration by in-line coupled large volume single drop microextraction and sweeping in capillary electrophoresis.

Single drop microextraction (SDME) is a convenient and powerful preconcentration method for CE before injection. By simple combination of sample-handling sequences without modification of the CE apparatus, a drop of an aqueous acceptor phase covered with a thin organic layer was formed at the tip of a capillary; 10 min SDME of fluorescein and 6-carboxyfluorescein from a donor phase of pH 1 to an acceptor phase of pH 9 provided 110-fold enrichments without stirring the donor phase. To improve the concentration effect further, SDME was coupled with an on-line (after injection) sample preconcentration method, sweeping, in which analytes in a long sample zone are accumulated at the boundary of a pseudostationary phase penetrating into the sample zone. It is thus necessary to inject a sample of much larger volume than that of a drop in typical SDME. A Teflon sleeve over the capillary inlet allowed a large volume drop to be held stably during extraction. By in-line coupling 10 min SDME and sweeping of a 30 nL sample using a cationic surfactant dodecyltrimethylammonium, enrichment factors of the double preconcentration were increased up to 32,000.