Pub Date : 2025-10-03DOI: 10.1016/j.jcoa.2025.100267
Paula Albendea, Aleksandra Gorska, Giorgia Purcaro
Mineral oil hydrocarbons (MOH) are a group of lipophilic contaminants composed of thousands of different compounds. The human toxicokinetics (absorption, metabolization, distribution and elimination) of these contaminants depends on their molecular structure but information are still lacking. The primary gap to be filled is the optimization and validation of a method to treat a very small amount of samples, as the ones produced during in-vitro studies.
The aim of this study is to develop and validate a miniaturized microwave-assisted saponification and extraction (MASE) method for the evaluation of MOH subclasses in low amounts of biological samples (<400 mg). The method was validated using an LC/GC×GC-FID/MS system in mono- and two-dimensional modes. The results demonstrated excellent recovery (≥94 %), repeatability, and compliance with JRC (Joint Research Center) guidance criteria for internal standard ratios. Furthermore, the method achieved a limit of quantification as low as 0.4 µg of MOH in the sample, outperforming conventional extraction methods.
{"title":"Optimization of a miniaturized microwave-assisted saponification and extraction method to evaluate mosh and MOAH in biological samples","authors":"Paula Albendea, Aleksandra Gorska, Giorgia Purcaro","doi":"10.1016/j.jcoa.2025.100267","DOIUrl":"10.1016/j.jcoa.2025.100267","url":null,"abstract":"<div><div>Mineral oil hydrocarbons (MOH) are a group of lipophilic contaminants composed of thousands of different compounds. The human toxicokinetics (absorption, metabolization, distribution and elimination) of these contaminants depends on their molecular structure but information are still lacking. The primary gap to be filled is the optimization and validation of a method to treat a very small amount of samples, as the ones produced during in-vitro studies.</div><div>The aim of this study is to develop and validate a miniaturized microwave-assisted saponification and extraction (MASE) method for the evaluation of MOH subclasses in low amounts of biological samples (<400 mg). The method was validated using an LC/GC×GC-FID/MS system in mono- and two-dimensional modes. The results demonstrated excellent recovery (≥94 %), repeatability, and compliance with JRC (Joint Research Center) guidance criteria for internal standard ratios. Furthermore, the method achieved a limit of quantification as low as 0.4 µg of MOH in the sample, outperforming conventional extraction methods.</div></div>","PeriodicalId":93576,"journal":{"name":"Journal of chromatography open","volume":"8 ","pages":"Article 100267"},"PeriodicalIF":3.2,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145265454","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-03DOI: 10.1016/j.jcoa.2025.100266
Tuğçe Öztepe, Cemil Can Eylem, Tuba Reçber, Emirhan Nemutlu
Metabolomics, a dynamic and rapidly evolving omics discipline, provides critical insights into the biochemical state of biological systems and holds transformative potential in fields such as biomarker discovery, disease mechanisms, and precision medicine. As the demand for high-throughput, unbiased, and reproducible metabolite profiling grows, particularly in clinical and translational settings, there is an urgent need for analytical platforms that can simultaneously address chemical diversity, sensitivity, and standardization.
Traditional single-column chromatographic systems often fall short in capturing the full spectrum of metabolites due to limited polarity range and separation capacity, leading to analytical blind spots and suboptimal data integration. In response to these limitations, dual-column systems have emerged as a promising solution by integrating orthogonal separation chemistries (e.g., reversed-phase; RP and hydrophilic interaction chromatography; HILIC) within a single analytical workflow. These systems offer superior performance by enabling the concurrent analysis of both polar and nonpolar metabolites, thereby reducing analysis time, improving sensitivity, and enhancing coverage, particularly in hybrid designs that unify targeted and untargeted metabolomics approaches.
However, despite their growing adoption, a comprehensive assessment of dual-column platforms, including design, performance, and advantages for metabolomics applications, remains limited. This review addresses this gap by systematically covering dual-column LC-MS applications in metabolomics reported between 1984 and 2025, comparing targeted and untargeted approaches and evaluating platform configurations, analytical capabilities, and contributions to workflow standardization. Furthermore, by discussing key methodological aspects, such as sample preparation, column setup, detection strategies, and data analysis, we highlight how these systems impact data quality and facilitate robust biological interpretation.
{"title":"Advances in dual-column chromatography for metabolomics: Opportunities, challenges, and applications","authors":"Tuğçe Öztepe, Cemil Can Eylem, Tuba Reçber, Emirhan Nemutlu","doi":"10.1016/j.jcoa.2025.100266","DOIUrl":"10.1016/j.jcoa.2025.100266","url":null,"abstract":"<div><div>Metabolomics, a dynamic and rapidly evolving omics discipline, provides critical insights into the biochemical state of biological systems and holds transformative potential in fields such as biomarker discovery, disease mechanisms, and precision medicine. As the demand for high-throughput, unbiased, and reproducible metabolite profiling grows, particularly in clinical and translational settings, there is an urgent need for analytical platforms that can simultaneously address chemical diversity, sensitivity, and standardization.</div><div>Traditional single-column chromatographic systems often fall short in capturing the full spectrum of metabolites due to limited polarity range and separation capacity, leading to analytical blind spots and suboptimal data integration. In response to these limitations, dual-column systems have emerged as a promising solution by integrating orthogonal separation chemistries (e.g., reversed-phase; RP and hydrophilic interaction chromatography; HILIC) within a single analytical workflow. These systems offer superior performance by enabling the concurrent analysis of both polar and nonpolar metabolites, thereby reducing analysis time, improving sensitivity, and enhancing coverage, particularly in hybrid designs that unify targeted and untargeted metabolomics approaches.</div><div>However, despite their growing adoption, a comprehensive assessment of dual-column platforms, including design, performance, and advantages for metabolomics applications, remains limited. This review addresses this gap by systematically covering dual-column LC-MS applications in metabolomics reported between 1984 and 2025, comparing targeted and untargeted approaches and evaluating platform configurations, analytical capabilities, and contributions to workflow standardization. Furthermore, by discussing key methodological aspects, such as sample preparation, column setup, detection strategies, and data analysis, we highlight how these systems impact data quality and facilitate robust biological interpretation.</div></div>","PeriodicalId":93576,"journal":{"name":"Journal of chromatography open","volume":"8 ","pages":"Article 100266"},"PeriodicalIF":3.2,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145324679","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-01DOI: 10.1016/j.jcoa.2025.100265
Alice Telle , Giulia Mastromatteo , Miriam Maiellaro, Lorenzo Sciuto, Grazia Bottillo, Emanuela Camera
Free fatty acids (FFAs) are essential components of skin surface lipids (SSLs), which consist of a blend of epidermal and sebaceous lipids. The sebum component of SSLs is abundant in seborrheic body areas, which are rich in sebaceous glands (SGs). Sebum FFAs exhibit unique characteristics, including a prevalence of C14-C18 chain lengths, terminal branching, and a double bond at the Δ6 position. Notably, over one-third of the sebaceous FAs contain a single double bond. Deregulated synthesis of monounsaturated FAs (MUFAs) can lead to changes in sebum composition, which contribute to the pathogenesis of inflammatory skin conditions such as acne vulgaris, rosacea, psoriasis, and seborrheic dermatitis. A comprehensive characterization of the different isomers of unbound MUFAs distinguished by the position of the carbon-carbon double bond, has not been achieved in human sebum. Therefore, we aimed to develop an analytical strategy to differentiate the various carbon-carbon double bond positions in FFAs. Our methodology combines the Paternò-Büchi reaction, utilizing 2-acpy, with liquid chromatography and tandem mass spectrometry (LC-MS/MS). This strategy was initially optimized on standard FFAs and subsequently applied to human sebum. The method enabled the characterization of seventeen MUFAs with C14-C18 chain lengths and two C18 polyunsaturated FAs, namely linoleic and sebaleic acid. The prevalence of sapienic acid (C16:1n-10) among MUFAs supports the dominance of the Δ6 desaturation pathway catalyzed by the FADS2 enzyme in human SG. Additionally, the assay proved comparable abundance of MUFAs and PUFAs in sebum from males and females.
{"title":"Characterization of double bond position in free fatty acids of human sebum","authors":"Alice Telle , Giulia Mastromatteo , Miriam Maiellaro, Lorenzo Sciuto, Grazia Bottillo, Emanuela Camera","doi":"10.1016/j.jcoa.2025.100265","DOIUrl":"10.1016/j.jcoa.2025.100265","url":null,"abstract":"<div><div>Free fatty acids (FFAs) are essential components of skin surface lipids (SSLs), which consist of a blend of epidermal and sebaceous lipids. The sebum component of SSLs is abundant in seborrheic body areas, which are rich in sebaceous glands (SGs). Sebum FFAs exhibit unique characteristics, including a prevalence of C14-C18 chain lengths, terminal branching, and a double bond at the Δ6 position. Notably, over one-third of the sebaceous FAs contain a single double bond. Deregulated synthesis of monounsaturated FAs (MUFAs) can lead to changes in sebum composition, which contribute to the pathogenesis of inflammatory skin conditions such as acne vulgaris, rosacea, psoriasis, and seborrheic dermatitis. A comprehensive characterization of the different isomers of unbound MUFAs distinguished by the position of the carbon-carbon double bond, has not been achieved in human sebum. Therefore, we aimed to develop an analytical strategy to differentiate the various carbon-carbon double bond positions in FFAs. Our methodology combines the Paternò-Büchi reaction, utilizing 2-acpy, with liquid chromatography and tandem mass spectrometry (LC-MS/MS). This strategy was initially optimized on standard FFAs and subsequently applied to human sebum. The method enabled the characterization of seventeen MUFAs with C14-C18 chain lengths and two C18 polyunsaturated FAs, namely linoleic and sebaleic acid. The prevalence of sapienic acid (C16:1n-10) among MUFAs supports the dominance of the Δ6 desaturation pathway catalyzed by the FADS2 enzyme in human SG. Additionally, the assay proved comparable abundance of MUFAs and PUFAs in sebum from males and females.</div></div>","PeriodicalId":93576,"journal":{"name":"Journal of chromatography open","volume":"8 ","pages":"Article 100265"},"PeriodicalIF":3.2,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145219050","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sulfolane is a water-miscible industrial solvent that has been widely utilized in various industries. Once released into the environment, it can infiltrate fractured rock structures in groundwater zones, leading to long-term contamination. Benzene, toluene, ethylbenzene, and xylenes (collectively known as BTEX) are commonly associated with sulfolane in the petrochemical industry. The presence and mobility of sulfolane can influence the distribution and transport behavior of BTEX at contaminated sites. Therefore, simultaneous analysis and monitoring of these compounds are crucial for understanding the source and fate of contamination.
In this study, a gas chromatography-mass spectrometry (GC–MS) method was developed and validated for the simultaneous quantitation of sulfolane and BTEX in rock samples. To address the slow release of sulfolane observed in certain samples when left in methanol and to facilitate rapid analysis, a microwave-assisted extraction (MAE) method was optimized for sample preparation. Analysis of spiked samples yielded recoveries of 86 % to 115 % for the various analytes, with relative standard deviations of up to 7.9 %. The validated method was then applied to rock samples collected from a contaminated site in central Alberta, Canada. All analyzed samples were contaminated with toluene. Only one sample was found to be contaminated with sulfolane. Ethylbenzene, o-xylene, m/p-xylene and benzene were detected in 82 %, 44 %, 40 %, and 4 % of the samples, respectively. Although the concentrations of these contaminants were all below the Alberta Tier 1 Soil Remediation Guidelines, the sampling covered only a limited depth. Therefore, additional sampling and investigation are necessary to conclusively determine the status of persistent contamination at the site.
{"title":"Simultaneous analysis of sulfolane and BTEX in rock samples by gas chromatography–mass spectrometry","authors":"Merrik Kobarfard , Jennie Hansson , Beth Parker , Tadeusz Górecki","doi":"10.1016/j.jcoa.2025.100264","DOIUrl":"10.1016/j.jcoa.2025.100264","url":null,"abstract":"<div><div>Sulfolane is a water-miscible industrial solvent that has been widely utilized in various industries. Once released into the environment, it can infiltrate fractured rock structures in groundwater zones, leading to long-term contamination. Benzene, toluene, ethylbenzene, and xylenes (collectively known as BTEX) are commonly associated with sulfolane in the petrochemical industry. The presence and mobility of sulfolane can influence the distribution and transport behavior of BTEX at contaminated sites. Therefore, simultaneous analysis and monitoring of these compounds are crucial for understanding the source and fate of contamination.</div><div>In this study, a gas chromatography-mass spectrometry (GC–MS) method was developed and validated for the simultaneous quantitation of sulfolane and BTEX in rock samples. To address the slow release of sulfolane observed in certain samples when left in methanol and to facilitate rapid analysis, a microwave-assisted extraction (MAE) method was optimized for sample preparation. Analysis of spiked samples yielded recoveries of 86 % to 115 % for the various analytes, with relative standard deviations of up to 7.9 %. The validated method was then applied to rock samples collected from a contaminated site in central Alberta, Canada. All analyzed samples were contaminated with toluene. Only one sample was found to be contaminated with sulfolane. Ethylbenzene, <em>o-</em>xylene, <em>m</em>/<em>p-</em>xylene and benzene were detected in 82 %, 44 %, 40 %, and 4 % of the samples, respectively. Although the concentrations of these contaminants were all below the Alberta Tier 1 Soil Remediation Guidelines, the sampling covered only a limited depth. Therefore, additional sampling and investigation are necessary to conclusively determine the status of persistent contamination at the site.</div></div>","PeriodicalId":93576,"journal":{"name":"Journal of chromatography open","volume":"8 ","pages":"Article 100264"},"PeriodicalIF":3.2,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145219109","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-24DOI: 10.1016/j.jcoa.2025.100263
Anthony S. Ransdell, Melora Reed, John Herrington, Maria Hougland, Ronan M. Kelly
The evolution of biotherapeutics to effectively treat diseases has resulted in complex molecule scaffolds compared to traditional monoclonal antibodies (mAbs). While Protein A (ProA) affinity chromatography continues to be the predominant choice for affinity-based purification of traditional mAbs, it has also been deployed for primary capture of Fc-fusions, Fabs, single variable domain on a variable heavy chain (VHHs), and heteromAbs. When binding these different molecular scaffolds, ProA offers a high-affinity interaction with the Fc-domain, or in the case of antibody-like fragments such as Fabs and VHHs, a weaker affinity interaction with certain human VH3 sequences. As a result of the dual binding mechanism of ProA, the removal of product-related impurities, such as homodimer and half-body, can be challenging when deployed in the primary purification step of a heteromAb process. To address some of the key challenges faced in the purification of these novel scaffolds, we evaluated the unique purification capabilities of the MabSelect™ VH3 affinity resin compared to MabSelect PrismA™ for the capture of VHHs, Fab-VHH fusions, mAbs, and an asymmetric antibody. Our studies showed that the purification of mAb-based scaffolds with the new MabSelect VH3 resin has similar binding capacity with comparable product quality, decreased host cell protein (HCP) content of the eluted product pool, and increased binding affinity for human VH3 sequence compared to MabSelect PrismA. Furthermore, we highlighted the advantages of utilizing this novel selective affinity resin to remove molecule-specific impurities to purify an asymmetric antibody.
与传统的单克隆抗体（mab）相比，生物治疗技术的发展导致了复杂的分子支架。虽然Protein A （ProA）亲和色谱法仍然是传统单克隆抗体亲和纯化的主要选择，但它也被用于fc -fusion、fab、可变重链（vhs）上的单变量结构域和异源单克隆抗体的主要捕获。当结合这些不同的分子支架时，ProA与fc结构域具有高亲和力的相互作用，或者在抗体样片段如fab和VHHs的情况下，与某些人类VH3序列具有较弱的亲和力相互作用。由于ProA的双重结合机制，在异源单抗工艺的初级纯化步骤中，去除与产品相关的杂质（如同型二聚体和半体）可能具有挑战性。为了解决这些新型支架纯化中面临的一些关键挑战，我们评估了MabSelect™VH3亲和树脂在捕获vhh、Fab-VHH融合物、单克隆抗体和不对称抗体方面的独特纯化能力，并与MabSelect PrismA™进行了比较。我们的研究表明，与MabSelect PrismA相比，使用新的MabSelect VH3树脂纯化mab基支架具有相似的结合能力和相当的产品质量，洗脱产物池中宿主细胞蛋白（HCP）含量降低，对人VH3序列的结合亲和力增加。此外，我们强调了利用这种新型选择性亲和树脂去除分子特异性杂质以纯化不对称抗体的优点。
{"title":"Selective purification of antibody-based protein scaffolds with a novel variable heavy chain 3 affinity resin","authors":"Anthony S. Ransdell, Melora Reed, John Herrington, Maria Hougland, Ronan M. Kelly","doi":"10.1016/j.jcoa.2025.100263","DOIUrl":"10.1016/j.jcoa.2025.100263","url":null,"abstract":"<div><div>The evolution of biotherapeutics to effectively treat diseases has resulted in complex molecule scaffolds compared to traditional monoclonal antibodies (mAbs). While Protein A (ProA) affinity chromatography continues to be the predominant choice for affinity-based purification of traditional mAbs, it has also been deployed for primary capture of Fc-fusions, Fabs, single variable domain on a variable heavy chain (VHHs), and heteromAbs. When binding these different molecular scaffolds, ProA offers a high-affinity interaction with the Fc-domain, or in the case of antibody-like fragments such as Fabs and VHHs, a weaker affinity interaction with certain human VH3 sequences. As a result of the dual binding mechanism of ProA, the removal of product-related impurities, such as homodimer and half-body, can be challenging when deployed in the primary purification step of a heteromAb process. To address some of the key challenges faced in the purification of these novel scaffolds, we evaluated the unique purification capabilities of the MabSelect™ VH3 affinity resin compared to MabSelect PrismA™ for the capture of VHHs, Fab-VHH fusions, mAbs, and an asymmetric antibody. Our studies showed that the purification of mAb-based scaffolds with the new MabSelect VH3 resin has similar binding capacity with comparable product quality, decreased host cell protein (HCP) content of the eluted product pool, and increased binding affinity for human VH3 sequence compared to MabSelect PrismA. Furthermore, we highlighted the advantages of utilizing this novel selective affinity resin to remove molecule-specific impurities to purify an asymmetric antibody.</div></div>","PeriodicalId":93576,"journal":{"name":"Journal of chromatography open","volume":"8 ","pages":"Article 100263"},"PeriodicalIF":3.2,"publicationDate":"2025-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145264955","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-24DOI: 10.1016/j.jcoa.2025.100262
Renata Biba , Karla Košpić , Blaž Ivšić , Lucija Vujević , Amela Hozić , Marijana Erk , Irena Đapić , Mario Cindrić
Micro solid-phase extraction (µSPE) is a simple and efficient method for peptide separation, purification, and fractionation prior to mass spectrometry (MS) in bottom-up proteomics workflows. Here, we introduce a positive-pressure (PP)-µSPE platform for offline multidimensional peptide fractionation. Six one-dimensional (1D) fractionation protocols were optimized at low pH reversed-phase (RP), high pH RP, strong cation exchange (SCX), hydrophilic-lipophilic balance (HLB), quaternary methyl-ammonium (QMA), and mixed strong anion exchange/reversed-phase (MAX) using bovine serum albumin (BSA) tryptic peptides. Each protocol yielded six fractions, which were evaluated by peptide size, isoelectric point, and hydrophilicity. Peptide fractions were separated on nano-C18 RP column and analyzed by nanoESI-QTOF-MS, and fractionation performance was subsequently evaluated for each fractionation mode. The data were then paired to quantify orthogonality in projected multidimensional fractionation by employing information theory. QMA yielded the highest entropy, indicating the greatest peptide dispersion in 1D. Conversely, high pH RP fractionation had the lowest entropy and led to increased peptide modification and aggregation, compromising downstream analysis. Joint entropy and mutual information analysis identified the most orthogonal pairings (QMA–low pH RP, MAX–QMA, HLB–QMA) and highlighted redundancy among methods sharing similar separation mechanisms. Workflow’s practical utility was demonstrated on the fragment antigen-binding part of Cetuximab, where QMA fractionation enabled identification of a previously undetected heavy chain peptide, achieving complete sequence coverage. These results demonstrate that PP-µSPE enables repeatable and combinable peptide fractionation across diverse sorbents and complex proteins, and supports targeted workflows by facilitating selective peptide isolation based on their physicochemical properties, streamlining experimental design in multidimensional proteomic analyses.
{"title":"Orthogonality of separation and sorbent evaluation in offline multidimensional peptide fractionation using automated positive pressure micro solid phase extraction","authors":"Renata Biba , Karla Košpić , Blaž Ivšić , Lucija Vujević , Amela Hozić , Marijana Erk , Irena Đapić , Mario Cindrić","doi":"10.1016/j.jcoa.2025.100262","DOIUrl":"10.1016/j.jcoa.2025.100262","url":null,"abstract":"<div><div>Micro solid-phase extraction (µSPE) is a simple and efficient method for peptide separation, purification, and fractionation prior to mass spectrometry (MS) in bottom-up proteomics workflows. Here, we introduce a positive-pressure (PP)-µSPE platform for offline multidimensional peptide fractionation. Six one-dimensional (1D) fractionation protocols were optimized at low pH reversed-phase (RP), high pH RP, strong cation exchange (SCX), hydrophilic-lipophilic balance (HLB), quaternary methyl-ammonium (QMA), and mixed strong anion exchange/reversed-phase (MAX) using bovine serum albumin (BSA) tryptic peptides. Each protocol yielded six fractions, which were evaluated by peptide size, isoelectric point, and hydrophilicity. Peptide fractions were separated on nano-C18 RP column and analyzed by nanoESI-QTOF-MS, and fractionation performance was subsequently evaluated for each fractionation mode. The data were then paired to quantify orthogonality in projected multidimensional fractionation by employing information theory. QMA yielded the highest entropy, indicating the greatest peptide dispersion in 1D. Conversely, high pH RP fractionation had the lowest entropy and led to increased peptide modification and aggregation, compromising downstream analysis. Joint entropy and mutual information analysis identified the most orthogonal pairings (QMA–low pH RP, MAX–QMA, HLB–QMA) and highlighted redundancy among methods sharing similar separation mechanisms. Workflow’s practical utility was demonstrated on the fragment antigen-binding part of Cetuximab, where QMA fractionation enabled identification of a previously undetected heavy chain peptide, achieving complete sequence coverage. These results demonstrate that PP-µSPE enables repeatable and combinable peptide fractionation across diverse sorbents and complex proteins, and supports targeted workflows by facilitating selective peptide isolation based on their physicochemical properties, streamlining experimental design in multidimensional proteomic analyses.</div></div>","PeriodicalId":93576,"journal":{"name":"Journal of chromatography open","volume":"8 ","pages":"Article 100262"},"PeriodicalIF":3.2,"publicationDate":"2025-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145218999","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-19DOI: 10.1016/j.jcoa.2025.100261
Brian R. van ‘t Veer , Jasper P.H. Smeets , Clementina Vitali , Anna K. Undas , Sander Affourtit , Hans-Gerd Janssen
To understand the health impact of micro- and nanoplastics (MNPs), methods to quantify the levels of the various particle size ranges as well as techniques to chemically characterize them are needed. Current analytical strategies largely focus either on the size or the chemical composition of these MNPs, without correlating the two. A novel hyphenated method is presented that combines hydrodynamic chromatography (HDC) with pyrolysis-gas chromatography-mass spectrometry (Py–GC–MS) for integrated size- and composition characterization. Because HDC does not use membranes or stationary phases, the need for mobile phase additives is significantly reduced, simplifying combination with Py–GC–MS identification. In the newly developed method, the effluent of the HDC size separation is fractionated and individual fractions are subjected to Py–GC–MS identification. Introduction of the fractions and subsequent pyrolysis is done using a programmable temperature vaporization (PTV) inlet. The PTV introduction step employs a three-stage temperature program: an initial low-temperature hold for water removal, followed by an additive-removal step at moderate temperature, and finally a treatment at high temperature to pyrolyze the MNPs. The PTV Py–GC–MS method showed a good performance in the analysis of various plastics including PET, PA12, PTFE, PTHF and PE. For other plastics like PAN, PVDC and PVC the identification capability of the method was also good, but quantification was compromised by partial losses of the material during additive removal. Application of the HDC–Py–GC–MS method to a PS nanoplastic mixture, as well as to a representative industrial acrylate/styrene nanoplastic sample, highlighted the method’s potential for correlating size and chemical composition.
{"title":"Integrated size- and composition analysis of nanoplastics by hyphenating hydrodynamic chromatography to pyrolysis-gas chromatography-mass spectrometry","authors":"Brian R. van ‘t Veer , Jasper P.H. Smeets , Clementina Vitali , Anna K. Undas , Sander Affourtit , Hans-Gerd Janssen","doi":"10.1016/j.jcoa.2025.100261","DOIUrl":"10.1016/j.jcoa.2025.100261","url":null,"abstract":"<div><div>To understand the health impact of micro- and nanoplastics (MNPs), methods to quantify the levels of the various particle size ranges as well as techniques to chemically characterize them are needed. Current analytical strategies largely focus either on the size or the chemical composition of these MNPs, without correlating the two. A novel hyphenated method is presented that combines hydrodynamic chromatography (HDC) with pyrolysis-gas chromatography-mass spectrometry (Py–GC–MS) for integrated size- and composition characterization. Because HDC does not use membranes or stationary phases, the need for mobile phase additives is significantly reduced, simplifying combination with Py–GC–MS identification. In the newly developed method, the effluent of the HDC size separation is fractionated and individual fractions are subjected to Py–GC–MS identification. Introduction of the fractions and subsequent pyrolysis is done using a programmable temperature vaporization (PTV) inlet. The PTV introduction step employs a three-stage temperature program: an initial low-temperature hold for water removal, followed by an additive-removal step at moderate temperature, and finally a treatment at high temperature to pyrolyze the MNPs. The PTV Py–GC–MS method showed a good performance in the analysis of various plastics including PET, PA12, PTFE, PTHF and PE. For other plastics like PAN, PVDC and PVC the identification capability of the method was also good, but quantification was compromised by partial losses of the material during additive removal. Application of the HDC–Py–GC–MS method to a PS nanoplastic mixture, as well as to a representative industrial acrylate/styrene nanoplastic sample, highlighted the method’s potential for correlating size and chemical composition.</div></div>","PeriodicalId":93576,"journal":{"name":"Journal of chromatography open","volume":"8 ","pages":"Article 100261"},"PeriodicalIF":3.2,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145158054","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-17DOI: 10.1016/j.jcoa.2025.100260
Erika Maria Ricci , Enrico Dainese , Vincenzo De Laurenzi , Giulio Lovato , Gianluca Sala , Marcello Locatelli , Miryam Perrucci
Since 1913, when Paul Ehrlich introduced the concept of “selective toxicity”, new strategies to obtain quality control, quantification of the drug were mandatory. For this reason, the introduction of antibody drug conjugates (ADC) has shown many critical points, being these systems made of small- and high-molecular weight compounds. Being ADCs a novelty for showing therapeutic action, they were subjected to a grow up in studies. There are in literature several approaches to follow ADCs for in vivo and in vitro studies because type of conjugation, release of the drug, and body-distribution are characteristic for each of them, resulting in many critical steps.
Liquid chromatography (LC) and capillary electrophoresis (CE) are the most common analytical technique used in order to obtain the aim. For the characterization of ADCs, there are several chromatographic techniques. HPLC finds application in both small and large molecules, related to the availability of various modes of separation (reversed-phase, size exclusion, ion exchange, mixed-mode etc.). Capillary electrophoresis (CE) finds its main application in large molecule therapeutics, where its electrophoretic separation mechanism offers a distinct, and often superior, separation of macromolecules compared to classic chromatographic techniques. Several modes of CE, including capillary electrophoresis sodium dodecyl sulphate (CE-SDS), capillary zone electrophoresis (CZE), and capillary isoelectric focusing (CIEF) or imaged capillary isoelectric focusing (iCIEF) are commonly utilized in characterization of the critical quality attributes (CQAs) of monoclonal antibody drugs such as charge variants, size variants, and positional isomers/purity etc.
In this review, analytical methods for physicochemical characterization of ADCs will be reported and analysed in order to highlight as the chromatographic procedures allow obtaining a complete ADCs evaluation and characterization.
{"title":"New analytical challenges in characterization of antibody-drug conjugates","authors":"Erika Maria Ricci , Enrico Dainese , Vincenzo De Laurenzi , Giulio Lovato , Gianluca Sala , Marcello Locatelli , Miryam Perrucci","doi":"10.1016/j.jcoa.2025.100260","DOIUrl":"10.1016/j.jcoa.2025.100260","url":null,"abstract":"<div><div>Since 1913, when Paul Ehrlich introduced the concept of “selective toxicity”, new strategies to obtain quality control, quantification of the drug were mandatory. For this reason, the introduction of antibody drug conjugates (ADC) has shown many critical points, being these systems made of small- and high-molecular weight compounds. Being ADCs a novelty for showing therapeutic action, they were subjected to a grow up in studies. There are in literature several approaches to follow ADCs for <em>in vivo</em> and <em>in vitro</em> studies because type of conjugation, release of the drug, and body-distribution are characteristic for each of them, resulting in many critical steps.</div><div>Liquid chromatography (LC) and capillary electrophoresis (CE) are the most common analytical technique used in order to obtain the aim. For the characterization of ADCs, there are several chromatographic techniques. HPLC finds application in both small and large molecules, related to the availability of various modes of separation (reversed-phase, size exclusion, ion exchange, mixed-mode etc.). Capillary electrophoresis (CE) finds its main application in large molecule therapeutics, where its electrophoretic separation mechanism offers a distinct, and often superior, separation of macromolecules compared to classic chromatographic techniques. Several modes of CE, including capillary electrophoresis sodium dodecyl sulphate (CE-SDS), capillary zone electrophoresis (CZE), and capillary isoelectric focusing (CIEF) or imaged capillary isoelectric focusing (iCIEF) are commonly utilized in characterization of the critical quality attributes (CQAs) of monoclonal antibody drugs such as charge variants, size variants, and positional isomers/purity etc.</div><div>In this review, analytical methods for physicochemical characterization of ADCs will be reported and analysed in order to highlight as the chromatographic procedures allow obtaining a complete ADCs evaluation and characterization.</div></div>","PeriodicalId":93576,"journal":{"name":"Journal of chromatography open","volume":"8 ","pages":"Article 100260"},"PeriodicalIF":3.2,"publicationDate":"2025-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145158055","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-15DOI: 10.1016/j.jcoa.2025.100258
Zuzana Vosáhlová , Jana Poláchová , Jiří Svoboda , Michal Kohout , Květa Kalíková
Two commercially available zwitterionic columns, one diol-based column and two newly developed experimental zwitterionic columns for hydrophilic interaction liquid chromatography (HILIC) were characterized and compared in terms of their interaction abilities. The novel HILIC columns offer mixed-mode cation-exchange and anion-exchange interactions together with minor hydrophobic contribution of alkyl spacers between the units.
Tanaka testing protocol, which provides complex information about column retentivity, hydrophobicity, hydrophilicity and cation/anion-exchange characters was performed. The results revealed the differences between individual zwitterionic stationary phases and clear predominance of cation-exchange character over anion-exchange character was observed for all tested columns. The measurements at different pH at various buffer salt concentrations revealed that the predominance of negative charge on the stationary phase surface occurs at pH 4.7 and higher (probably due to the dissociation of residual silanols). At lower pH (pH 3.0) the anion-exchange mechanism also applies. The cation-exchange character (applying across the whole pH range) has been further studied using other simple chromatographic tests and compared with >20 other commercially available columns. The application potential of all the tested columns was verified for analysis of tryptophan and its derivatives and also for cytochrome C tryptic digests.
{"title":"Interaction properties and separation potential of zwitterionic stationary phases in hydrophilic interaction liquid chromatography","authors":"Zuzana Vosáhlová , Jana Poláchová , Jiří Svoboda , Michal Kohout , Květa Kalíková","doi":"10.1016/j.jcoa.2025.100258","DOIUrl":"10.1016/j.jcoa.2025.100258","url":null,"abstract":"<div><div>Two commercially available zwitterionic columns, one diol-based column and two newly developed experimental zwitterionic columns for hydrophilic interaction liquid chromatography (HILIC) were characterized and compared in terms of their interaction abilities. The novel HILIC columns offer mixed-mode cation-exchange and anion-exchange interactions together with minor hydrophobic contribution of alkyl spacers between the units.</div><div>Tanaka testing protocol, which provides complex information about column retentivity, hydrophobicity, hydrophilicity and cation/anion-exchange characters was performed. The results revealed the differences between individual zwitterionic stationary phases and clear predominance of cation-exchange character over anion-exchange character was observed for all tested columns. The measurements at different pH at various buffer salt concentrations revealed that the predominance of negative charge on the stationary phase surface occurs at pH 4.7 and higher (probably due to the dissociation of residual silanols). At lower pH (pH 3.0) the anion-exchange mechanism also applies. The cation-exchange character (applying across the whole pH range) has been further studied using other simple chromatographic tests and compared with >20 other commercially available columns. The application potential of all the tested columns was verified for analysis of tryptophan and its derivatives and also for cytochrome C tryptic digests.</div></div>","PeriodicalId":93576,"journal":{"name":"Journal of chromatography open","volume":"8 ","pages":"Article 100258"},"PeriodicalIF":3.2,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145104205","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-15DOI: 10.1016/j.jcoa.2025.100259
Jiarong Cui , Fengshou Dong
Pesticide residue analysis is fundamental to food safety and environmental protection, where sample pretreatment critically determines detection accuracy. This review examines the evolution from traditional methods (Soxhlet extraction, liquid-liquid extraction) to contemporary mainstream technologies (solid-phase extraction, QuEChERS, microextraction), and explores future prospects. Modern techniques like microextraction (SPME, DLLME) and magnetic solid-phase extraction (MSPE) have significantly enhanced efficiency while reducing solvent consumption by 90–95 %. The QuEChERS methodology has become the gold standard for multiresidue analysis in complex matrices. Future advancements focus on the miniaturization of analytical systems, suitable greener methodologies, and the development of advanced techniques for complex matrices and ultra-trace analysis.
{"title":"Research advances in sample pretreatment techniques for pesticide residues","authors":"Jiarong Cui , Fengshou Dong","doi":"10.1016/j.jcoa.2025.100259","DOIUrl":"10.1016/j.jcoa.2025.100259","url":null,"abstract":"<div><div>Pesticide residue analysis is fundamental to food safety and environmental protection, where sample pretreatment critically determines detection accuracy. This review examines the evolution from traditional methods (Soxhlet extraction, liquid-liquid extraction) to contemporary mainstream technologies (solid-phase extraction, QuEChERS, microextraction), and explores future prospects. Modern techniques like microextraction (SPME, DLLME) and magnetic solid-phase extraction (MSPE) have significantly enhanced efficiency while reducing solvent consumption by 90–95 %. The QuEChERS methodology has become the gold standard for multiresidue analysis in complex matrices. Future advancements focus on the miniaturization of analytical systems, suitable greener methodologies, and the development of advanced techniques for complex matrices and ultra-trace analysis.</div></div>","PeriodicalId":93576,"journal":{"name":"Journal of chromatography open","volume":"8 ","pages":"Article 100259"},"PeriodicalIF":3.2,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145158056","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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