Pub Date : 2025-11-20DOI: 10.1016/j.chroma.2025.466559
Ji-young Park , Dayeong Lee , Ranhee Park , Hyehyun Kim , Jeongkwon Kim
A rapid and efficient analytical method was developed for the determination of plutonium in large-volume seawater to overcome the limitations of conventional coprecipitation techniques, which typically require an analysis time of 1–2 d and involve multiple labor-intensive steps. The proposed method integrates microwave-assisted acid digestion, CaF2/LaF3 coprecipitation, and TEVA extraction chromatography with actinide-selective resin (AN-resin) to enable high recovery and significantly reduce the analysis time. Additionally, a key challenge was overcome through method optimization using seawater pre-equilibrated with 239Pu and 242Pu as an internal tracer, namely the efficient and rapid separation of Pu from the AN-resin eluate while maintaining its oxidation state. Validation using 20 L seawater samples spiked with 239Pu achieved recoveries of 86.2 ± 4.4% and 90.5 ± 4.5% for 242Pu, with an analytical accuracy of 96.7% and precision (relative standard deviation) of 3.1%. The total pretreatment time, including a 2 h preconcentration step, was reduced to ∼7 h, while achieving detection limits of 0.03–0.05 μBq/kg. Interlaboratory comparisons confirmed the robustness of the developed method, while field application to Korean coastal waters yielded 239+240Pu activities and isotopic ratios consistent with regional monitoring data. This study presents a high-throughput and reliable alternative to conventional methods, offering a versatile platform for emergency response and environmental monitoring of plutonium and other actinides in marine systems.
{"title":"Method development for plutonium detection in seawater: Selective extraction and sample preparation using actinide resin","authors":"Ji-young Park , Dayeong Lee , Ranhee Park , Hyehyun Kim , Jeongkwon Kim","doi":"10.1016/j.chroma.2025.466559","DOIUrl":"10.1016/j.chroma.2025.466559","url":null,"abstract":"<div><div>A rapid and efficient analytical method was developed for the determination of plutonium in large-volume seawater to overcome the limitations of conventional coprecipitation techniques, which typically require an analysis time of 1–2 d and involve multiple labor-intensive steps. The proposed method integrates microwave-assisted acid digestion, CaF<sub>2</sub>/LaF<sub>3</sub> coprecipitation, and TEVA extraction chromatography with actinide-selective resin (AN-resin) to enable high recovery and significantly reduce the analysis time. Additionally, a key challenge was overcome through method optimization using seawater pre-equilibrated with <sup>239</sup>Pu and <sup>242</sup>Pu as an internal tracer, namely the efficient and rapid separation of Pu from the AN-resin eluate while maintaining its oxidation state. Validation using 20 L seawater samples spiked with <sup>239</sup>Pu achieved recoveries of 86.2 ± 4.4% and 90.5 ± 4.5% for <sup>242</sup>Pu, with an analytical accuracy of 96.7% and precision (relative standard deviation) of 3.1%. The total pretreatment time, including a 2 h preconcentration step, was reduced to ∼7 h, while achieving detection limits of 0.03–0.05 μBq/kg. Interlaboratory comparisons confirmed the robustness of the developed method, while field application to Korean coastal waters yielded <sup>239+240</sup>Pu activities and isotopic ratios consistent with regional monitoring data. This study presents a high-throughput and reliable alternative to conventional methods, offering a versatile platform for emergency response and environmental monitoring of plutonium and other actinides in marine systems.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1766 ","pages":"Article 466559"},"PeriodicalIF":4.0,"publicationDate":"2025-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145652948","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}
Pub Date : 2025-11-19DOI: 10.1016/j.chroma.2025.466555
Lin Li , Bing Feng , Lin Yang , Mengying Zhao , Zhiqiang Kong , Minmin Li
The use of preservatives can effectively delay the spoilage of fruits and vegetables and reduce post-harvest losses; however, due to their variety and complex substrates, they pose challenges for food safety supervision. In our study, we developed a fast and simple method, which is described as a modified QuEChERS method coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), for the simultaneous detection of 15 preservatives and 5 main metabolites in 20 kinds of fruits and vegetables in 6 min. The method's accuracy, stability, and reproducibility, including recoveries ranging from 69.35 % to 119.67 % at three spiked levels, were demonstrated with relative standard deviations (RSD) < 7.99 %. Analysis of 339 samples indicated widespread multi-residue contamination ranging from 1.0 × 10–3 to 9.9 mg/kg. Considering the consumption frequency and human health risk, prochloraz and difenoconazole were chosen for storage simulation experiment on garlic sprouts and ginger. The degradation in garlic sprouts and ginger followed first-order kinetics. After 28 d storage under 4 °C, prochloraz possessed residue level of 2.02 mg/kg (1.5-fold) and 3.56 mg/kg (5-fold) on garlic sprouts and 1.45 mg/kg (1.5-fold) and 3.04 mg/kg (5-fold) on ginger, meanwhile, difenoconazole possessed residue level of 1.28 mg/kg (1.5-fold) and 3.81 mg/kg (5-fold) on garlic sprouts and 1.17 mg/kg (1.5-fold) and 3.37 mg/kg (5-fold) on ginger. Prochloraz exhibited acute risk with HQa (acute hazard quotient) of 2.27. This study underscores the need for continuous monitoring, MRL revisions, and harmonized pesticide guidelines to safeguard consumer health. The optimized analytical framework benefits residue analysis in complex matrices.
{"title":"Multi-Residue analysis and health risk assessment of preservatives in vegetables and fruits across major chinese provinces: Implications for regulatory standards","authors":"Lin Li , Bing Feng , Lin Yang , Mengying Zhao , Zhiqiang Kong , Minmin Li","doi":"10.1016/j.chroma.2025.466555","DOIUrl":"10.1016/j.chroma.2025.466555","url":null,"abstract":"<div><div>The use of preservatives can effectively delay the spoilage of fruits and vegetables and reduce post-harvest losses; however, due to their variety and complex substrates, they pose challenges for food safety supervision. In our study, we developed a fast and simple method, which is described as a modified QuEChERS method coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), for the simultaneous detection of 15 preservatives and 5 main metabolites in 20 kinds of fruits and vegetables in 6 min. The method's accuracy, stability, and reproducibility, including recoveries ranging from 69.35 % to 119.67 % at three spiked levels, were demonstrated with relative standard deviations (RSD) < 7.99 %. Analysis of 339 samples indicated widespread multi-residue contamination ranging from 1.0 × 10<sup>–3</sup> to 9.9 mg/kg. Considering the consumption frequency and human health risk, prochloraz and difenoconazole were chosen for storage simulation experiment on garlic sprouts and ginger. The degradation in garlic sprouts and ginger followed first-order kinetics. After 28 d storage under 4 °C, prochloraz possessed residue level of 2.02 mg/kg (1.5-fold) and 3.56 mg/kg (5-fold) on garlic sprouts and 1.45 mg/kg (1.5-fold) and 3.04 mg/kg (5-fold) on ginger, meanwhile, difenoconazole possessed residue level of 1.28 mg/kg (1.5-fold) and 3.81 mg/kg (5-fold) on garlic sprouts and 1.17 mg/kg (1.5-fold) and 3.37 mg/kg (5-fold) on ginger. Prochloraz exhibited acute risk with HQa (acute hazard quotient) of 2.27. This study underscores the need for continuous monitoring, MRL revisions, and harmonized pesticide guidelines to safeguard consumer health. The optimized analytical framework benefits residue analysis in complex matrices.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1766 ","pages":"Article 466555"},"PeriodicalIF":4.0,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145585637","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}
Pub Date : 2025-11-19DOI: 10.1016/j.chroma.2025.466560
Stilianos G. Roussis, Claus Rentel
Significant recent interest has developed in synthetic antisense and small interfering RNA (siRNA) oligonucleotides due to their high therapeutic potential. Characterization and monitoring of low-level impurities produced during oligonucleotide manufacturing is challenging, especially for impurities that co-elute and are isobaric with the parent oligonucleotide. To improve the separation of such impurities, the effects of pH under ion-pair reversed phase (IP-RP) chromatography conditions have been examined here. Effective separation of co-eluting, near-isobaric deamination (DA) impurities from the parent oligonucleotide based on ionization (pKa) differences has been achieved under acidic conditions. The method produces a single chromatographic peak encompassing all individual positional isomers of the DA impurity, which greatly simplifies quantitative analysis. Optimum separation of other co-eluting impurities based on electronegativity/polarity differences (PO) was accomplished under basic conditions. Operation of the mass spectrometer (MS) in the selected ion monitoring (SIM) mode counters the lower sensitivity typically associated with negative ion MS analysis of oligonucleotides under low pH conditions. Analysis of stressed samples (80 °C, 0–7 days) for the extent of deamination over time produced linear relationships with both UV and MS methods of detection. The separation of other near-isobaric impurities from each other, for example, n-dMeC from n-T, and n-MOE MeC from n-MOE T, is demonstrated. The new pH-enabled method is faster and simpler than other methods requiring sample desulfurization or HRMS experiments measuring differences in isotopic peak distributions.
{"title":"pH effects on the separation of oligonucleotides by ion-pair reserved phase liquid chromatography mass spectrometry","authors":"Stilianos G. Roussis, Claus Rentel","doi":"10.1016/j.chroma.2025.466560","DOIUrl":"10.1016/j.chroma.2025.466560","url":null,"abstract":"<div><div>Significant recent interest has developed in synthetic antisense and small interfering RNA (siRNA) oligonucleotides due to their high therapeutic potential. Characterization and monitoring of low-level impurities produced during oligonucleotide manufacturing is challenging, especially for impurities that co-elute and are isobaric with the parent oligonucleotide. To improve the separation of such impurities, the effects of pH under ion-pair reversed phase (IP-RP) chromatography conditions have been examined here. Effective separation of co-eluting, near-isobaric deamination (DA) impurities from the parent oligonucleotide based on ionization (pKa) differences has been achieved under acidic conditions. The method produces a single chromatographic peak encompassing all individual positional isomers of the DA impurity, which greatly simplifies quantitative analysis. Optimum separation of other co-eluting impurities based on electronegativity/polarity differences (PO) was accomplished under basic conditions. Operation of the mass spectrometer (MS) in the selected ion monitoring (SIM) mode counters the lower sensitivity typically associated with negative ion MS analysis of oligonucleotides under low pH conditions. Analysis of stressed samples (80 °C, 0–7 days) for the extent of deamination over time produced linear relationships with both UV and MS methods of detection. The separation of other near-isobaric impurities from each other, for example, n-d<sup>Me</sup>C from n-T, and n-MOE <sup>Me</sup>C from n-MOE T, is demonstrated. The new pH-enabled method is faster and simpler than other methods requiring sample desulfurization or HRMS experiments measuring differences in isotopic peak distributions.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1766 ","pages":"Article 466560"},"PeriodicalIF":4.0,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145595638","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}
Pub Date : 2025-11-19DOI: 10.1016/j.chroma.2025.466553
Gaojin Zhou , Zhaojin Zhang , Subhan Mahmood , Jie Tang , Shun Yao
For the first time, two kinds of green solvents including ionic liquid (IL) and deep eutectic solvent (DES) were combined for achieving flavonoids and terpenoids simultaneously from Ginkgo leaves. They were applied in the form of micelles and the complex with natural carbon dots (CDs) as extraction and separation mediums, respectively. Especially, the latter was prepared from the lignocellulosic residue of Ginkgo leaves after extraction, which achieved the comprehensive utilization of natural resources. Critical micelle concentrations (CMC) of the IL were determined as 4.70–5.38 in the range from 5 to 45 °C, and the main particle size of CDs was found between 20 and 25 nm. After comprehensive characterization on them, a biphasic system composed of IL micelles and CDs@DES was constructed, with the former in the upper layer and the latter in the lower layer. Then it was successfully applied for obtaining flavonoids and terpenoids from the leaves of Ginkgo biloba with extraction efficiency of 96.2 % and 93.2 % respectively, which were analyzed by liquid chromatography-mass spectrum (LC-MS). Moreover, the IL and DES could be repeatedly used through the post-treatment.
{"title":"Combined use of ionic liquid micelles and natural carbon dots@deep eutectic solvent (CDs@DES) to extract and separate flavonoids and terpenoids from Ginkgo biloba leaves","authors":"Gaojin Zhou , Zhaojin Zhang , Subhan Mahmood , Jie Tang , Shun Yao","doi":"10.1016/j.chroma.2025.466553","DOIUrl":"10.1016/j.chroma.2025.466553","url":null,"abstract":"<div><div>For the first time, two kinds of green solvents including ionic liquid (IL) and deep eutectic solvent (DES) were combined for achieving flavonoids and terpenoids simultaneously from Ginkgo leaves. They were applied in the form of micelles and the complex with natural carbon dots (CDs) as extraction and separation mediums, respectively. Especially, the latter was prepared from the lignocellulosic residue of Ginkgo leaves after extraction, which achieved the comprehensive utilization of natural resources. Critical micelle concentrations (CMC) of the IL were determined as 4.70–5.38 in the range from 5 to 45 °C, and the main particle size of CDs was found between 20 and 25 nm. After comprehensive characterization on them, a biphasic system composed of IL micelles and CDs@DES was constructed, with the former in the upper layer and the latter in the lower layer. Then it was successfully applied for obtaining flavonoids and terpenoids from the leaves of <em>Ginkgo biloba</em> with extraction efficiency of 96.2 % and 93.2 % respectively, which were analyzed by liquid chromatography-mass spectrum (LC-MS). Moreover, the IL and DES could be repeatedly used through the post-treatment.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1766 ","pages":"Article 466553"},"PeriodicalIF":4.0,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145570954","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}
Pub Date : 2025-11-19DOI: 10.1016/j.chroma.2025.466558
Arash Mirzahosseini , Annamária Sepsey , Ali Mhammad , Gergely Dombi , Simon Horváth , Eliza Tóth , Gábor Németh , Oliver Trapp , Attila Felinger , Gergő Tóth
Many drugs are chiral, and their enantiomers often display distinct pharmacological activities. Accurate determination of enantiomerization kinetics during chromatographic separations is therefore of critical importance. Batman peaks (plateau regions arising between partially resolved enantiomer peaks) carry valuable information about on-column interconversion dynamics. We developed an automated R framework that integrates both the unified equation and a stochastic model to extract kinetic and thermodynamic constants from chromatographic data. The workflow applies robust optimization algorithms to enhance flexibility, accelerate fitting, and improve accuracy relative to manual approaches. Quetiapine chromatograms were analyzed over a wide range of flow rates () and temperatures (10 °C to 40 °C). Both the unified equation and the stochastic model produced consistent rate constants, which were in good agreement with values obtained from optical rotation experiments. Notably, the stochastic model remained applicable even under extreme conditions where peaks coalesced. Linearized Eyring–Polányi analysis afforded the determination of thermodynamic parameters. The presented workflow provides a robust, flexible, and customizable platform for determining enantiomerization kinetics from Batman peaks. By combining the unified equation with stochastic modeling, it delivers reliable results across diverse chromatographic conditions. This framework is readily adaptable for routine analysis of dynamic enantiomerization in pharmaceutical and analytical chemistry applications.
许多药物是手性的，它们的对映体通常表现出不同的药理活性。因此，色谱分离过程中对映异构化动力学的准确测定是至关重要的。蝙蝠侠峰（在部分分解的对映体峰之间产生的高原区域）携带有关列内相互转化动力学的宝贵信息。我们开发了一个自动化的R框架，集成了统一方程和随机模型，从色谱数据中提取动力学和热力学常数。该工作流应用稳健的优化算法来增强灵活性，加速拟合，并提高相对于手动方法的准确性。喹硫平色谱分析在宽流速（0.1mL/min ~ 3.0 ml /min）和温度（10℃~ 40℃）范围内进行。统一方程和随机模型都得到了一致的速率常数，与旋光实验结果吻合较好。值得注意的是，即使在峰值合并的极端条件下，随机模型仍然适用。线性化Eyring-Polányi分析给出了热力学参数的确定。提出的工作流程提供了一个强大的，灵活的，可定制的平台，以确定从蝙蝠侠峰对映异构化动力学。通过将统一方程与随机建模相结合，它可以在不同的色谱条件下提供可靠的结果。这个框架很容易适用于药物和分析化学应用中动态对映异构化的常规分析。
{"title":"Automated R workflow for Batman peak fitting using unified equation and stochastic modeling","authors":"Arash Mirzahosseini , Annamária Sepsey , Ali Mhammad , Gergely Dombi , Simon Horváth , Eliza Tóth , Gábor Németh , Oliver Trapp , Attila Felinger , Gergő Tóth","doi":"10.1016/j.chroma.2025.466558","DOIUrl":"10.1016/j.chroma.2025.466558","url":null,"abstract":"<div><div>Many drugs are chiral, and their enantiomers often display distinct pharmacological activities. Accurate determination of enantiomerization kinetics during chromatographic separations is therefore of critical importance. Batman peaks (plateau regions arising between partially resolved enantiomer peaks) carry valuable information about on-column interconversion dynamics. We developed an automated <span>R</span> framework that integrates both the unified equation and a stochastic model to extract kinetic and thermodynamic constants from chromatographic data. The workflow applies robust optimization algorithms to enhance flexibility, accelerate fitting, and improve accuracy relative to manual approaches. Quetiapine chromatograms were analyzed over a wide range of flow rates (<span><math><mrow><mn>0</mn><mo>.</mo><mn>1</mn><mspace></mspace><mstyle><mi>m</mi><mi>L</mi></mstyle><mo>/</mo><mstyle><mo>min</mo></mstyle><mspace></mspace><mtext>to</mtext><mspace></mspace><mn>3</mn><mo>.</mo><mn>0</mn><mspace></mspace><mstyle><mi>m</mi><mi>L</mi></mstyle><mo>/</mo><mstyle><mo>min</mo></mstyle></mrow></math></span>) and temperatures (10<!--> <!-->°C to 40<!--> <!-->°C). Both the unified equation and the stochastic model produced consistent rate constants, which were in good agreement with values obtained from optical rotation experiments. Notably, the stochastic model remained applicable even under extreme conditions where peaks coalesced. Linearized Eyring–Polányi analysis afforded the determination of thermodynamic parameters. The presented workflow provides a robust, flexible, and customizable platform for determining enantiomerization kinetics from Batman peaks. By combining the unified equation with stochastic modeling, it delivers reliable results across diverse chromatographic conditions. This framework is readily adaptable for routine analysis of dynamic enantiomerization in pharmaceutical and analytical chemistry applications.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1766 ","pages":"Article 466558"},"PeriodicalIF":4.0,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145571028","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}
Pub Date : 2025-11-19DOI: 10.1016/j.chroma.2025.466552
Zihan Li , Xiaoyan Dong , Linling Yu , Yan Sun , Qinghong Shi
The coronavirus disease 2019 (COVID-19) pandemic has posed not only a severe threat to human health and the global economy but also greater requirements for the efficient isolation of highly variable viruses. To address this dilemma, a strategy for the development of affibody ligands targeting the conserved region of the receptor binding domain (RBD) on the spike (S) protein was proposed, and a virtual affibody library (148 molecules) was constructed on the basis of key residue identification of the 3–2A2–4 nanobody that binds to the RBD. The most potential candidate, Q30, was obtained after two rounds of docking and molecular dynamics (MD) simulations. Competitive ELISA confirmed that Q30 bound to the conserved region of the RBD, rather than to the human angiotensin-converting enzyme 2-binding region. Furthermore, Q30 had high affinities of 0.23 μmol/L for the wild-type RBD and 0.81 μmol/L for the Omicron variant, indicating good broad-spectrum affinity. By coupling Q30 onto Sepharose Fast Flow (SepFF), the affinity gel (Q30-SepFF) could efficiently adsorb both the RBD and its Omicron variant, validating the broad-spectrum of the chromatography. Affinity chromatography was successfully applied for the purification of the RBD (a purity of 87.52 %), the RBD omicron variant (a purity of 89.41 %) and the Omicron vaccine (a purity of 84.62 %), indicating great applicability in vaccine purification.
2019冠状病毒病（COVID-19）大流行不仅对人类健康和全球经济构成严重威胁，而且对高效分离高变异病毒提出了更高要求。为了解决这一难题，本文提出了一种针对刺突蛋白受体结合域（RBD）保守区域的附着体配体开发策略，并在结合RBD的3-2A2-4纳米体的关键残基鉴定的基础上构建了一个包含148个分子的虚拟附着体文库。经过两轮对接和分子动力学（MD）模拟，获得了最有潜力的候选分子Q30。竞争性ELISA证实Q30结合到RBD的保守区域，而不是人血管紧张素转换酶2结合区域。Q30对野生型RBD和Omicron变体的亲和度分别为0.23 μmol/L和0.81 μmol/L，具有良好的广谱亲和性。通过将Q30偶联到Sepharose Fast Flow （SepFF）上，亲和凝胶（Q30-SepFF）可以有效吸附RBD及其Omicron变体，验证了色谱的广谱性。亲和层析法成功纯化了RBD（纯度为87.52%）、RBD的omicron变体（纯度为89.41%）和omicron疫苗（纯度为84.62%），在疫苗纯化中具有很强的适用性。
{"title":"Discovery of a broad-spectrum affibody ligand for COVID-19 vaccine purification","authors":"Zihan Li , Xiaoyan Dong , Linling Yu , Yan Sun , Qinghong Shi","doi":"10.1016/j.chroma.2025.466552","DOIUrl":"10.1016/j.chroma.2025.466552","url":null,"abstract":"<div><div>The coronavirus disease 2019 (COVID-19) pandemic has posed not only a severe threat to human health and the global economy but also greater requirements for the efficient isolation of highly variable viruses. To address this dilemma, a strategy for the development of affibody ligands targeting the conserved region of the receptor binding domain (RBD) on the spike (S) protein was proposed, and a virtual affibody library (148 molecules) was constructed on the basis of key residue identification of the 3–2A2–4 nanobody that binds to the RBD. The most potential candidate, Q30, was obtained after two rounds of docking and molecular dynamics (MD) simulations. Competitive ELISA confirmed that Q30 bound to the conserved region of the RBD, rather than to the human angiotensin-converting enzyme 2-binding region. Furthermore, Q30 had high affinities of 0.23 μmol/L for the wild-type RBD and 0.81 μmol/L for the Omicron variant, indicating good broad-spectrum affinity. By coupling Q30 onto Sepharose Fast Flow (SepFF), the affinity gel (Q30-SepFF) could efficiently adsorb both the RBD and its Omicron variant, validating the broad-spectrum of the chromatography. Affinity chromatography was successfully applied for the purification of the RBD (a purity of 87.52 %), the RBD omicron variant (a purity of 89.41 %) and the Omicron vaccine (a purity of 84.62 %), indicating great applicability in vaccine purification.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1766 ","pages":"Article 466552"},"PeriodicalIF":4.0,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145585591","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}
Pub Date : 2025-11-19DOI: 10.1016/j.chroma.2025.466556
Weilong Zhao , Baixin Ye , Feng Liu , Fei Xu , Chunmiao Bo
At present, aflatoxin M1 (AFM1) and tetracycline (TC) have caused contamination to milk. Therefore, simultaneous detection of these two compounds is of certain significance in the field of food safety. A dual-functional Janus-MIP-Apt materials featuring boronic acid-affinitive molecularly imprinted sites and aptamer (Apt) recognition has been designed for the simultaneous detection of TC and AFM1. Owing to the anisotropy of Janus, molecularly imprinted sites facilitate TC recognition via hydrophobic interactions, π-π stacking, and boronic acid affinity, while AFM1 recognition is achieved through an immobilized Apt. Thus, Janus-MIP-Apt exhibits high adsorption capacity and favorable selectivity for both TC and AFM1, serving as an effective solid-phase extraction (SPE) adsorbent for subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In this study, a method integrating SPE with LC-MS/MS was developed using Janus-MIP-Apt as the adsorbent for detecting TC and AFM1 in milk. This method demonstrates a broad linear range, satisfactory recoveries (86.7 − 105.8 %), and limits of detection were 0.1 μg/L (TC) and 0.2 μg/L (AFM1).
{"title":"Simultaneous detection of tetracycline and aflatoxin M1 using constructing Janus particles functionalized by molecular imprinting and aptamer","authors":"Weilong Zhao , Baixin Ye , Feng Liu , Fei Xu , Chunmiao Bo","doi":"10.1016/j.chroma.2025.466556","DOIUrl":"10.1016/j.chroma.2025.466556","url":null,"abstract":"<div><div>At present, aflatoxin M1 (AFM1) and tetracycline (TC) have caused contamination to milk. Therefore, simultaneous detection of these two compounds is of certain significance in the field of food safety. A dual-functional Janus-MIP-Apt materials featuring boronic acid-affinitive molecularly imprinted sites and aptamer (Apt) recognition has been designed for the simultaneous detection of TC and AFM1. Owing to the anisotropy of Janus, molecularly imprinted sites facilitate TC recognition via hydrophobic interactions, π-π stacking, and boronic acid affinity, while AFM1 recognition is achieved through an immobilized Apt. Thus, Janus-MIP-Apt exhibits high adsorption capacity and favorable selectivity for both TC and AFM1, serving as an effective solid-phase extraction (SPE) adsorbent for subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In this study, a method integrating SPE with LC-MS/MS was developed using Janus-MIP-Apt as the adsorbent for detecting TC and AFM1 in milk. This method demonstrates a broad linear range, satisfactory recoveries (86.7 − 105.8 %), and limits of detection were 0.1 μg/L (TC) and 0.2 μg/L (AFM1).</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1766 ","pages":"Article 466556"},"PeriodicalIF":4.0,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145585642","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}
Pub Date : 2025-11-19DOI: 10.1016/j.chroma.2025.466557
Shriarjun Shastry , Arianna Minzoni , Morgan R. Hurst , Crystal Collazo , Eduardo Barbieri , Wenning Chu , Joseph Taris , Patrick Gilbert , Christopher Major , Michael Daniele , Stefano Menegatti
Adeno-associated virus (AAV) vectors are revolutionizing gene therapy with remarkable therapeutic potential. Yet, their clinical translation is limited by inadequate biomanufacturing technologies that compromise product quality and accessibility. Addressing these issues, this study systematically investigates the conditions used for midstream and downstream AAV processing – namely, HEK293 cell lysis, tangential flow filtration (TFF), and chromatographic purification – and their impact on product yield and quality using AAV8 as a model serotype. We evaluated a panel of detergents (Triton X-100, sodium deoxycholate, Deviron C16/S9–13, CTAB, AAV-MAX) and salts (KCl, NaCl), and found that, while the capsid yields were similar across the various lysates, the subsequent process steps exhibited a strong dependence on lysis conditions. In particular, residual detergents in the lysates affected the efficiency of the TFF step by reducing permeate flux and necessitating the use of membranes with higher molecular weight cut-offs or additional buffer exchange prior to affinity capture. Triton X-100 and sodium deoxycholate afforded efficient cell lysis and high vector yields, but compromised TFF throughput. Conversely, lysates produced using salts or CTAB enabled robust TFF, with high permeate flux, and maintained capsid recoveries above 75 %. The subsequent affinity capture of AAV8 from selected lysates using AvXcel resin afforded yields of 45 - 70 %, >100-fold reduction in host cell impurities, and a 1.9-fold enrichment of cell-transducing units. Product polishing by anion-exchange chromatography reached final titers of 5.0 - 7.7 × 1011 vg/mL, full capsid ratios up to 85 %, and transduction activities of 3.2 × 105 TU/mL, while reducing host cell protein and DNA levels to < 2 ng/mL and < 100 pg/mL, meeting current regulatory guidelines for gene therapy. These results delineate the interplay between midstream and downstream operation conditions, providing practical guidance for AAV purification workflows suitable for large-scale manufacturing.
{"title":"Integrating the affinity and anion-exchange purification of AAV8: how the selective enrichment of full capsids at the affinity capture step enhances the downstream processing of gene therapies","authors":"Shriarjun Shastry , Arianna Minzoni , Morgan R. Hurst , Crystal Collazo , Eduardo Barbieri , Wenning Chu , Joseph Taris , Patrick Gilbert , Christopher Major , Michael Daniele , Stefano Menegatti","doi":"10.1016/j.chroma.2025.466557","DOIUrl":"10.1016/j.chroma.2025.466557","url":null,"abstract":"<div><div>Adeno-associated virus (AAV) vectors are revolutionizing gene therapy with remarkable therapeutic potential. Yet, their clinical translation is limited by inadequate biomanufacturing technologies that compromise product quality and accessibility. Addressing these issues, this study systematically investigates the conditions used for midstream and downstream AAV processing – namely, HEK293 cell lysis, tangential flow filtration (TFF), and chromatographic purification – and their impact on product yield and quality using AAV8 as a model serotype. We evaluated a panel of detergents (Triton X-100, sodium deoxycholate, Deviron C16/S9–13, CTAB, AAV-MAX) and salts (KCl, NaCl), and found that, while the capsid yields were similar across the various lysates, the subsequent process steps exhibited a strong dependence on lysis conditions. In particular, residual detergents in the lysates affected the efficiency of the TFF step by reducing permeate flux and necessitating the use of membranes with higher molecular weight cut-offs or additional buffer exchange prior to affinity capture. Triton X-100 and sodium deoxycholate afforded efficient cell lysis and high vector yields, but compromised TFF throughput. Conversely, lysates produced using salts or CTAB enabled robust TFF, with high permeate flux, and maintained capsid recoveries above 75 %. The subsequent affinity capture of AAV8 from selected lysates using AvXcel resin afforded yields of 45 - 70 %, >100-fold reduction in host cell impurities, and a 1.9-fold enrichment of cell-transducing units. Product polishing by anion-exchange chromatography reached final titers of 5.0 - 7.7 × 10<sup>11</sup> vg/mL, full capsid ratios up to 85 %, and transduction activities of 3.2 × 10<sup>5</sup> TU/mL, while reducing host cell protein and DNA levels to < 2 ng/mL and < 100 pg/mL, meeting current regulatory guidelines for gene therapy. These results delineate the interplay between midstream and downstream operation conditions, providing practical guidance for AAV purification workflows suitable for large-scale manufacturing.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1765 ","pages":"Article 466557"},"PeriodicalIF":4.0,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145620978","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}
For the analysis of micropollutants in biota samples such as insects with aquatic life stages with inherently low body weight, miniaturization of common extraction methods is necessary. For QuEChERS extractions, miniaturization down to a few individual organisms was reported for invertebrates. For the investigation of interindividual differences in insects, even further miniaturization is necessary. Our salt-free SWIEET extraction method recently developed as an alternative to QuEChERS was originally optimized for a total volume of 5 mL. To enable the determination of carbendazim in midges, that weigh between 0.3 and 0.5 mg, the method required miniaturization. Using a model analyte mix, we investigated how reducing the total extraction volume affected analyte recoveries and investigated matrix effects using Chironomus riparius as model organisms. The miniaturized SWIEET extraction was then applied to the analysis of the pesticide carbendazim in adult midges of Chironomus riparius from an exposure experiment at the larval stage. Since matrix effects played a significant role in the extraction of carbendazim, particularly for samples from lower exposure concentrations, sample preparation and calibration modes had to be further optimized. With the final protocol, we were able to successfully extract carbendazim from single midges. The effects of pooling midges were investigated to better understand matrix effects and effects on robustness. With the optimized and miniaturized method, we achieved repeatable results, making it suitable for determining carbendazim loads in midges and correlating them to the exposure concentrations.
{"title":"Miniaturization of the SWIEET extraction method to assess the body burden of carbendazim in Chironomus riparius midges after metamorphosis","authors":"Nadja Kalinke , Johanna Bock , Lea Dovodja , Jörg Oehlmann , Carolin Huhn","doi":"10.1016/j.chroma.2025.466554","DOIUrl":"10.1016/j.chroma.2025.466554","url":null,"abstract":"<div><div>For the analysis of micropollutants in biota samples such as insects with aquatic life stages with inherently low body weight, miniaturization of common extraction methods is necessary. For QuEChERS extractions, miniaturization down to a few individual organisms was reported for invertebrates. For the investigation of interindividual differences in insects, even further miniaturization is necessary. Our salt-free SWIEET extraction method recently developed as an alternative to QuEChERS was originally optimized for a total volume of 5 mL. To enable the determination of carbendazim in midges, that weigh between 0.3 and 0.5 mg, the method required miniaturization. Using a model analyte mix, we investigated how reducing the total extraction volume affected analyte recoveries and investigated matrix effects using <em>Chironomus riparius</em> as model organisms. The miniaturized SWIEET extraction was then applied to the analysis of the pesticide carbendazim in adult midges of <em>Chironomus riparius</em> from an exposure experiment at the larval stage. Since matrix effects played a significant role in the extraction of carbendazim, particularly for samples from lower exposure concentrations, sample preparation and calibration modes had to be further optimized. With the final protocol, we were able to successfully extract carbendazim from single midges. The effects of pooling midges were investigated to better understand matrix effects and effects on robustness. With the optimized and miniaturized method, we achieved repeatable results, making it suitable for determining carbendazim loads in midges and correlating them to the exposure concentrations.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1765 ","pages":"Article 466554"},"PeriodicalIF":4.0,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145621021","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}
Small interfering RNA (siRNA) therapeutics represent one of the most rapidly advancing pharmaceutical modalities. Since the approval of the first siRNA-based drug in 2018, seven siRNA therapeutics have been approved to date. During manufacturing, “non-optimal duplexes” and “single-stranded impurities” can arise from synthetic byproducts such as shortmers and longmers, or from minor imbalances in strand quantification and mixing. Furthermore, phosphorothioate linkages introduce chirality, generating complex diastereomeric mixtures. The use of delivery systems, including lipid nanoparticles or N-acetylgalactosamine (GalNAc) conjugates, further increases the structural complexity and heterogeneity of the final product. Compared to single-stranded oligonucleotides, siRNA—being double-stranded—yields more complex chromatographic and mass spectral profiles, making analytical characterization particularly challenging. Therefore, the development of robust and reliable analytical methods is essential to ensure the quality of siRNA therapeutics. This review summarizes key analytical techniques for the quality evaluation of siRNA therapeutics. Liquid chromatography (LC) is one of the most widely used approaches, with separation modes such as ion-pair reversed-phase (IP-RP), anion exchange (AEX), size exclusion chromatography (SEC), and hydrophilic interaction chromatography (HILIC) being actively explored. Mass spectrometry (MS) is another powerful tool that provides molecular mass-based structural information not accessible via UV detection alone. The combination of LC and MS offers a highly effective platform for characterizing siRNA therapeutics, enabling both qualitative and quantitative assessment of impurities and isomers. Furthermore, tandem MS (MS/MS) can provide sequence-specific information for the identification of active pharmaceutical ingredients. In addition to quality assessment, the application of LC and MS techniques in the pharmacokinetic analysis of siRNA therapeutics is also briefly discussed.
{"title":"Current trends in liquid chromatography-mass spectrometry analysis of small interfering RNAs: A short review","authors":"Hiroyuki Togawa , Takao Yamaguchi , Junji Kawakami , Satoshi Obika","doi":"10.1016/j.chroma.2025.466550","DOIUrl":"10.1016/j.chroma.2025.466550","url":null,"abstract":"<div><div>Small interfering RNA (siRNA) therapeutics represent one of the most rapidly advancing pharmaceutical modalities. Since the approval of the first siRNA-based drug in 2018, seven siRNA therapeutics have been approved to date. During manufacturing, “non-optimal duplexes” and “single-stranded impurities” can arise from synthetic byproducts such as shortmers and longmers, or from minor imbalances in strand quantification and mixing. Furthermore, phosphorothioate linkages introduce chirality, generating complex diastereomeric mixtures. The use of delivery systems, including lipid nanoparticles or <em>N</em>-acetylgalactosamine (GalNAc) conjugates, further increases the structural complexity and heterogeneity of the final product. Compared to single-stranded oligonucleotides, siRNA—being double-stranded—yields more complex chromatographic and mass spectral profiles, making analytical characterization particularly challenging. Therefore, the development of robust and reliable analytical methods is essential to ensure the quality of siRNA therapeutics. This review summarizes key analytical techniques for the quality evaluation of siRNA therapeutics. Liquid chromatography (LC) is one of the most widely used approaches, with separation modes such as ion-pair reversed-phase (IP-RP), anion exchange (AEX), size exclusion chromatography (SEC), and hydrophilic interaction chromatography (HILIC) being actively explored. Mass spectrometry (MS) is another powerful tool that provides molecular mass-based structural information not accessible <em>via</em> UV detection alone. The combination of LC and MS offers a highly effective platform for characterizing siRNA therapeutics, enabling both qualitative and quantitative assessment of impurities and isomers. Furthermore, tandem MS (MS/MS) can provide sequence-specific information for the identification of active pharmaceutical ingredients. In addition to quality assessment, the application of LC and MS techniques in the pharmacokinetic analysis of siRNA therapeutics is also briefly discussed.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1766 ","pages":"Article 466550"},"PeriodicalIF":4.0,"publicationDate":"2025-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145585608","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}
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