ELECTROPHORESIS最新文献

mRNA-based vaccines and self-amplifying mRNA (saRNA) have gained growing attention for disease prevention and treatment, but precise detection of low-concentration RNA (including mRNA-lipid nanoparticles, mRNA-LNPs) stability and integrity remains challenging-limiting quality control of RNA-based therapeutics. Capillary electrophoresis (CE)-based instruments show potential, yet suitable concentration strategies for low-abundance, labile RNAs are lacking. This study established two distinct concentration methods (freeze-drying and ultrafiltration) applicable to mRNA, mRNA-LNPs, and saRNA formulations, enabling the conversion of dilute solutions to high-concentration preparations while preserving the structural and molecular integrity of the target nucleic acids. Subsequent stability evaluations and conventional high-performance liquid chromatography analyses of the concentrated products verified their superior storage stability and strong practical applicability. This article aims to reduce the difficulty of RNA integrity assessment and improve the accuracy of detecting various low-concentration RNA samples that may arise in the future.

The growing concern over drug abuse, particularly involving amphetamine-type substances (ATS), has led to their monitoring in environmental waters. These chiral compounds, commonly found in river water and wastewater treatment plant (WWTP) effluents, require analytical methods capable of enantiomeric discrimination, as their enantiomers can exhibit different pharmacological and environmental behaviours. A method based on capillary electrophoresis (CE) coupled with tandem mass spectrometry (MS/MS) was developed using a dual cyclodextrin (CD) system consisting of 0.1% 2-hydroxypropyl-β-CD and 0.1% γ-CD in the background electrolyte (BGE), which enabled baseline resolution of the target enantiomers of the ATS under study. Samples were pretreated with solid-phase extraction using a mixed-mode cation exchange sorbent, ExtraBond SCX. Samples of 100 mL for WWTP influent and 250 mL for river water and WWTP effluent were extracted and then eluted with 5 mL of 5% NH₄OH in methanol. Recoveries ranged between 40% and 67% for all amphetamines studied, with detection limits between 0.1 and 0.8 µg/L. Analysis of environmental samples from the Ebre River and WWTPs in Reus and Tarragona (Catalonia, Spain) confirmed the presence of some of the target compounds. Both enantiomers of 3,4-methylenedioxymethamphetamine (MDMA) were determined in WWTP influents and effluents, whereas R-amphetamine was quantified in an influent sample. No target compounds were detected in the analysed river water samples. These findings demonstrate the potential of the developed chiral CE-MS/MS method for robust enantiomeric profiling in environmental waters, attributed to efficient separation based on differences in effective mobility (µ_eff), electroosmotic flow (µ_eo) and selective interactions with CD chiral selectors. The method showed moderate adherence to green analytical principles, achieving an AGREE score of 0.47. Its environmental advantages include the use of CE, minimal solvent consumption and non-toxic chiral selectors, offering a more suitable alternative to more traditional LC-based methods.

This study describes the use of microchip micellar electrokinetic chromatography (MEKC) integrated with capacitively coupled contactless conductivity detection (C⁴D) for the determination of monoethylene glycol (MEG) in gas condensate samples. The samples were subjected to a liquid-liquid extraction step and then analyzed by chip-based MEKC-C⁴D. For this purpose, sodium dodecyl sulfate (SDS) was used as a surfactant at a concentration of 30 mmol L^-1 added in 50 mmol L^-1 phosphate (pH = 9.0). Samples were introduced into microchannels through floating injection mode by applying a voltage of 600 V during 10 s. Separations were performed under an electric field of 82 V cm^-1 and monitored by C⁴D measurements recorded applying a 1200-kHz frequency sinusoidal wave with 20-V_pp excitation voltage. The proposed methodology employing MEKC-C⁴D revealed a linear behavior (r² ≥ 0.99) in the MEG concentration range between 150-450 µmol L^-1 and LOD equal to 33 µmol L^-1. Three gas condensate samples were then analyzed, and the achieved MEG concentration values ranged from 173 to 213 µmol L^-1. Recovery experiments provided values between 89 and 102%. Based on the results reported in this study, MEKC-C⁴D devices have demonstrated to be a promising and ecological analytical tool for MEG analysis with huge potential for in-field applications.

This study includes the synthesis of monomethyl auristatin E (MMAE) payload conjugated Trastuzumab (TRA) by an enzyme-mediated glycan-remodelling reaction, followed by comprehensive multilevel characterization of conjugation end products. Intact proteoforms of TRA and TRA-MMAE antibody-drug conjugates (ADC) were separated, quantitated, and identified by microfluidic chip-based ultraviolet imaged channel isoelectric focusing mass spectrometry (icIEF-UV/MS). Isoelectric point and deconvoluted intact mass shifts observed with TRA-MMAE allowed for the determination of the drug-to-antibody ratio (DAR) and detection of trace levels of enzymatic and conjugation intermediates. Parallel analysis by reversed-phase high-performance liquid chromatography (RP-HPLC) peptide mapping with electron-activated dissociation (EAD) fragmentation was also performed. Peptide level results corroborated the putative intact identifications, localized posttranslational modifications (PTMs) on the underivatized TRA structure, and validated site-specific conjugation of the MMAE payload to the Asn-300 attached glycan structure of the ADC. Combined icIEF-UV/MS and RP-HPLC peptide map with EAD fragmentation effectively confirmed the high yield of TRA-MMAE DAR 2 produced by the evaluated chemoenzymatic conjugation reaction. Overall, these results establish a streamlined production and analytical workflow capable of providing well-characterized, highly homogeneous ADC structures for downstream preclinical screening and eventual scale-up.

Most current forensic applications concerning DNA mixtures primarily focus on individual identification, specifically determining the presence of a person of interest (POI). However, when the target individual is unavailable due to death or disappearance, traditional methods often prove inadequate. Our previous exploratory research has demonstrated that integrating Bayesian network algorithms with the novel genetic marker-microhaplotype-typing technology can effectively facilitate forensic DNA mixture analysis across various scenarios. However, it is important to note that short tandem repeat (STR) typing based on capillary electrophoresis (CE) remains the most widely employed method in forensic practice, and the profiling data for the majority of DNA mixed samples are still derived from this established technology. Therefore, further investigation into various scenarios devoid of POIs based on traditional STR genotyping data is warranted. In this study, we undertook an investigation into three scenarios involving DNA mixtures in the absence of POIs, leveraging relatedness information through Bayesian network algorithms. The analyses were based on traditional CE-based STR genotyping data derived from artificially synthesized and simulated mixed samples. The Bayesian network framework offers considerable flexibility, enabling the incorporation of kinship information for various interpretive purposes, including assessing the potential contribution of a missing pedigree member to a DNA mixture and evaluating the relatedness among contributors within or between mixed DNA profiles. The aforementioned research offers a referable experimental framework for addressing complex DNA mixtures within the realm of forensic practice.

Recent advances in membrane protein biochemistry have enabled the isolation of complexes in detergent-free, near-native states using synthetic amphipathic copolymers such as styrene-maleic acid (SMA) and diisobutylene maleic acid (DIBMA). However, these polymers often interfere with conventional protein detection methods, particularly in SDS-PAGE and Clear Native PAGE (CN-PAGE), hindering visualization and quantification. Here, we systematically evaluated 15 staining and detection methods-including Coomassie Brilliant Blue, silver, zinc, copper, Ponceau S, and SYPRO fluorescent dyes-on proteins solubilized by 13 different agents, including detergent n-dodecyl-β-d-maltoside (DDM), five SMA variants, and three DIBMA variants, from bovine heart mitochondria and cyanobacterial thylakoids. A photochemical, stain-free detection method using trichloroethanol (TCE) and UV activation proved to be optimal. This method covalently labels solvent-accessible tryptophan and tyrosine residues, generating robust fluorescence signals that are unaffected by polymer interference. TCE-modified proteins display dual emission peaks at ∼460 nm and a shoulder near 490 nm, likely corresponding to tyrosine and tryptophan adducts, respectively. The polymer-insensitive nature of TCE labeling allows sharp band resolution, particularly for low molecular weight proteins, and is compatible with high-throughput microplate analysis. This approach significantly enhances the qualitative and quantitative assessment of membrane proteins solubilized in polymer nanodiscs, enabling improved detection sensitivity, reduced background, and precise visualization of subunits. By facilitating accurate biochemical characterization of membrane proteins in their native-like lipid environments, this method provides a powerful tool for structural and functional proteomics across diverse biological systems.

This study investigated the synthesis and chiral separation of a series of bio-based amide derivatives. Moreover, the greenness of both separation techniques, that is, high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC), was compared. The synthesis employed a two-step, solvent-free, one-pot procedure at 60°C, yielding esters quantitatively and amides with yields of 31%-70%. Chiral separations were performed on polysaccharide stationary phases under the most optimal green conditions. Using a 70/30 heptane/ethanol mobile phase in HPLC, racemate 13 exhibited the highest resolution (R_s = 11.8) and retention time (72.5 min); however, using a 40% ethanol phase reduced both resolution and retention times. SFC, using a 50/50 CO₂/ethanol mobile phase, provided baseline resolution (R_s = 1.51-9.13) for all analytes and significantly reduced analysis times (maximum retention time of 12.6 min for the second eluting enantiomer of racemate 13). Greenness evaluation was carried out using the AGREE and Analytical Method Greenness Score (AMGS) tools. The AGREE score averaged at 0.55 for HPLC and 0.64 for SFC, indicating a slight preference for SFC due to reduced solvent hazards and waste. The AMGS scores confirmed this trend: HPLC showed an average of 40.9 (up to 128 for racemate 13), whereas SFC averaged 14.9 (maximum 31.1). Instrument energy consumption was the dominant contributor to the AMGS score for both techniques, accounting for over 95% of the score for HPLC and over 93% for SFC. For racemate 13, this proportion increased to 99% and 96%, respectively.

Following the valsartan scandal in 2018, the testing of drug substances and drug products for N-nitrosamines has become a critical and mandatory quality control measure. The European Pharmacopoeia chapter 2.5.42 currently describes three analytical methods for this purpose: HPLC-MS/MS, GC-MS, and GC-MS/MS. The US Pharmacopeia monograph 〈1469〉 adds four other methods, LC-high-resolution mass spectrometry (HRMS), headspace GC-MS, LC-MS/MS, and GC-MS/MS. In addition, our group has developed a universal method on the basis of supercritical fluid chromatography (SFC), capable of separating 16 different N-nitrosamines within just 4 min. These eight methods differ significantly in terms of sustainability, with particular emphasis on the reagents used, the separation techniques employed, and their performance characteristics. When assessing the sustainability of such analytical methods, it is essential to consider not only ecological but also economic factors.