Plant-derived compounds interact with DNA and proteins, producing changes in molecular mobility, conformation, and complex formation. Capillary and gel electrophoresis detect and quantify these changes to determine binding affinity, interaction strength, specificity, stoichiometry, and mechanism. ABSTRACT Plant-derived compounds are increasingly recognized as promising therapeutic agents due to their broad biological activities and structural diversity, making their interactions with DNA and proteins central to understanding mechanisms of action and guiding drug discovery. Electrophoretic techniques probe these interactions by tracking changes in electrophoretic mobility, conformation, complex formation, and biopolymer integrity. This review surveys electrophoretic strategies for characterizing plant-derived compound-DNA and -protein interactions, covering fundamental DNA-binding modes (intercalation, groove binding, electrostatic association, cleavage), representative phytochemicals and extracts, and analytical challenges of complex plant matrices, including chemical heterogeneity, synergistic and antagonistic effects, and active-constituent identification. Particular attention is given to capillary electrophoresis-based approaches (affinity CE, CE, and pressure-assisted frontal analysis) and gel-based methods (agarose gel electrophoresis, native PAGE, SDS-PAGE). The literature shows capillary electrophoresis excels at quantifying binding affinity, stoichiometry, and specificity, whereas gel electrophoresis better visualizes ligand-induced changes in DNA topology and protein behavior. HSA and BSA dominate as model proteins, while advanced methods remain underapplied to chemically complex extracts, gaps pointing toward broader protein targets and natural-product systems. Integrating mass spectrometry, advanced detection, and data-driven approaches could further strengthen the mechanistic and quantitative reach of electrophoretic analysis in this field.

This study investigated site-specific N-glycosylation alterations in thyroglobulin from patients with papillary thyroid carcinoma using nano-LC–MS/MS. Advanced bioinformatics and machine learning identified these unique glycan signatures, establishing the Asn1365-HexNAc(3)Hex(6) glycoform as a highly predictive diagnostic biomarker (AUC = 0.83). ABSTRACT Thyroglobulin (Tg) is a clinically established biomarker for thyroid cancer; however, its diagnostic specificity remains limited due to confounding benign conditions. This study investigated changes in the site-specific N-glycosylation profiles of thyroglobulin protein derived from papillary thyroid cancer and adjacent healthy tissues. Using a bottom-up mass spectrometry-based glycoproteomic approach, we analyzed site-specific glycan compositions in retrospective patient tissue samples. Nine distinct N-glycosylation sites were identified and quantified in this study. Statistical analysis revealed significant differences in the site-specific glycosylation profiles. The glycan compositions HexNAc(3)Hex(5) and HexNAc(3)Hex(6) at the Asn1365 site exhibited a discriminatory potential. Dimensionality reduction and hierarchical clustering based on the top four glycopeptide variants showed observable grouping of tumor versus normal samples. Furthermore, supervised machine learning models were used to evaluate these features, with a neural network achieving the highest diagnostic performance (area under the curve [AUC] = 0.83). Feature importance analysis indicated that Asn1365-HexNAc(3)Hex(6) was the strongest contributor to the predictive power of the model. The observed alterations in specific glycan compositions, combined with machine learning evaluations, may offer improved diagnostic specificity and provide promising avenues for personalized thyroid cancer management.

ABSTRACT Monitoring of critical quality attributes (CQAs) is essential for the development of biotherapeutics. One example of a CQA is molecular fragmentation, which is often analyzed by capillary gel electrophoresis with sodium dodecyl sulfate (SDS). Sialylation is a post-translational modification and form of glycosylation that can impact purity profiles of biotherapeutics, resulting in complex structure elucidation. Here, we studied the heterogeneity of Biotherapeutic 1 as a result of O-linked glycosylation with sialylation. Biotherapeutic 1 displayed a second unidentified peak in SDS–capillary gel electrophoresis (SDS-CGE) under reducing conditions that directly impacted peak integration practices and, therefore, method validation. The two peaks were highly reproducible in SDS-CGE as well as complementary LabChip experiments. The apparent molecular weights were calculated using molecular weight ladders with known protein standards. A combination of ion exchange chromatography (IEX), hydrophilic interaction chromatography mass spectrometry (HILIC-MS), and ultra-high-performance size exclusion chromatography (UP-SEC) were used to identify O-linked glycosylation as responsible for the production of reduced peak 1 and reduced peak 2 in SDS-CGE. Specifically, reduced peak 2 contained sialylation that was not observed in reduced peak 1, resulting in two distinct migration times due to impacts in SDS binding efficacy. Enzymatic removal of the sialic acids simplified the heterogeneity into a single uniform peak (reduced peak 1). This work and methodologies highlight the impact a single O-linked glycan can have on SDS-CGE and is applicable to analyzing future biotherapeutics involving complex structure profiles resulting from sialylation.

ABSTRACT 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.

Combining high-recovery ChocoTip desalting with DMNG-assisted reconstitution improved peptide recovery in low-input proteomics. The workflow increased peptide identifications and enhanced the recovery of longer and more hydrophobic peptides from approximately 50-cell-equivalent HeLa digests. ABSTRACT Low-input mass spectrometry (MS)-based proteomics is vulnerable to peptide loss during final sample preparation. We evaluated whether ChocoTip desalting and decyl maltose neopentyl glycol (DMNG)-assisted reconstitution improve peptide recovery from HeLa digests corresponding to 10 ng of protein (∼50 cells). ChocoTip reduces irreversible peptide adsorption into mesopores of chromatographic particles during microscale desalting, whereas DMNG is expected to suppress hydrophobic-interaction-mediated nonspecific adsorption to plasticware during redissolution, transfer, and injection. After selecting DMNG as the nonionic detergent additive, we compared conventional StageTip desalting, ChocoTip desalting, DMNG-assisted reconstitution, and their combination. ChocoTip and DMNG method each improved peptide recovery but affected the peptide populations eluting in different regions of the nanoflow reversed-phase liquid chromatography (nanoRPLC) gradient. Their combination yielded the highest number of peptide identifications and increased peptide intensities across almost the entire nanoRPLC gradient, including commonly identified peptides. Peptides uniquely recovered by the combined workflow covered a broad hydrophobicity range and included longer sequences. These results indicate that suppressing peptide loss at both desalting and post-desalting stages provides a simple strategy to improve sensitivity and peptide coverage in low-input MS-based proteomics.

A rapid and environment-friendly one-step extraction method was developed for the determination of chiral and achiral pesticides in plant-based beverages by UHPLC–HRMS/MS. The proposed workflow provides a simple and efficient alternative for residue analysis in these increasingly consumed food matrices, such as hazelnut-based beverages. ABSTRACT The increasing consumption of plant-based beverages (PBBs), driven by ethical and health-related reasons, highlights the need for robust analytical methods assessing their chemical safety. Among these products, hazelnut-based beverages are of particular interest in Italy, where hazelnut production is economically relevant. A rapid one-step extraction procedure was developed for the multiresidue determination of selected chiral and achiral pesticides in PBBs by ultra-high-performance liquid chromatography–high resolution mass spectrometry (UHPLC–HRMS). The method combines matrix precipitation and analyte extraction in a single step, providing a simple and efficient workflow. Validation, carried out in hazelnut beverage, included recovery, precision, linearity, decision limit (CCα), and detection capability (CCβ) evaluation. As no European Union maximum residue levels (MRLs) are available for PBBs, provisional MRLs for hazelnut milk were derived from raw hazelnut MRLs using a recipe-based processing factor, according to Regulation (EC) No 396/2005. The method achieved recoveries of 60%–90%, intra- and inter-day precision generally below 20%, CCα values of 0.001–0.016 µg L−1, CCβ values of 0.002–0.027 µg L−1, and good linearity for most analytes (R 2 > 0.99). The proposed procedure is suitable for pesticide residue analysis in PBBs, such as hazelnut-based beverages, and represents a valuable approach for monitoring emerging food matrices, for which residue data and regulatory limits are still lacking.

CE–MS enabled rapid, derivatisation-free quantification of 19 amino acids and related derivatives in Lucilia sericata larval excretions/secretions. The validated workflow revealed reproducible amino acid profiles, providing new quantitative insight into the low-molecular-weight composition of maggot secretions relevant to wound-healing research. ABSTRACT A capillary electrophoresis–mass spectrometry (CE–MS) method was developed and validated for the determination of 19 amino acids and related compounds in excretions/secretions (ES) of Lucilia sericata larvae. Sample preparation consisted of dilution of ES with water, filtration and direct CE–MS analysis. Separation was performed with 50 mM formic acid as the background electrolyte, and methanol–water (1:1, v/v) containing 1% formic acid was used as sheath liquid. The total analysis time was 20 min. The method showed acceptable linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), sensitivity, apparent recovery and short-term applicability for freshly prepared samples. Correlation coefficients for the calibration curves ranged from 0.983 to 0.998. Precision was within 18% at the lowest quality control level and within 15% at medium and high levels. Accuracy ranged from 83% to 118% at the lowest level and from 85% to 115% at the other levels. In larval ES, the most abundant compounds were threonine (565 ± 7 µM), tryptophan (455 ± 6 µM), anserine (404 ± 7 µM), histidine (330 ± 14 µM) and cystine (279 ± 4 µM). The method offers a simple workflow for targeted profiling of amino acids and related compounds in larval ES.

The application of discrete spacers—2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS) and histidine—significantly enhances electrophoretic resolution of thiamine and pyridoxine in the unconventional hydrodynamically closed 2D-CITP–MS/MS system. The method demonstrates high reproducibility, minimal signal suppression, and strong analytical greenness, as confirmed by the presented scoring metrics. ABSTRACT The effect of specific spacers added to the separation environment of capillary isotachophoresis in a hydrodynamically closed two-dimensional device hyphenated with tandem mass spectrometry is reported in this short communication. Combination of two spacers, here, 2-amino-2-hydroxymethyl-propane-1,3-diol and histidine, led to enhanced resolution of two model analytes (thiamine and pyridoxine) in model and real pharmaceutical samples (tablet drug dosage form). The addition of the spacers at relatively high concentration level (i.e., 20 µg/mL) caused no significant interference and suppression of the analytical signal in the mass spectrometry detection step. Eco-friendliness, practicality, and performance of the method were confirmed using the metric tools of the “White Analytical Chemistry.” High repeatability of the separation process could broaden the application possibilities of the hydrodynamically closed capillary electrophoresis system.

Natural compound phosphonothrixin's herbicidal activity resides exclusively in its (S)-enantiomer, yet existing enantioselective syntheses are cumbersome, and direct chiral LC separation is hampered by the compound's high polarity, lack of aromatic groups, and metal chelation–induced peak tailing. By employing a zwitterionic sulfohexyl quinidine carbamate, stationary phase reliable analytical and preparative chiral separations were achieved, enantiomers isolated, and absolute configurations assigned by optical rotation and ECD spectroscopy. ABSTRACT Phosphonothrixin (PPX), a phosphonate natural product known for its potent herbicidal activity, has attracted renewed interest because of the need for alternative herbicides. Its biological activity is confined to the (S)-enantiomer. Although the only reported enantioselective synthesis is complex, labor-intensive, and low yielding, direct chiral liquid chromatographic separation is likewise challenging because of its high polarity and lack of aromatic moieties, which limit interactions with commonly used chiral stationary phases (CSPs). In addition, phosphonic acid-containing compounds tend to chelate metal ions and adsorb to metal surfaces, further complicating chromatographic separation by causing pronounced peak tailing. Here, we describe a reliable and reproducible analytical and preparative method for the direct chiral separation of PPX using a zwitterionic sulfohexyl quinidine and quinine carbamate CSPs, as well as two synthetic phosphonate derivatives using a cellulose tris(3,5-dichlorophenylcarbamate) stationary phase as an alternative approach. The best separation of PPX was achieved with the zwitterionic sulfohexyl quinidine CSP (Rs = 2.4). The separated enantiomers were isolated, and their chemical and stereochemical identities were confirmed by mass spectrometry, optical rotation, and electronic circular dichroism spectroscopy. This approach enabled the successful isolation of enantiomerically pure (S)-(−)- and (R)-(+)-PPX.

The separation of selected mycosporine-like amino acids and two biosynthetic precursors, in total 13 compounds, by HILIC-UPLC is described for the first time. With an analysis time of only 5 min, it surpasses all currently known methods for analysis of these promising natural products. ABSTRACT Mycosporine-like amino acids (MAAs) are photoprotective compounds primarily produced by marine organisms, especially red algae. Chemically, MAAs and their precursors are low-molecular-weight natural products that absorb ultraviolet radiation in the range of 270–360 nm without generating free radicals. Owing to these properties, MAAs have attracted considerable interest for potential pharmaceutical and cosmetic applications as natural sunscreen agents. In this study, the first hydrophilic interaction liquid chromatography-ultra-performance liquid chromatography (HILIC-UPLC) method, coupled with both diode array detector (DAD) and tandem mass spectrometry (MS/MS), was developed for the determination of 11 MAAs and 2 MAA precursors in various algal species. As column, a YMC-Triart Diol-HILIC (particle size: 1.9 µm) was used, and the separation of the MAAs realized in under 5 min. The validation of the method was carried out following International Council for Harmonisation (ICH) guidelines, demonstrating linearity, selectivity, precision, and accuracy. The analysis of extracts prepared from well-known species, such as Porphyra sp. and Chondrus crispus, confirmed practical applicability. Demonstrating feasibility of MS/MS-based quantification of co-eluting compounds, the method represents an appealing alternative that surpasses established procedures not only in terms of analysis time but also by providing a substantially increased informative value.

ABSTRACT Capillary electrophoresis (CE) coupled with capacitively coupled contactless conductivity detection (CE–C4D) has emerged as a sensitive, versatile, cost-effective, and environmentally friendly technique for pharmaceutical analysis. The primary objective of this review is to critically evaluate pharmaceutical applications of CE–C4D, including active pharmaceutical ingredient (API) quantification, chiral separations, counter-ion analysis, and impurity and degradation product profiling. A concise and selective overview of CE–C4D fundamentals and key stages of its development—from early oscillometric detectors to advanced dual-axial designs—is provided solely to establish essential detection principles and to support discussion of method optimization, particularly the critical role of background electrolyte (BGE) selection. Summary tables compile BGE compositions and limits of detection (LODs), offering a comparative resource for researchers. Compared with conventional detectors such as ultraviolet (UV), fluorescence, and mass spectrometry (MS), CE–C4D enables universal and direct analysis of both UV-active and non-UV-active compounds, with additional advantages in portability and miniaturization.

ABSTRACT Spontaneous isolated superior mesenteric artery dissection (SISMAD) is a rare but increasingly recognized vascular disorder that can lead to intestinal dysfunction with severe complications. Currently, no plasma biomarkers, particularly metabolomics-based markers, have been identified for SISMAD. To identify novel plasma biomarkers and potential therapeutic targets for SISMAD and its associated intestinal dysfunction, we analyzed plasma samples from 30 patients with SISMAD and 30 healthy controls. Blood was collected within 72 h of diagnosis. Ultra-high-performance liquid chromatography coupled with orbitrap mass spectrometry was used to profile metabolites, followed by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) to analyze the data. A total of 123 and 128 metabolites were significantly changed in the positive and negative ion modes, respectively. Notably, tryptophan-related metabolites, including methyl 2-(1H-indol-3-yl) acetate and indole-3-pyruvic acid (IPA), were significantly reduced in the SISMAD group (p 

ABSTRACT 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.

ABSTRACT 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.

ABSTRACT This work focuses on the separation of oligonucleotides (ONs) in a free solution by capillary electrophoresis—mass spectrometry (CE-MS). Specifically, we evaluated a combination of separation in acidic background electrolytes (BGEs), nanospray ionization, and time-of-flight mass spectrometry in positive mode. A mixture of synthetic ONs, ranging in length from 15 to 78 nt, was employed as the test compounds. The key to a good separation selectivity of ONs lies in the different protonation of individual nucleobases. To this end, we assessed the acidity constant (pK a) of the nucleobases in nucleotides experimentally as 3.3 for adenine, 4.4 for cytosine, 2.5 for guanine, and  40 nt.

ABSTRACT Didelphis aurita is a synanthropic marsupial widely distributed in southeastern Brazil, known for its resistance to venom and its relevance in biomedical research. This study aimed to characterize the serum proteinogram of free-living D. aurita individuals. Blood samples from 27 animals, classified as “healthy” or “diseased,” were analyzed. Eighteen protein bands were identified, with molecular weights ranging from 24 to 242 kDa. Among these, variations in specific bands were associated with health status (band J), sex (bands D, M, N, and P), and age (bands N and P). Mass spectrometry (liquid chromatography–tandem mass spectrometry [LC–MS/MS]) identified seven proteins, including DM64, ceruloplasmin, von Willebrand factor A (VWFA) domain protein, alpha-2-macroglobulin, fibronectin, and actin depolymerizing factor. These results highlight the influence of biological factors on serum protein profiles and reinforce the potential of D. aurita as a model for immunological and proteomic studies.

Abstract Heparan sulphates (HSs), a specific class of glycosaminoglycans (GAGs), are important participants of cellular signalling. Analytical characterization of GAGs requires a complex sample preparation workflow. Although a detailed stability and recovery study is available for the chondroitin sulphate GAG class, the literature concerning HS is incomplete in this regard. Therefore, our aim was to systematically investigate various parameters that could potentially influence the stability and recovery of HS samples when performing disaccharide analysis using high-performance liquid chromatography–mass spectrometry. First, effects concerning vacuum evaporation and freezing were investigated. Next, the storage stability of the HS disaccharides was analysed under several conditions such as temperature, pH, digestion buffers, injection solvents and storage vessels. We have identified several critical parameters influencing the stability and recovery of HS disaccharides. We concluded that major sample loss is expected when Tris–HCl is used as digestion buffer, followed by vacuum evaporation at elevated temperatures, or samples are stored under alkaline conditions. Following the practical considerations of this paper can contribute to increasing the reliability of future analytical measurements.

ABSTRACT In diabetes management, oral formulation of insulin (INS) has the potential to improve safety, convenience, and patient-centered care compared to subcutaneous injections. However, its bioavailability remains limited, necessitating improved delivery strategies. Recent clinical trials indicate that taurocholic acid (TCA) can enhance the bioavailability of oral INS as an absorption enhancer. In this work, electrospray ionization mass spectrometry (ESI-MS) analysis revealed the formation of 1:1–1:4 INS–TCA complexes. MS/MS was used to explore the fragmentation pathway of complex ions and confirm binding stability in the gas phase. Circular dichroism spectra showed no clear conformational change in INS upon TCA binding, even though TCA enhanced INS's structural stability. Using Taylor dispersion analysis (TDA), we determined the diffusion coefficient and hydrodynamic radius of INS and its complexes. TCA binding was observed to increase INS size in both the 1:1 and 1:2 INS–TCA complexes. The binding constant of INS and TCA (1.3 × 103 L/mol) with approximately five binding sites was obtained via pressure-assisted capillary electrophoresis frontal analysis. Molecular docking simulations indicated that TCA binds to external binding sites on the INS B chain (near Ser-B9, Glu-B13, and Phe-B24 residues), consistent with ESI-MS and TDA results. These findings suggest that TCA binding may enhance INS absorption and increase the bioavailability of oral INS therapy.