Pub Date : 2025-11-19DOI: 10.1016/j.jchromb.2025.124861
Siqi Wang , Min Si , Qihuan Liao , Yifei Li , Wenyu Yang , Huaizhen He , Cheng Wang
The transmembrane protease serine 2 (TMPRSS2) plays a crucial role in the cellular entry of coronaviruses, making the search for its inhibitors pertinent for developing novel antiviral drugs. Cell membrane chromatography (CMC) is a novel methodology that immobilizes membrane receptors on silica gel, utilizing chromatographic techniques to discover new drugs. To enhance the accuracy of this method, this study employed styrene-maleic acid (SMA) copolymers for protein extraction His-tag for protein immobilization, which would minimize alterations to the biological structure of TMPRSS2. Methodological validation demonstrated that this model offers improved reproducibility and longer column lifespan compared to traditional CMC columns, alongside superior screening performance. Utilizing this model, a screening campaign was conducted against a commercial small molecule library containing 3010 compounds. Preliminary activity validation revealed that the screened famotidine and TS0665 effectively inhibited pseudovirus infection of cells. These findings provide an experimental foundation for the subsequent development of antiviral therapeutics.
{"title":"Identification of potential TMPRSS2 inhibitors via high-throughput screening based on oriented immobilized cell membrane chromatography technology","authors":"Siqi Wang , Min Si , Qihuan Liao , Yifei Li , Wenyu Yang , Huaizhen He , Cheng Wang","doi":"10.1016/j.jchromb.2025.124861","DOIUrl":"10.1016/j.jchromb.2025.124861","url":null,"abstract":"<div><div>The transmembrane protease serine 2 (TMPRSS2) plays a crucial role in the cellular entry of coronaviruses, making the search for its inhibitors pertinent for developing novel antiviral drugs. Cell membrane chromatography (CMC) is a novel methodology that immobilizes membrane receptors on silica gel, utilizing chromatographic techniques to discover new drugs. To enhance the accuracy of this method, this study employed styrene-maleic acid (SMA) copolymers for protein extraction His-tag for protein immobilization, which would minimize alterations to the biological structure of TMPRSS2. Methodological validation demonstrated that this model offers improved reproducibility and longer column lifespan compared to traditional CMC columns, alongside superior screening performance. Utilizing this model, a screening campaign was conducted against a commercial small molecule library containing 3010 compounds. Preliminary activity validation revealed that the screened famotidine and TS0665 effectively inhibited pseudovirus infection of cells. These findings provide an experimental foundation for the subsequent development of antiviral therapeutics.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1269 ","pages":"Article 124861"},"PeriodicalIF":2.8,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145546402","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-17DOI: 10.1016/j.jchromb.2025.124859
Mehdi Hosseini , Ebaa Adnan Azooz
Accurate determination of gallic acid (GA), a potent antioxidant present in grapevine leaves, is essential for optimizing extraction processes in pharmaceutical and food applications. Therefore, developing highly selective and sensitive methods for its analysis in complex plant-derived matrices is critical. To address this, a bio-inspired ionic liquid based on the gallic acid structure was designed, specifically the ionic liquid 1-[(3,4,5-trihydroxybenzyl)methyl]pyridinium chloride ([GaPy][Cl]). This tailored ionic liquid enables precise and selective microextraction of gallic acid via a simple and well-defined mechanism, thus enhancing the accuracy of its quantification in complex plant-derived grapevine leaf matrices. The ionic liquid was successfully synthesized and characterized by various techniques, including NMR, FTIR, and elemental analysis. The extraction mechanism of gallic acid from grapevine leaf samples was elucidated through Density Functional Theory (DFT) calculations, which revealed that strong hydrogen bonding at multiple sites is responsible for the highly efficient interactions during the extraction process. Based on systematic experimental evaluations conducted using the developed microextraction method, the proposed approach successfully quantified gallic acid at very low concentrations, achieving a limit of detection (LOD) of 0.13 ng mL−1. The method exhibits excellent precision, with intra-day and inter-day relative standard deviations (RSDs, n = 7) of 1.79 % and 2.74 %, respectively. Furthermore, recovery studies performed on various real samples—including grape leaves, grapes, grapevine stems, and selected aqueous matrices—yielded recoveries ranging from 94.6 % to 99.4 %, confirming the high accuracy and robust analytical performance of the method for the determination of gallic acid in complex matrices.
没食子酸（GA）是葡萄藤叶中一种有效的抗氧化剂，准确测定其含量对优化制药和食品中的提取工艺至关重要。因此，开发高选择性和敏感的方法来分析复杂的植物源性基质是至关重要的。为了解决这个问题，设计了一种基于没食子酸结构的仿生离子液体，即离子液体1-[（3,4,5-三羟基苄基）甲基]氯化吡啶（[GaPy][Cl]）。这种定制的离子液体能够通过简单而明确的机制精确和选择性地微提取没食子酸，从而提高其在复杂植物衍生葡萄藤叶基质中的定量准确性。成功地合成了离子液体，并通过核磁共振、红外光谱和元素分析等多种技术对其进行了表征。利用密度泛函理论（DFT）分析了葡萄叶样品中没食子酸的提取机理，结果表明，在提取过程中，多位点的强氢键是高效相互作用的原因。采用微萃取方法进行了系统的实验评估，该方法在极低浓度下成功地定量了没食子酸，检出限（LOD）为0.13 ng mL-1。方法精密度高，日内、日间相对标准偏差（rsd, n = 7）分别为1.79%和2.74%。此外，对各种实际样品（包括葡萄叶、葡萄、葡萄藤茎和选定的水基质）进行了回收率研究，回收率从94.6%到99.4%不等，证实了该方法在复杂基质中测定没食子酸的高精度和稳健的分析性能。
{"title":"Bio-inspired ionic liquid design: An advanced and environmentally friendly microextraction method for the selective separation and precise quantification of gallic acid in complex plant-derived matrices","authors":"Mehdi Hosseini , Ebaa Adnan Azooz","doi":"10.1016/j.jchromb.2025.124859","DOIUrl":"10.1016/j.jchromb.2025.124859","url":null,"abstract":"<div><div>Accurate determination of gallic acid (GA), a potent antioxidant present in grapevine leaves, is essential for optimizing extraction processes in pharmaceutical and food applications. Therefore, developing highly selective and sensitive methods for its analysis in complex plant-derived matrices is critical. To address this, a bio-inspired ionic liquid based on the gallic acid structure was designed, specifically the ionic liquid 1-[(3,4,5-trihydroxybenzyl)methyl]pyridinium chloride ([GaPy][Cl]). This tailored ionic liquid enables precise and selective microextraction of gallic acid via a simple and well-defined mechanism, thus enhancing the accuracy of its quantification in complex plant-derived grapevine leaf matrices. The ionic liquid was successfully synthesized and characterized by various techniques, including NMR, FTIR, and elemental analysis. The extraction mechanism of gallic acid from grapevine leaf samples was elucidated through Density Functional Theory (DFT) calculations, which revealed that strong hydrogen bonding at multiple sites is responsible for the highly efficient interactions during the extraction process. Based on systematic experimental evaluations conducted using the developed microextraction method, the proposed approach successfully quantified gallic acid at very low concentrations, achieving a limit of detection (LOD) of 0.13 ng mL<sup>−1</sup>. The method exhibits excellent precision, with intra-day and inter-day relative standard deviations (RSDs, <em>n</em> = 7) of 1.79 % and 2.74 %, respectively. Furthermore, recovery studies performed on various real samples—including grape leaves, grapes, grapevine stems, and selected aqueous matrices—yielded recoveries ranging from 94.6 % to 99.4 %, confirming the high accuracy and robust analytical performance of the method for the determination of gallic acid in complex matrices.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1268 ","pages":"Article 124859"},"PeriodicalIF":2.8,"publicationDate":"2025-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145558941","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-15DOI: 10.1016/j.jchromb.2025.124849
Rose Mathew , P. Lijina , H.S. Varshitha , Gnanesh Kumar Belur Shivappa
Native glycosyl sucrose derivatives in plants represents primarily the trisaccharide isomers having different monosaccharides composition and linkage specificity. They can occur as complex mixtures wherein the separation and identification of individual trisaccharide becomes challenging. Herein, we employed porous graphitic carbon (PGC) liquid chromatography coupled to mass spectrometry to evaluate the elution order of major glycosyl sucrose trisaccharides and determined the structural feature through tandem mass spectrometry (MS/MS). Major trisaccharides comprised of galactosyl-, glucosyl- and fructosyl sucrose derivatives along with few reducing trisaccharides revealed distinct elution order. Furthermore, the application of this approach to profile the oligosaccharides in grape seeds showed the separation and identification of multiple native trisaccharides in the complex mixture that were hitherto unknown.
{"title":"Distinguishing native trisaccharides having differential monosaccharide composition and linkage using PGC-LC MS/MS","authors":"Rose Mathew , P. Lijina , H.S. Varshitha , Gnanesh Kumar Belur Shivappa","doi":"10.1016/j.jchromb.2025.124849","DOIUrl":"10.1016/j.jchromb.2025.124849","url":null,"abstract":"<div><div>Native glycosyl sucrose derivatives in plants represents primarily the trisaccharide isomers having different monosaccharides composition and linkage specificity. They can occur as complex mixtures wherein the separation and identification of individual trisaccharide becomes challenging. Herein, we employed porous graphitic carbon (PGC) liquid chromatography coupled to mass spectrometry to evaluate the elution order of major glycosyl sucrose trisaccharides and determined the structural feature through tandem mass spectrometry (MS/MS). Major trisaccharides comprised of galactosyl-, glucosyl- and fructosyl sucrose derivatives along with few reducing trisaccharides revealed distinct elution order. Furthermore, the application of this approach to profile the oligosaccharides in grape seeds showed the separation and identification of multiple native trisaccharides in the complex mixture that were hitherto unknown.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1269 ","pages":"Article 124849"},"PeriodicalIF":2.8,"publicationDate":"2025-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145575172","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Human Serum Albumin (HSA) is an important protein that helps regulate oncotic pressure and transport of diverse molecules. A high-performance adsorbent for the separation of albumin is strategic to improve efficiency and selectivity, which can result in a low-cost and simplified process for obtaining high-purity albumin. The present study aimed to elucidate the adsorption of HSA on modified bentonite and represent the adsorption capacity. Various characterizations confirm the modification and structural changes of bentonite after surface modification with dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DTSACL). A central composite design (CCD) for the response surface methodology (RSM) was used to investigate and optimize the effect of the pH, concentration of HSA, and sorbent dosage on the adsorption. The findings demonstrated that electrostatic interaction and functionalization of the surface were implemented to enhance the adsorption capacity of the modified bentonite. Thermodynamic investigations indicate an exothermic adsorption process and preferred ambient temperatures, while isothermal examinations confirm that monolayer adsorption was a better fit for the Langmuir model. The maximum adsorption capacity (qe) of 430 mg /g was obtained with a desirability of 0.98 at optimized pH = 6.2, initial HSA concentration = 418.9 mg/L, and DTSACL-bentonite dosage = 30.1 mg in aqueous solution of HSA. The adsorption of HSA from real human serum samples achieved an adsorption efficiency of 79.8 % and a recovery rate of 92.5 %. High-performance liquid chromatography (HPLC) and SDS-PAGE analyses confirmed that DTSACL-bentonite exhibits high selectivity and efficiency, highlighting its potential as a promising adsorbent for the separation and purification of HSA.
{"title":"Bentonite modified with quaternary ammonium for enhanced human serum albumin adsorption","authors":"Saeed Barzegar , Mehran Javanbakht , Behrouz Akbari-Adergani","doi":"10.1016/j.jchromb.2025.124850","DOIUrl":"10.1016/j.jchromb.2025.124850","url":null,"abstract":"<div><div>Human Serum Albumin (HSA) is an important protein that helps regulate oncotic<!--> <!-->pressure and transport of diverse molecules. A high-performance adsorbent for the separation of albumin is strategic to improve efficiency and selectivity,<!--> <!-->which can result in a low-cost and simplified process for obtaining high-purity albumin. The present study aimed to elucidate the adsorption of HSA on modified bentonite and represent the adsorption capacity. Various characterizations confirm the modification and structural changes of bentonite after surface<!--> <!-->modification with dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DTSACL). A central composite design (CCD) for the response surface methodology (RSM) was used to investigate and optimize the effect of the pH, concentration of HSA, and sorbent dosage on the adsorption. The<!--> <!-->findings demonstrated that electrostatic interaction and functionalization of the surface were implemented to enhance the adsorption capacity of the modified bentonite. Thermodynamic investigations indicate an exothermic adsorption<!--> <!-->process and preferred ambient temperatures, while isothermal examinations confirm that monolayer adsorption was a better fit for the Langmuir model. The maximum adsorption capacity (q<sub>e</sub>) of 430 mg /g was obtained with a desirability of 0.98 at optimized pH = 6.2,<!--> <!-->initial HSA concentration = 418.9 mg/L, and DTSACL-bentonite dosage = 30.1 mg in aqueous solution of HSA. The adsorption of HSA from real human serum samples achieved an adsorption efficiency of 79.8 % and a recovery rate of 92.5 %. High-performance liquid chromatography (HPLC) and SDS-PAGE analyses confirmed that DTSACL-bentonite exhibits high selectivity and efficiency, highlighting its potential as a promising adsorbent for the separation and purification of HSA.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1268 ","pages":"Article 124850"},"PeriodicalIF":2.8,"publicationDate":"2025-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145524974","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-09DOI: 10.1016/j.jchromb.2025.124848
Magnus Sture , Stig Valdersnes , Stepan Boitsov , Bjørn Einar Grøsvik , Aasim Ali
This study adopted and validated a quantitative LC-MS/MS method for the rapid detection of five ultra-short-chain (USC) and three short chain (SC) per- and polyfluoroalkyl substances (PFAS) in diverse environmental matrices. The method was applied to soil from an active fire training site, as well as to freshwater and seawater, at and near Bergen airport, Norway, and was further validated for blue mussels. The total oxidizable precursor assay, applied to soil and aqueous film-forming foam (AFFF), provided evidence that AFFF-derived precursors in foam and contaminated soil degrade to USC PFAS, indicating the AFFF as continuing secondary source.
USC PFAS concentrations in freshwater ranged from 13 to 725 ng/L, with the highest levels detected at the fire training site. Seawater near the airport contained 10.4–14.9 ng/L, with concentration decreasing offshore. Depth profiling showed USC PFAS to be surface-associated. USC PFAS were also detected in mussels for the first time, both from urban and remote areas, indicating potential for bioaccumulation.
This study addresses an important methodological gap by validating and applying a reliable analytical approach for monitoring USC PFAS in complex matrices, thereby supporting advancements in environmental monitoring and regulation. The results reveal AFFF impacted sites as major point sources of USC-/SC-PFAS and highlight their high mobility, persistence, and potential bioaccumulation in mussels.
{"title":"Source and fate of ultra-short-chain PFAS in water and biota from an AFFF impacted site","authors":"Magnus Sture , Stig Valdersnes , Stepan Boitsov , Bjørn Einar Grøsvik , Aasim Ali","doi":"10.1016/j.jchromb.2025.124848","DOIUrl":"10.1016/j.jchromb.2025.124848","url":null,"abstract":"<div><div>This study adopted and validated a quantitative LC-MS/MS method for the rapid detection of five ultra-short-chain (USC) and three short chain (SC) <em>per</em>- and polyfluoroalkyl substances (PFAS) in diverse environmental matrices. The method was applied to soil from an active fire training site, as well as to freshwater and seawater, at and near Bergen airport, Norway, and was further validated for blue mussels. The total oxidizable precursor assay, applied to soil and aqueous film-forming foam (AFFF), provided evidence that AFFF-derived precursors in foam and contaminated soil degrade to USC PFAS, indicating the AFFF as continuing secondary source.</div><div>USC PFAS concentrations in freshwater ranged from 13 to 725 ng/L, with the highest levels detected at the fire training site. Seawater near the airport contained 10.4–14.9 ng/L, with concentration decreasing offshore. Depth profiling showed USC PFAS to be surface-associated. USC PFAS were also detected in mussels for the first time, both from urban and remote areas, indicating potential for bioaccumulation.</div><div>This study addresses an important methodological gap by validating and applying a reliable analytical approach for monitoring USC PFAS in complex matrices, thereby supporting advancements in environmental monitoring and regulation. The results reveal AFFF impacted sites as major point sources of USC-/SC-PFAS and highlight their high mobility, persistence, and potential bioaccumulation in mussels.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1268 ","pages":"Article 124848"},"PeriodicalIF":2.8,"publicationDate":"2025-11-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145524961","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-07DOI: 10.1016/j.jchromb.2025.124841
Ruijun Cai , Zhongnan Mao , Tao An , Yunbo Zhang , Li Zhou
Byakangelicin is a furanocoumarin derived from the roots of Angelica dahurica. It exhibits pharmacological properties, including anti-tumor activity, as well as against liver injury and fibrosis. In this study, Ultra-High-Performance Liquid Chromatography with Q-Exactive Orbitrap Mass Spectrometry technology was employed to investigate the metabolic profile of byakangelicin in rats. Following intragastric administration the suspension of byakangelicin, plasma and tissue specimens were harvested. According to high-resolution extracted ion chromatograms, the metabolites of byakangelicin were identified by comparing accurate mass, diagnostic fragment ions, and chromatographic retention times. Data collection was performed in positive ion mode to facilitate metabolite characterization of byakangelicin. Overall, 46 metabolites were successfully characterised. The primary metabolic pathways included hydroxylation, dehydroxylation, methylation, demethylation, reduction, glucuronidation, glycination, and cysteinylation. This systematic investigation of the metabolic characteristics and pathways of byakangelicin in rats provides a valuable reference for future pharmacodynamic evaluations, pharmacological research, and drug development.
{"title":"Identification of byakangelicin metabolites in rats via the UHPLC-Q-exactive orbitrap mass spectrometer","authors":"Ruijun Cai , Zhongnan Mao , Tao An , Yunbo Zhang , Li Zhou","doi":"10.1016/j.jchromb.2025.124841","DOIUrl":"10.1016/j.jchromb.2025.124841","url":null,"abstract":"<div><div>Byakangelicin is a furanocoumarin derived from the roots of Angelica dahurica. It exhibits pharmacological properties, including anti-tumor activity, as well as against liver injury and fibrosis. In this study, Ultra-High-Performance Liquid Chromatography with Q-Exactive Orbitrap Mass Spectrometry technology was employed to investigate the metabolic profile of byakangelicin in rats. Following intragastric administration the suspension of byakangelicin, plasma and tissue specimens were harvested. According to high-resolution extracted ion chromatograms, the metabolites of byakangelicin were identified by comparing accurate mass, diagnostic fragment ions, and chromatographic retention times. Data collection was performed in positive ion mode to facilitate metabolite characterization of byakangelicin. Overall, 46 metabolites were successfully characterised. The primary metabolic pathways included hydroxylation, dehydroxylation, methylation, demethylation, reduction, glucuronidation, glycination, and cysteinylation. This systematic investigation of the metabolic characteristics and pathways of byakangelicin in rats provides a valuable reference for future pharmacodynamic evaluations, pharmacological research, and drug development.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1268 ","pages":"Article 124841"},"PeriodicalIF":2.8,"publicationDate":"2025-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145497991","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-07DOI: 10.1016/j.jchromb.2025.124846
Yeşim Somay Selbes
Optimal detection of Erythropoietin receptor agonists (ERAs) in anti-doping analyses relies heavily on efficient immunopurification (IP) strategies. While antibody-based IP is routinely applied, aptamers have emerged as promising alternatives. This proof-of-principle study investigated the applicability of an anti-EPO DNA aptamer for ERA enrichment in serum, followed by Sarkosyl-Polyacrylamide Gel Electrophoresis (SAR-PAGE) analysis.
The aptamer-based method successfully captured recombinant ERA variants (rEPO, dEPO, EPO-Fc, CERA) with defined limits of detection (LOD) in serum: 10 mIU/mL for rEPO, 10 pg/mL for dEPO, and 25 pg/mL for EPO-Fc and CERA. In comparison, the antibody-based method achieved lower serum LODs of 5 mIU/mL for rEPO, 5 pg/mL for dEPO, and 12.5 pg/mL for EPO-Fc and CERA. Both methods demonstrated good selectivity, as no non-specific bands were detected in blank serum matrices. However, endogenous blood EPO (bEPO) was not enriched by the aptamer, likely reflecting glycosylation-related structural differences between endogenous and recombinant isoforms.
These findings highlight that aptamers can provide highly specific recognition of recombinant ERAs without cross-reactivity to bEPO, which may simplify interpretation in doping control analyses. Nonetheless, further optimization—such as refining immobilization chemistries, incorporating differently glycosylated EPO forms during SELEX, and sequence improvements—is required to enhance capture of native isoforms. Overall, this study establishes a foundation for aptamer-based enrichment as a complementary alternative to antibody-based methods in anti-doping applications.
{"title":"Aptamer enrichment strategy for the detection of erythropoietin-receptor agonists in blood samples: A potential alternative to antibody-based assays","authors":"Yeşim Somay Selbes","doi":"10.1016/j.jchromb.2025.124846","DOIUrl":"10.1016/j.jchromb.2025.124846","url":null,"abstract":"<div><div>Optimal detection of Erythropoietin receptor agonists (ERAs) in anti-doping analyses relies heavily on efficient immunopurification (IP) strategies. While antibody-based IP is routinely applied, aptamers have emerged as promising alternatives. This proof-of-principle study investigated the applicability of an anti-EPO DNA aptamer for ERA enrichment in serum, followed by Sarkosyl-Polyacrylamide Gel Electrophoresis (SAR-PAGE) analysis.</div><div>The aptamer-based method successfully captured recombinant ERA variants (rEPO, dEPO, EPO-Fc, CERA) with defined limits of detection (LOD) in serum: 10 mIU/mL for rEPO, 10 pg/mL for dEPO, and 25 pg/mL for EPO-Fc and CERA. In comparison, the antibody-based method achieved lower serum LODs of 5 mIU/mL for rEPO, 5 pg/mL for dEPO, and 12.5 pg/mL for EPO-Fc and CERA. Both methods demonstrated good selectivity, as no non-specific bands were detected in blank serum matrices. However, endogenous blood EPO (bEPO) was not enriched by the aptamer, likely reflecting glycosylation-related structural differences between endogenous and recombinant isoforms.</div><div>These findings highlight that aptamers can provide highly specific recognition of recombinant ERAs without cross-reactivity to bEPO, which may simplify interpretation in doping control analyses. Nonetheless, further optimization—such as refining immobilization chemistries, incorporating differently glycosylated EPO forms during SELEX, and sequence improvements—is required to enhance capture of native isoforms. Overall, this study establishes a foundation for aptamer-based enrichment as a complementary alternative to antibody-based methods in anti-doping applications.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1268 ","pages":"Article 124846"},"PeriodicalIF":2.8,"publicationDate":"2025-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145484533","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-06DOI: 10.1016/j.jchromb.2025.124847
Hong-yan Ge , Ya-jun Zhang , Jun-qin Qiao , Li Fan , He-liang Fu , Hong-zhen Lian
In this work, a dual-parameter quantification protocol was successfully developed for determining the enzymatic activity of human urinary kallidinogenase (KN) using reversed-phase high-performance liquid chromatography (RP-HPLC). KN serves as a hydrolytic enzyme to cleave the substrate S-2266 (H-D-Val-Leu-Arg-pNA). Effective separation of the product, substrate, and KN was achieved under optimized chromatographic conditions. By minimizing matrix interference through HPLC separation and using multi-wavelength detection, both selectivity and sensitivity were significantly improved. Moreover, the dual-parameter quantification method, which simultaneously measured the formation of reaction products and the consumption of substrates to reflect KN enzyme activity, markedly improved the reliability of the determination. This method was successfully applied to the activity assay of actual KN samples. The established method has been demonstrated to be effective and reliable with comparable greenness. It provides a powerful tool for the quantitative monitoring of KN activity in biological products and paves the way for its application in complex biological matrices.
{"title":"A reversed-phase HPLC-based dual-parameter assay for the human urinary kallidinogenase enzyme activity","authors":"Hong-yan Ge , Ya-jun Zhang , Jun-qin Qiao , Li Fan , He-liang Fu , Hong-zhen Lian","doi":"10.1016/j.jchromb.2025.124847","DOIUrl":"10.1016/j.jchromb.2025.124847","url":null,"abstract":"<div><div>In this work, a dual-parameter quantification protocol was successfully developed for determining the enzymatic activity of human urinary kallidinogenase (KN) using reversed-phase high-performance liquid chromatography (RP-HPLC). KN serves as a hydrolytic enzyme to cleave the substrate S-2266 (H-D-Val-Leu-Arg-pNA). Effective separation of the product, substrate, and KN was achieved under optimized chromatographic conditions. By minimizing matrix interference through HPLC separation and using multi-wavelength detection, both selectivity and sensitivity were significantly improved. Moreover, the dual-parameter quantification method, which simultaneously measured the formation of reaction products and the consumption of substrates to reflect KN enzyme activity, markedly improved the reliability of the determination. This method was successfully applied to the activity assay of actual KN samples. The established method has been demonstrated to be effective and reliable with comparable greenness. It provides a powerful tool for the quantitative monitoring of KN activity in biological products and paves the way for its application in complex biological matrices.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1268 ","pages":"Article 124847"},"PeriodicalIF":2.8,"publicationDate":"2025-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145524977","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-05DOI: 10.1016/j.jchromb.2025.124845
Tingyu Lang , Xiaolei Liang , Yongxiu Yang
Background
Currently, contradictions remain regarding the causal relationship between telomere length (TL) and ovarian tumors (OT). To elucidate the potential causal relationship, we conducted a two-sample bidirectional Mendelian randomization (MR) study.
Method
All the data were from European populations and obtained from the Genome-Wide Association Study, the FinnGen study, and the Ovarian Cancer Association Consortium. We used inverse variance weighting (IVW) as the primary method, supplemented by other five methods to calculate odds ratios (OR) and 95 % confidence intervals (95 % CI) and checked for pleiotropy. Cochran's Q was used to detect heterogeneity.
Results
The IVW method supported TL as a risk factor for malignant (OR = 1.273, 95 % CI = 1.012–1.602) and benign ovarian tumors (BOTs) (OR = 1.337, 95 % CI = 1.093–1.637). Besides, ovarian cancer subtypes evaluated the strong causal relationship between TL and low malignant potential serous ovarian cancer (LMSOC) by applying the IVW method after Bonferroni correction. There was suggestive evidence for low malignant potential mucinous ovarian cancer, invasive mucinous ovarian cancer, and low-grade serous ovarian cancer. No causal relationships were found for other ovarian cancer subtypes.
Conclusion
Our MR study provides strong genetic evidence that longer telomere length increases risk of specific ovarian tumor subtypes—most robustly for benign ovarian tumors (BOTs) and low malignant potential serous ovarian cancer (LMSOC). For other subtypes, observed associations were suggestive but inconclusive after multiplicity correction, highlighting the etiological heterogeneity of ovarian tumors To optimize treatment regimens and preventive measures for OT, we will continue to thoroughly investigate the potential mechanism of action of these associations in future studies.
目前，关于端粒长度（TL）与卵巢肿瘤（OT）之间的因果关系仍存在矛盾。为了阐明潜在的因果关系，我们进行了一项双样本双向孟德尔随机化（MR）研究。方法所有数据均来自欧洲人群，分别来自全基因组关联研究、FinnGen研究和卵巢癌协会联盟。我们以逆方差加权（IVW）为主要方法，辅以其他5种方法计算比值比（OR）和95%置信区间（95% CI），并检查多效性。Cochran’s Q用于检测异质性。结果IVW方法支持TL是卵巢恶性肿瘤（OR = 1.273, 95% CI = 1.012 ~ 1.602）和良性肿瘤（bot）的危险因素（OR = 1.337, 95% CI = 1.093 ~ 1.637）。此外，通过Bonferroni校正后的IVW方法，卵巢癌亚型评估TL与低恶性潜在浆液性卵巢癌（LMSOC）之间的强因果关系。提示潜在的低恶性粘液性卵巢癌、浸润性粘液性卵巢癌和低级别浆液性卵巢癌。其他卵巢癌亚型没有发现因果关系。结论磁共振研究提供了强有力的遗传学证据，表明端粒长度较长会增加特定卵巢肿瘤亚型的风险，其中良性卵巢肿瘤（bot）和低恶性潜势浆液性卵巢癌（LMSOC）的风险最大。对于其他亚型，观察到的关联是提示性的，但经过多重校正后不确定，突出了卵巢肿瘤的病因异质性。为了优化治疗方案和预防措施，我们将在未来的研究中继续深入研究这些关联的潜在作用机制。
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Pub Date : 2025-11-05DOI: 10.1016/j.jchromb.2025.124844
Yao Ma , Qing-nan Chen , Rui Liao , Yun-e Bai , Jian-kuan Li , Shuang Hu , Jian-ping Gao
In this study, a cyclodextrin-dispersive liquid-liquid microextraction-HPLC-based method was developed to quantitatively study four components of Codonopsis Radix (CR): carboxymethyl-β-cyclodextrin (CM-β-CD) was incorporated into the DES, then added to the sample solution, and the mixture was vortexed and centrifuged, the upper hydrophobic phase was then collected for HPLC analysis. The primary elements influencing the extraction efficiency were optimized to choose the circumstances for this experiment, including the type and consumption of cyclodextrin, the NaCl concentration, the pH level of the sample phase and the extraction time. The EFs for four target components extracted by cyclodextrin-DES were between 8.4 and 71.2, which were all higher than those extracted only by DES. In their respective linearity ranges, the four analytes exhibited good linearity (R2 ≥ 0.99), with detection limits of 1.2 × 10−2 μg/mL, 5.3 × 10−3 μg/mL, 3 × 10−4 μg/mL, and 1.1 × 10−4 μg/mL. The precision and accuracy of the method ranged from 0.8 % to 10.0 % and 90.0 % to 105.2 %. Finally, this method was applied to the quantification of fresh-cut CR and traditional-processed CR. The results showed that the content of tangshenoside I and lobetyolinin of fresh-cut CR was significantly higher than in traditionally processed CR, but there were no significant differences between fresh-cut CR and traditionally processed CR for lobetyolin and lobetyol. The use of CM-β-CD in combination with DES provides rapid, simple, and reliable quantification of the target analytes. By this method, differences between the two processing methods were compared at the level of small-molecule content, providing evidence for the feasibility of the fresh-cutting processing method and demonstrating a theoretical basis for the quality evaluation method of the fresh-cut process.
{"title":"A cyclodextrin-deep eutectic solvent-based dispersive liquid-liquid microextraction method for quantitative determination of small molecules in fresh-cut Codonopsis Radix","authors":"Yao Ma , Qing-nan Chen , Rui Liao , Yun-e Bai , Jian-kuan Li , Shuang Hu , Jian-ping Gao","doi":"10.1016/j.jchromb.2025.124844","DOIUrl":"10.1016/j.jchromb.2025.124844","url":null,"abstract":"<div><div>In this study, a cyclodextrin-dispersive liquid-liquid microextraction-HPLC-based method was developed to quantitatively study four components of Codonopsis Radix (CR): carboxymethyl-<em>β</em>-cyclodextrin (CM-<em>β</em>-CD) was incorporated into the DES, then added to the sample solution, and the mixture was vortexed and centrifuged, the upper hydrophobic phase was then collected for HPLC analysis. The primary elements influencing the extraction efficiency were optimized to choose the circumstances for this experiment, including the type and consumption of cyclodextrin, the NaCl concentration, the pH level of the sample phase and the extraction time. The <em>EFs</em> for four target components extracted by cyclodextrin-DES were between 8.4 and 71.2, which were all higher than those extracted only by DES. In their respective linearity ranges, the four analytes exhibited good linearity (R<sup>2</sup> ≥ 0.99), with detection limits of 1.2 × 10<sup>−2</sup> μg/mL, 5.3 × 10<sup>−3</sup> μg/mL, 3 × 10<sup>−4</sup> μg/mL, and 1.1 × 10<sup>−4</sup> μg/mL. The precision and accuracy of the method ranged from 0.8 % to 10.0 % and 90.0 % to 105.2 %. Finally, this method was applied to the quantification of fresh-cut CR and traditional-processed CR. The results showed that the content of tangshenoside I and lobetyolinin of fresh-cut CR was significantly higher than in traditionally processed CR, but there were no significant differences between fresh-cut CR and traditionally processed CR for lobetyolin and lobetyol. The use of CM-<em>β</em>-CD in combination with DES provides rapid, simple, and reliable quantification of the target analytes. By this method, differences between the two processing methods were compared at the level of small-molecule content, providing evidence for the feasibility of the fresh-cutting processing method and demonstrating a theoretical basis for the quality evaluation method of the fresh-cut process.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1268 ","pages":"Article 124844"},"PeriodicalIF":2.8,"publicationDate":"2025-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145508706","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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