Journal of Chromatography A最新文献

{"title":"Simultaneous quantification of cytidine, methylcytidine, and hydroxymethylcytidine by isotope-dilution LC–MS/MS with application to mouse liver samples","authors":"Pooja Mishra ,&nbsp;Jing Ma ,&nbsp;Huan Xie ,&nbsp;Fang Li ,&nbsp;Robert Y.L. Tsai ,&nbsp;Dong Liang","doi":"10.1016/j.chroma.2026.466721","DOIUrl":"10.1016/j.chroma.2026.466721","url":null,"abstract":"<div><div>DNA methylation and hydroxymethylation are important epigenetic modifications that play key roles in cancer development and aging processes by regulating gene expression and genome stability. Traditionally, bisulfite conversion-based or antibody-based enzyme-linked immunosorbent assays are used to find DNA methylation. These tests are non specific, tedious, and not able to differentiate the difference between methylation and hydroxymethylation. To address these issues, we developed a sensitive, reproducible, and specific LC–MS/MS method for simultaneous quantification of two major DNA methylation products, 5-methyl-2′-deoxycytidine (5-mdC) and 5-hydroxymethyl-2′-deoxycytidine (5-hmdC), as well as 2′-deoxycytidine (2-dC), using corresponding stable isotope-labeled internal standards: 5-methyl-2′-deoxycytidine-d₃, 5-(hydroxymethyl)-2′-deoxycytidine-d₃, and 2′-deoxycytidine-¹³C,¹⁵N₂. We purified DNA samples from mouse liver tissue, broke them down with enzymes, filtered them, added internal standards, and then run them through a SCIEX 6500+ Triple Quad LC–MS/MS system with an Atlantis T3 C18 column under a binary gradient. The method showed great chromatographic separation and specificity, with MRM transitions of m/z 228.154 to 112.1 for 2-dC, 242.143 to 126.2 for 5-mdC, and 258.135 to 142.1 for 5-hmdC.Peak area ratio of analyte to internal standard exhibited linearity across calibration ranges of 5–5000 ng /mL for 2-dC, 0.5–500 ng/ mL for 5-mdC, and 0.05–10 ng/mL for 5-hmdC (R² &gt; 0.999), using 2 µL injection and a total runtime of 9 min. The 5-hmdC level in female mouse liver significantly increased with aging from two to sixteen months old (0.0958 % to 0.1984 %; <em>P</em> <em>&lt;</em> <em>0.001</em>), whereas 5-mdC remained unchanged (3.47 % to 3.56 %; n.s.). These data confirm the accurate and reproducible quantification of DNA methylation and hydroxymethylation in tissue samples using the developed LC-MS/MS assay and indicate a broad application to cell culture and clinical biomarker studies.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1769 ","pages":"Article 466721"},"PeriodicalIF":4.0,"publicationDate":"2026-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146045931","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Synthesis of modified siloxane-based three-dimensional porous polymer as adsorbent for high-efficiency enrichment of phenyl urea herbicides from vegetables","authors":"Miao Jing ,&nbsp;Juntao Wang ,&nbsp;Ruiyang Ma ,&nbsp;Yaxing Guo ,&nbsp;Zichen Zhao ,&nbsp;Qiuhua Wu ,&nbsp;Zhi Wang","doi":"10.1016/j.chroma.2025.466664","DOIUrl":"10.1016/j.chroma.2025.466664","url":null,"abstract":"<div><div>A porous hyper-crosslinked polymer (OPS/BCM-HCP) was synthesized for the first time using cage-shaped octaphenyl POSS as the monomer and biphenyl-p-dichlorobenzene as the cross-linker for the efficient enrichment of phenylurea herbicides (chlorotoluron, isoproturon, monolinuron and buturon). High performance extraction for the phenylureas was realized by using the OPS/BCM-HCP as the SPE adsorbent from water, cabbage and white radish samples. Under the optimized conditions, the newly established method by coupling the SPE with HPLC-UV had a good linear response for phenylureas in the range of 0.06–100.0 ng mL⁻¹ for water, 2.10–200.0 ng g⁻¹ for cabbage, and 1.20–200.0 ng g⁻¹ for white radish samples, with the determination coefficient larger than 0.9912. The limits of detection (LODs, S/<em>N</em> = 3) and limits of quantification (LOQs, S/<em>N</em> = 9) were 0.02–0.03 and 0.06–0.09 ng mL⁻¹ for water, 0.70–0.95 and 2.10–2.85 ng g⁻¹ for cabbage, and 0.40–0.85 and 1.20–2.55 ng g⁻¹ for white radish, respectively. The method recoveries were 80.0 %-116 % with the RSDs from 3.6 % to 8.9 %. The experimental results showed that OPS/BCM-HCP had an excellent adsorption effect on phenylurea herbicides, and the method provided an alternative way for the effective quantification of the herbicides in vegetable samples.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1769 ","pages":"Article 466664"},"PeriodicalIF":4.0,"publicationDate":"2026-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145883754","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Reversed-phase chromatography of peptides carrying non-canonical proline analogues, with special focus on 4R/4S-fluoroproline","authors":"Alexandre Prefontaine ,&nbsp;Justin Liba ,&nbsp;Vic Spicer ,&nbsp;Suvrat Chowdhary ,&nbsp;Christin Treiber-Kleinke ,&nbsp;Vladimir Kubyshkin ,&nbsp;Beate Koksch ,&nbsp;Ned Budisa ,&nbsp;Oleg V. Krokhin","doi":"10.1016/j.chroma.2026.466704","DOIUrl":"10.1016/j.chroma.2026.466704","url":null,"abstract":"<div><div>We investigated the reversed-phase chromatographic behaviour of peptides carrying nine non-canonical proline analogues using LC-MS/MS bottom-up proteomics analysis of <em>Escherichia coli</em> (<em>E.coli)</em> following co-translational incorporation. Of particular interest were the epimers 4<em>R</em>- and 4<em>S</em>-fluoroproline. On average, peptide interactions with the hydrophobic C18 phase increased in the order: 4<em>R</em>-Flp &lt; Pro &lt; 4<em>S</em>-Flp. The mean retention shifts were found to be -0.11 and +0.06% acetonitrile for 4<em>R</em>- and 4<em>S</em>-Flp, respectively (0.1% formic acid eluent additive). This diminutive average difference in hydrophobic properties belies the unpredictable chromatographic behaviour of modified peptides. The incorporation of either fluorinated epimer, each relatively amphipathic, can make certain peptides more hydrophobic, while for others, the peptide becomes more hydrophilic. Incorporation of a single 4<em>R</em>-fluoroproline produces a shift ranging from (2.18 to +2.55) %ACN, while <em>4S-</em>fluoroproline produces shifts ranging from (-4.82 to +4.67) %ACN on the RP HPLC elution scale. In addition to common composition-dependent factors affecting peptide retention, such as peptide length, we identified several sequence-specific features that explain deviations from the average retention characteristics. These include nearest neighbour effects and amphipathic helicity. Other analogues investigated encompassed diverse chemical features, including hydroxylation (4<em>R</em>-hydroxyproline), dehydrogenation (3,4-dehydroproline), ring expansion (pipercolic acid), heteroatom inclusion (3-thia-proline &amp; 4-thia-proline) and bridge ring structures (4,5-<em>trans</em>-methanoproline &amp; 4,5-<em>cis</em>-methanoproline).</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1769 ","pages":"Article 466704"},"PeriodicalIF":4.0,"publicationDate":"2026-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145973922","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Ionic liquid-based aqueous two-phase extraction as a pretreatment strategy for the determination of 38 pharmaceuticals and personal care products in aquatic environments by HPLC-MS/MS","authors":"Shuo Xie ,&nbsp;Wenbin Zhang ,&nbsp;Yike Han ,&nbsp;Xinyan Dong ,&nbsp;Wanning Li ,&nbsp;Penghui Li ,&nbsp;Liyun Kong","doi":"10.1016/j.chroma.2026.466670","DOIUrl":"10.1016/j.chroma.2026.466670","url":null,"abstract":"<div><div>The accurate determination of pharmaceuticals and personal care products (PPCPs) in wastewater is crucial for comprehensive environmental risk assessment and human exposure evaluation. Given the complex matrices of aqueous environmental samples, efficient sample preparation remains a critical challenge in analytical procedures. This study presents an ionic liquid-based aqueous two-phase system (IL-ATPS) extraction methodology used as a pretreatment step prior to high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of 38 PPCPs. A mechanistic investigation revealed that target analytes with more hydrogen-bonding sites and higher hydrogen-bond donor (HBD) capacity exhibited enhanced solubility in ILs. In addition, IL anions with stronger hydrogen-bond acceptor (HBA) capacity further improved the solubility of the 38 PPCPs. These findings indicate that hydrogen bonding between analytes and ILs likely played a critical role in the dissolution mechanism. The established IL-salt ATPS extraction method utilized a 15 mL aqueous sample, 0.2 g tetraethylammonium acetate ([N₂₂₂₂][OAc]), and 12 g K₂HPO₄, with a 1-minute vortex mixing. Compared with traditional solid phase extraction (SPE), this approach significantly reduced the sample volume from 1 L to 15 mL, avoided the use of volatile organic solvents, simplified the overall processing, and enabled rapid (&lt;15 minutes) pretreatment. The method demonstrated excellent sensitivity with limits of detection (LODs) ranging from 0.00013 to 0.11 ng/mL. Method validation via standard addition experiments demonstrated satisfactory accuracy, yielding recoveries between 74.3 and 129.8 %. Precision was assessed using both intra-day and inter-day measurements. The intra-day relative standard deviations (RSDs) ranged from 0.7 % to 19.9 %, while the inter-day RSDs ranged from 1.4 % to 18.4 %, demonstrating that the method provides consistent and reproducible results under controlled laboratory conditions. These findings underscore the present IL-based ATPS extraction method, followed by HPLC-MS/MS analysis, as an eco-friendly, robust, and scalable approach for PPCPs monitoring in aqueous environmental samples, offering potential applications in environmental monitoring and risk assessment.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1769 ","pages":"Article 466670"},"PeriodicalIF":4.0,"publicationDate":"2026-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145909693","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"pH-dependent separation and identification of saponins from Beta vulgaris L. using high-speed countercurrent chromatography and high-resolution mass spectrometry","authors":"Anna Tekieli ,&nbsp;Mariusz Kowalczyk ,&nbsp;Sławomir Wybraniec ,&nbsp;Aneta Spórna-Kucab","doi":"10.1016/j.chroma.2025.466643","DOIUrl":"10.1016/j.chroma.2025.466643","url":null,"abstract":"<div><div>This study investigated the saponins from <em>Beta vulgaris</em> L. cv. Cylindra roots using a combination of semi-preparative high-speed countercurrent chromatography (HSCCC) and ultra high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS/MS). A new solvent system was developed for the HSCCC purification of saponins under varying pH conditions (3, 5, and 7), which significantly affected their separation. The solvent system consisted of 1-butanol, acetone, acetonitrile, and citrate-phosphate buffer (1.0:0.1:0.05:0.8; v/v/v/v).</div><div>The pH adjustment in HSCCC noticeably affected separation efficiency, with higher pH values generally leading to faster elution. The separation behavior of saponins depended primarily on the type, number, and arrangement of sugar substituents. In the HSCCC system, compounds bearing more sugar residues tend to be eluted earlier because their greater hydrophilicity enhances interactions with the mobile phase. Despite this overall trend, saponins containing terminal hexose or pentose units were eluted later, indicating that such sugars can significantly extend elution time. Additionally, saponins featuring dioxolane moieties showed a particularly strong pH dependence, with pH adjustments causing reversible shifts in the elution order of isomeric pairs.</div><div>Ten predominant saponins, including the highly concentrated betavulgaroside IV and betavulgaroside III, were quantified with high accuracy using UHPLC<img>-HRMS/MS. The total saponin content in Cylindra was found to be 7.2 g/kg dry extract (DE), significantly higher than previously reported for other <em>B. vulgaris</em> cultivars. Additionally, the study identified 47 saponins, including the novel aglycone norhederagenin (<em>m/z</em> 455.31), thus expanding the phytochemical profile of beetroot.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1769 ","pages":"Article 466643"},"PeriodicalIF":4.0,"publicationDate":"2026-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145899055","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Analysis of mRNA multimerisation (aggregation) using non-denaturing ion-pair reversed-phase liquid chromatography","authors":"Alexandra L.J. Webb ,&nbsp;Emma N. Welbourne ,&nbsp;Thomas C. Minshull ,&nbsp;Kate A. Loveday ,&nbsp;Prerna Bora ,&nbsp;Zoltán Kis ,&nbsp;Gunilla A. Nilsson ,&nbsp;Andal Murthy ,&nbsp;Eivor Örnskov ,&nbsp;Mark J. Dickman","doi":"10.1016/j.chroma.2025.466653","DOIUrl":"10.1016/j.chroma.2025.466653","url":null,"abstract":"<div><div>mRNA-based technology has emerged as a new class of medicines with a wide range of applications, including viral vaccines, cancer vaccines, and therapeutics for the treatment of metabolic diseases and cardiovascular conditions. Impurities, including double-stranded RNA (dsRNA), mRNA fragments, and mRNA multimers (aggregates) that result from the manufacturing of mRNA, as well as from subsequent purification, formulation, and storage, can potentially impact the safety and efficacy of mRNA medicines.</div><div>mRNA higher-order structures and mRNA multimers (aggregates) can affect translational efficiency and also impact the efficiency of formulation into lipid nanoparticles. mRNA purity is typically analysed using denaturing or partially denaturing methods, precluding the detection of mRNA multimers (aggregates). In this study, we developed and utilised ion-pair reversed-phase HPLC (IP-RP HPLC) under non-denaturing conditions to analyse mRNA multimers. The inclusion of 1 mM Mg²⁺ in the mobile phase stabilises mRNA higher-order structures, RNA:RNA interactions, and the formation of mRNA dimers/multimers, which can be readily separated from the mRNA monomers.</div><div>The ability to resolve mRNA monomers from mRNA dimers/multimers was demonstrated for a range of mRNA sequences and lengths. Moreover, we have shown that the relative abundance of mRNA dimers/multimers is concentration dependent. Using the relative percentage of dimer vs concentration of monomer, we were able to determine that the K_d of the interaction between two eGFP mRNA monomers was 82.93 nM. Characterisation and sizing of the mRNA multimers was performed using mass photometry analysis following the purification of mRNA monomer and dimer/multimer peaks using IP-RP HPLC.</div><div>Thus, non-denaturing IP-RP demonstrates significant advantages over current approaches for the analysis of mRNA multimers (aggregates). The high-throughput, temperature-dependent profiling of mRNA multimerisation using IP-RP HPLC will enable further comparative studies on the stability of mRNA multimers and provide important insights into potential factors influencing mRNA multimerisation.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1769 ","pages":"Article 466653"},"PeriodicalIF":4.0,"publicationDate":"2026-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145964696","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Solid phase extraction based on a coral like structure conjugated microporous polymer for the detection of preservatives in milk and water","authors":"Hai-Long Jiang ,&nbsp;Hong-Yan Liu ,&nbsp;Xiao-Li Wang ,&nbsp;Da-Feng Jiang ,&nbsp;Dong-Mei Liu ,&nbsp;Jin-Peng Yuan ,&nbsp;Xia Wang ,&nbsp;Ru-Song Zhao","doi":"10.1016/j.chroma.2025.466667","DOIUrl":"10.1016/j.chroma.2025.466667","url":null,"abstract":"<div><div>In this study, a coral like structure conjugated microporous polymer with abundant N/O functional groups was synthesized using a one-step method. The synthesized material was assembled into a solid-phase extraction (SPE) column, and it showed efficient extraction performance for paraben-based preservatives. Under optimized SPE conditions, a high performance liquid chromatographic (HPLC) method with good linear (r≥0.998), low detection limit (0.07-0.19 ng/mL) and high precision (1.8-6.7%, n=6) was developed. The applicability of this method was evaluated by analyzing real samples (milk and water), with spiked recovery rates ranging from 82.8% to 106%, further confirming the accuracy and reliability of the method. The adsorption mechanism was also discussed by simulation calculations, and it was mainly dominated by hydrogen bonding, π-π interactions and van der Waals forces.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1769 ","pages":"Article 466667"},"PeriodicalIF":4.0,"publicationDate":"2026-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145883756","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Preparation of a novel C18-sulfobetaine stationary phase based on silica monolith particles for multimodal high performance liquid chromatography","authors":"Ashraf Ali ,&nbsp;Ya Song ,&nbsp;Yongxing Hu ,&nbsp;Sarah Alharthi ,&nbsp;Eman Y. Santali ,&nbsp;Wenjie Zhao","doi":"10.1016/j.chroma.2026.466683","DOIUrl":"10.1016/j.chroma.2026.466683","url":null,"abstract":"<div><div>Mixed-mode stationary phases enhance separation flexibility for complex samples by integrating multiple retention mechanisms in high performance liquid chromatography (HPLC). In this study, a novel mixed-mode stationary phase was prepared by functionalizing sub-1 µm porous silica monolith particles (pore size ∼ 35 nm) with 4-vinyl-1,3-sulfopropyl pyridinium betaine (VSPB) and octadecyl silane (C18). The stationary phase (SMP-C18-SB) was characterized by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy, transmission electron microscopy, solid-state 13C NMR spectroscopy, Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) analysis. The resultant stationary phase was packed into stainless-steel column (150×4.6 mm) by slurry packing method with sequential pressure of 80-100 MPA. The SMP-C18-SB column demonstrated excellent separation performance for alkylbenzenes, nucleosides, zwitterionic compounds, peptides, and proteins, facilitated by hydrophobic, hydrogen bonding, and hydrophilic interactions from C18 and SB groups. SMP-C18-SB column exhibited superior separation performance in both reversed-phase and hydrophilic interaction modes. Moreover, the column back pressure was quite low owing to the irregular shape of SMPs. The SMP-C18-SB column also exhibited excellent repeatability, with intra-day relative standard deviations (RSDs) of &lt;0.11% for retention time and &lt;1.02% for peak area (n = 10 each) after nearly 1000 consecutive injection. Owing to its versatile mixed-mode capability, the SMP-C18-SB column could be used for the separation of complex mixtures containing polar, non-polar and zwitteionic compounds.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1769 ","pages":"Article 466683"},"PeriodicalIF":4.0,"publicationDate":"2026-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145973924","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Integrated HPLC–UV validation and LC–MS/MS optimization with molecular networking and chemometrics for advanced characterization of chlorogenic acids in Ilex guayusa","authors":"Thomas Garzón ,&nbsp;Jefferson V. Pastuña-Fasso ,&nbsp;Cristian Quiroz-Moreno ,&nbsp;Melanie Ochoa-Ocampo ,&nbsp;Evencio J. Medina-Villamizar ,&nbsp;Nina Espinosa de los Monteros-Silva ,&nbsp;Karel Diéguez-Santana ,&nbsp;Jessica L. Cooperstone ,&nbsp;Zulay Niño-Ruíz ,&nbsp;José R. Almeida ,&nbsp;Noroska G.S. Mogollón","doi":"10.1016/j.chroma.2026.466732","DOIUrl":"10.1016/j.chroma.2026.466732","url":null,"abstract":"<div><div>Chlorogenic acids (caffeoylquinic acid isomers, CQAs) are major phenolic constituents of <em>Ilex guayusa,</em> but their comprehensive profiling in complex plant matrices is hindered by co-elution, overlapping UV spectra, and isomeric similarity in MS/MS. Rather than aiming to fully resolve isomer-specific quantification by MS, here we present an integrated workflow that couples validated HPLC–UV quantification of the major CQA (5-CQA) with an optimized UPLC–MS/MS strategy designed to improve MS1 peak integrity and expand MS/MS coverage for higher-confidence structural annotation. The HPLC–UV method showed excellent performance for targeted quantification of 5-CQA, including strong linearity (r² = 0.998), selectivity, sensitivity (LOQ = 0.25 mg/L), precision, and recovery. For LC–MS/MS, FastDDA acquisition (top-5 vs. top-15 precursors) revealed the expected trade-off between fragmentation depth and MS1 peak quality; however, post-acquisition raw-data merging restored MS1 fidelity and increased the number of detected features by 43%, enabling high-confidence annotation rather than quantitative discrimination of 16 metabolites and the propagation of oxidized CQA-related derivatives using feature-based molecular networking. Multivariate analyses (PCA, volcano plots, HCA) indicated that geographic location exerted the strongest influence on the metabolite composition, followed by sunlight exposure and plant age. Overall, the proposed workflow provides a practical framework that integrates robust chromatographic quantification with MS acquisition and data-processing optimization, thereby enhancing structural characterization and biological interpretation, rather than complete isomer-resolved quantification, of chlorogenic-acid-related chemistry across complex plant-derived and natural product matrices</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1769 ","pages":"Article 466732"},"PeriodicalIF":4.0,"publicationDate":"2026-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146074172","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular dynamics simulations elucidate the structural determinants of size-exclusion chromatography behavior in dimeric G-quadruplex-RHAU","authors":"Min Zhu ,&nbsp;Long-yu Zhu ,&nbsp;Lu-yan An ,&nbsp;Ju Wang ,&nbsp;Jun-qin Qiao ,&nbsp;Han-yue Yang ,&nbsp;Wei-juan Zheng ,&nbsp;Hong-zhen Lian","doi":"10.1016/j.chroma.2026.466706","DOIUrl":"10.1016/j.chroma.2026.466706","url":null,"abstract":"<div><div>Research on G-quadruplexes (G4s)-RNA helicase associated with AU-rich element (RHAU) interaction has facilitated G4s-targeted therapeutic development. Despite this progress, the interaction between dimeric G-quadruplexes (<span>d</span>-G4s) and RHAU remain less explored compared to monomeric structures. Developing convenient and visual methods to elucidate the interaction mechanisms between different <span>d</span>-G4s structures and RHAU can provide new insights into binding modes, thus aiding in the design of molecular tools targeting <span>d</span>-G4s. In this study, we combined molecular dynamics (MD) simulations with size-exclusion chromatography (SEC) and isothermal titration calorimetry (ITC) to investigate structural dependencies in <span>d</span>-G4s-RHAU interactions for the first time. Diverse <span>d</span>-G4s included hybrid non-parallel (<span>d</span>-24TTG), intramolecular tandem parallel (dAGRO100, GGA8), interlocking parallel (93del), and intermolecular stacked parallel (T30695, T30177) structures were analyzed. MD simulations revealed that the distinct SEC retention behaviors in <span>d</span>-G4s-RHAU interactions were mainly governed by binding site accessibility, inter-site steric hindrance, and binding free energy gradients, trends supported by ITC measured affinities. Furthermore, energy decomposition analysis identified Glu26 in RHAU as a critical residue contributing to electrostatic interactions. Mutating to Arg26 substantially decreased the binding free energy (-94.05 ± 0.41 kJ/mol), emphasizing its functional importance. Thus, this work demonstrates that MD simulations are indispensable for revealing the causes of experimental phenomena and understanding the mechanisms underlying chromatographic behavior. This combined strategy not only discerns interaction patterns stemming from structural diversity but facilitates the rapid screening of G4s-targeting molecules.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1769 ","pages":"Article 466706"},"PeriodicalIF":4.0,"publicationDate":"2026-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145973921","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}