Pub Date : 2026-01-22DOI: 10.1016/j.chroma.2026.466726
Yue Deng , Shuang Feng , Xiaoyan Zhang , Jiarui Zhang , Jiale Liu , Xinyue Zhou , Shimeng Fu , Yong Zhang , Lei Yin , Meiyun Shi
This study presents an innovative analytical approach utilizing ultra-performance convergence chromatography tandem mass spectrometry (UPCC-MS/MS) for the systematic separation and quantification of 20 oligomers (n = 9–28) of polyethylene glycol 1000 (PEG1K) in rat urine. A key innovation involves the use of ammonium adducts ( [M+NH₄]⁺ and [M + 2NH₄]²⁺) as precursor ions, significantly enhancing sensitivity and selectivity, particularly for higher oligomers (n > 21), where doubly charged species predominated. Coupled with a post-column compensation strategy, ionization efficiency was improved by 1–10-fold. Chromatographic separation was achieved under isocratic conditions using a green mobile phase based on supercritical CO₂ with a methanol–formic acid co-solvent, enabling efficient baseline resolution within a short runtime while minimizing organic solvent consumption. The method was validated per FDA guidelines, demonstrating excellent linearity, precision, and accuracy. Its applicability was confirmed through excretion kinetics studies in rats tail vein injection of PEG1K, revealing rapid renal clearance with 23% of the dose excreted within 4 h and cumulative excretion reaching 64% at 48 h. Green analytical chemistry metric assessments (BAGI and GEMAM) highlighted the method's superior environmental profile. This work establishes a sensitive, efficient, and sustainable platform for polymer pharmacokinetic research.
{"title":"High-throughput and green UPCC-MS/MS assay for simultaneous quantification of 20 oligomers of PEG1K coupled with ammonium adduct and post-column infusion strategies to enhance sensitivity","authors":"Yue Deng , Shuang Feng , Xiaoyan Zhang , Jiarui Zhang , Jiale Liu , Xinyue Zhou , Shimeng Fu , Yong Zhang , Lei Yin , Meiyun Shi","doi":"10.1016/j.chroma.2026.466726","DOIUrl":"10.1016/j.chroma.2026.466726","url":null,"abstract":"<div><div>This study presents an innovative analytical approach utilizing ultra-performance convergence chromatography tandem mass spectrometry (UPCC-MS/MS) for the systematic separation and quantification of 20 oligomers (<em>n</em> = 9–28) of polyethylene glycol 1000 (PEG1K) in rat urine. A key innovation involves the use of ammonium adducts ( [M+NH₄]⁺ and [M + 2NH₄]²⁺) as precursor ions, significantly enhancing sensitivity and selectivity, particularly for higher oligomers (<em>n</em> > 21), where doubly charged species predominated. Coupled with a post-column compensation strategy, ionization efficiency was improved by 1–10-fold. Chromatographic separation was achieved under isocratic conditions using a green mobile phase based on supercritical CO₂ with a methanol–formic acid co-solvent, enabling efficient baseline resolution within a short runtime while minimizing organic solvent consumption. The method was validated per FDA guidelines, demonstrating excellent linearity, precision, and accuracy. Its applicability was confirmed through excretion kinetics studies in rats tail vein injection of PEG1K, revealing rapid renal clearance with 23% of the dose excreted within 4 h and cumulative excretion reaching 64% at 48 h. Green analytical chemistry metric assessments (BAGI and GEMAM) highlighted the method's superior environmental profile. This work establishes a sensitive, efficient, and sustainable platform for polymer pharmacokinetic research.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1770 ","pages":"Article 466726"},"PeriodicalIF":4.0,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146090510","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 : 2026-01-22DOI: 10.1016/j.chroma.2026.466732
Thomas Garzón , Jefferson V. Pastuña-Fasso , Cristian Quiroz-Moreno , Melanie Ochoa-Ocampo , Evencio J. Medina-Villamizar , Nina Espinosa de los Monteros-Silva , Karel Diéguez-Santana , Jessica L. Cooperstone , Zulay Niño-Ruíz , José R. Almeida , Noroska G.S. Mogollón
Chlorogenic acids (caffeoylquinic acid isomers, CQAs) are major phenolic constituents of Ilex guayusa, but their comprehensive profiling in complex plant matrices is hindered by co-elution, overlapping UV spectra, and isomeric similarity in MS/MS. Rather than aiming to fully resolve isomer-specific quantification by MS, here we present an integrated workflow that couples validated HPLC–UV quantification of the major CQA (5-CQA) with an optimized UPLC–MS/MS strategy designed to improve MS1 peak integrity and expand MS/MS coverage for higher-confidence structural annotation. The HPLC–UV method showed excellent performance for targeted quantification of 5-CQA, including strong linearity (r² = 0.998), selectivity, sensitivity (LOQ = 0.25 mg/L), precision, and recovery. For LC–MS/MS, FastDDA acquisition (top-5 vs. top-15 precursors) revealed the expected trade-off between fragmentation depth and MS1 peak quality; however, post-acquisition raw-data merging restored MS1 fidelity and increased the number of detected features by 43%, enabling high-confidence annotation rather than quantitative discrimination of 16 metabolites and the propagation of oxidized CQA-related derivatives using feature-based molecular networking. Multivariate analyses (PCA, volcano plots, HCA) indicated that geographic location exerted the strongest influence on the metabolite composition, followed by sunlight exposure and plant age. Overall, the proposed workflow provides a practical framework that integrates robust chromatographic quantification with MS acquisition and data-processing optimization, thereby enhancing structural characterization and biological interpretation, rather than complete isomer-resolved quantification, of chlorogenic-acid-related chemistry across complex plant-derived and natural product matrices
{"title":"Integrated HPLC–UV validation and LC–MS/MS optimization with molecular networking and chemometrics for advanced characterization of chlorogenic acids in Ilex guayusa","authors":"Thomas Garzón , Jefferson V. Pastuña-Fasso , Cristian Quiroz-Moreno , Melanie Ochoa-Ocampo , Evencio J. Medina-Villamizar , Nina Espinosa de los Monteros-Silva , Karel Diéguez-Santana , Jessica L. Cooperstone , Zulay Niño-Ruíz , José R. Almeida , Noroska G.S. Mogollón","doi":"10.1016/j.chroma.2026.466732","DOIUrl":"10.1016/j.chroma.2026.466732","url":null,"abstract":"<div><div>Chlorogenic acids (caffeoylquinic acid isomers, CQAs) are major phenolic constituents of <em>Ilex guayusa,</em> but their comprehensive profiling in complex plant matrices is hindered by co-elution, overlapping UV spectra, and isomeric similarity in MS/MS. Rather than aiming to fully resolve isomer-specific quantification by MS, here we present an integrated workflow that couples validated HPLC–UV quantification of the major CQA (5-CQA) with an optimized UPLC–MS/MS strategy designed to improve MS1 peak integrity and expand MS/MS coverage for higher-confidence structural annotation. The HPLC–UV method showed excellent performance for targeted quantification of 5-CQA, including strong linearity (r² = 0.998), selectivity, sensitivity (LOQ = 0.25 mg/L), precision, and recovery. For LC–MS/MS, FastDDA acquisition (top-5 vs. top-15 precursors) revealed the expected trade-off between fragmentation depth and MS1 peak quality; however, post-acquisition raw-data merging restored MS1 fidelity and increased the number of detected features by 43%, enabling high-confidence annotation rather than quantitative discrimination of 16 metabolites and the propagation of oxidized CQA-related derivatives using feature-based molecular networking. Multivariate analyses (PCA, volcano plots, HCA) indicated that geographic location exerted the strongest influence on the metabolite composition, followed by sunlight exposure and plant age. Overall, the proposed workflow provides a practical framework that integrates robust chromatographic quantification with MS acquisition and data-processing optimization, thereby enhancing structural characterization and biological interpretation, rather than complete isomer-resolved quantification, of chlorogenic-acid-related chemistry across complex plant-derived and natural product matrices</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1769 ","pages":"Article 466732"},"PeriodicalIF":4.0,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146074172","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-22DOI: 10.1016/j.chroma.2026.466733
Li Zhu , Miao Guo , Yingjia Liu , Lu Zhang , Suntao Li , Jiaqi Zhao , Jianzheng Zhu , Meihui Sang , Liqiang Qian , Yan Zhang , Hua Xiao
Membrane proteins (MPs) play crucial roles in cellular functions and are key targets for drug discovery and cancer research. However, due to their poor solubility, high hydrophobicity, and low abundance, MPs present significant analytical challenges, particularly with regard to their dissolution, isolation, and characterization. Efficiently extracting MPs from frozen tissue samples remains challenging, which limits our ability to study their functions in pathological processes. In this study, we developed a metal-organic frameworks (MOFs)-assisted bio-separation strategy using Zeolitic Imidazolate Framework-67 (ZIF-67) for efficient membrane proteome isolation from frozen mouse lung tissue. We optimized key parameters for MPs extraction, including protein extraction approaches (tissue grinding versus enzymolysis), material-to-protein ratio, and SDS concentration. Our results demonstrated that ZIF-67 strategy with enzymolysis dramatically improved the enrichment efficiency of MPs with multi-transmembrane domains (TMs≥2) than the commercial kit method. In particular, among the 500 most abundant MPs isolated by the optimized ZIF-67 strategy, 133 were MPs with TMs≥2, which was 2.18 times that of the kit method. We further applied this strategy to analyze two distinct types of clinical lung cancer tissue samples (ground glass opacity nodules and solid nodules). Quantitative proteomics analysis revealed distinct membrane protein profiles and dysregulated pathways in the two lung cancer nodules. The enhanced capability of the ZIF-67 strategy for analyzing multi-transmembrane proteome provided valuable biological information for investigating the underlying molecular mechanisms underlying distinct nodule types. The strategy’s compatibility with frozen tissues underscores its broad applicability in translational and biomedical studies, paving the way for in-depth exploration of MPs in disease mechanisms and therapeutic development.
{"title":"Transmembrane proteome analysis of frozen mouse lung tissues by LC-MS using metal organic framework-based protein extraction","authors":"Li Zhu , Miao Guo , Yingjia Liu , Lu Zhang , Suntao Li , Jiaqi Zhao , Jianzheng Zhu , Meihui Sang , Liqiang Qian , Yan Zhang , Hua Xiao","doi":"10.1016/j.chroma.2026.466733","DOIUrl":"10.1016/j.chroma.2026.466733","url":null,"abstract":"<div><div>Membrane proteins (MPs) play crucial roles in cellular functions and are key targets for drug discovery and cancer research. However, due to their poor solubility, high hydrophobicity, and low abundance, MPs present significant analytical challenges, particularly with regard to their dissolution, isolation, and characterization. Efficiently extracting MPs from frozen tissue samples remains challenging, which limits our ability to study their functions in pathological processes. In this study, we developed a metal-organic frameworks (MOFs)-assisted bio-separation strategy using Zeolitic Imidazolate Framework-67 (ZIF-67) for efficient membrane proteome isolation from frozen mouse lung tissue. We optimized key parameters for MPs extraction, including protein extraction approaches (tissue grinding versus enzymolysis), material-to-protein ratio, and SDS concentration. Our results demonstrated that ZIF-67 strategy with enzymolysis dramatically improved the enrichment efficiency of MPs with multi-transmembrane domains (TMs≥2) than the commercial kit method. In particular, among the 500 most abundant MPs isolated by the optimized ZIF-67 strategy, 133 were MPs with TMs≥2, which was 2.18 times that of the kit method. We further applied this strategy to analyze two distinct types of clinical lung cancer tissue samples (ground glass opacity nodules and solid nodules). Quantitative proteomics analysis revealed distinct membrane protein profiles and dysregulated pathways in the two lung cancer nodules. The enhanced capability of the ZIF-67 strategy for analyzing multi-transmembrane proteome provided valuable biological information for investigating the underlying molecular mechanisms underlying distinct nodule types. The strategy’s compatibility with frozen tissues underscores its broad applicability in translational and biomedical studies, paving the way for in-depth exploration of MPs in disease mechanisms and therapeutic development.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1771 ","pages":"Article 466733"},"PeriodicalIF":4.0,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146071101","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 : 2026-01-22DOI: 10.1016/j.chroma.2026.466731
Miroslav Lísa , Roman Hájek
Gangliosides are biologically significant molecules with essential physiological roles in the organism. They are increasingly studied as biomarkers for disease diagnosis and progression. Due to their extreme structural diversity and typically low abundance in biological samples, efficient and sensitive liquid chromatography–mass spectrometry (LC-MS) methods are required for their analysis. In this study, we present a comprehensive comparison of the performance of two widely used LC-MS approaches: reversed-phase (RP) and hydrophilic interaction liquid chromatography (HILIC). Both methods were independently optimized following an identical protocol, with a focus on chromatographic performance and MS response. Notably, salt concentration had a pronounced impact on ganglioside retention, peak shape, isomer separation, and MS signal intensity in both chromatographic modes. In HILIC, adjusting the initial gradient composition and profile enabled effective separation of gangliosides from highly abundant matrix lipids. The RP method demonstrated superior ganglioside signal intensity and a 3.5-fold reduction in analysis time. Both approaches allowed the separation of gangliosides based on the number of sialic acid and saccharide residues. Additionally, the RP method enabled separation according to ceramide composition. We established rules for RP separation based on the number of carbon atoms in the ceramide moiety, which can be used to predict ganglioside retention times. Based on our findings, each method offers distinct advantages for specific analytical goals. The HILIC approach is better suited for untargeted ganglioside profiling using accurate m/z and interclass separation, while the RP method is ideal for sensitive, high-throughput targeted analysis in complex biological matrices.
{"title":"Comprehensive evaluation of reversed-phase and hydrophilic interaction liquid chromatography – mass spectrometry for analysis of gangliosides","authors":"Miroslav Lísa , Roman Hájek","doi":"10.1016/j.chroma.2026.466731","DOIUrl":"10.1016/j.chroma.2026.466731","url":null,"abstract":"<div><div>Gangliosides are biologically significant molecules with essential physiological roles in the organism. They are increasingly studied as biomarkers for disease diagnosis and progression. Due to their extreme structural diversity and typically low abundance in biological samples, efficient and sensitive liquid chromatography–mass spectrometry (LC-MS) methods are required for their analysis. In this study, we present a comprehensive comparison of the performance of two widely used LC-MS approaches: reversed-phase (RP) and hydrophilic interaction liquid chromatography (HILIC). Both methods were independently optimized following an identical protocol, with a focus on chromatographic performance and MS response. Notably, salt concentration had a pronounced impact on ganglioside retention, peak shape, isomer separation, and MS signal intensity in both chromatographic modes. In HILIC, adjusting the initial gradient composition and profile enabled effective separation of gangliosides from highly abundant matrix lipids. The RP method demonstrated superior ganglioside signal intensity and a 3.5-fold reduction in analysis time. Both approaches allowed the separation of gangliosides based on the number of sialic acid and saccharide residues. Additionally, the RP method enabled separation according to ceramide composition. We established rules for RP separation based on the number of carbon atoms in the ceramide moiety, which can be used to predict ganglioside retention times. Based on our findings, each method offers distinct advantages for specific analytical goals. The HILIC approach is better suited for untargeted ganglioside profiling using accurate <em>m/z</em> and interclass separation, while the RP method is ideal for sensitive, high-throughput targeted analysis in complex biological matrices.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1770 ","pages":"Article 466731"},"PeriodicalIF":4.0,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146083746","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 : 2026-01-21DOI: 10.1016/j.chroma.2026.466722
Maby Moll Martínez-Garzón , José Martin Posada-Lotero , Milton Hernando Rosero-Moreano , Elisabeth Restrepo-Parra , Francy Nelly Jiménez-García
Environmental concern regarding the presence of steroid hormones in hospital wastewater treatment plants underscores the need for effective monitoring and removal strategies. Even at trace levels, steroid estrogens exhibit strong biological activity and bioaccumulation potential, classifying them as endocrine-disrupting compounds. In this study, a Montmorillonite/Hydroxyapatite (MMT/HAp) composite was proposed as an environmentally friendly and low-cost alternative for the extraction of estrone (E1), 17β-estradiol (E2), 17α-ethinylestradiol (EE2), and estriol (E3). Sample preparation was carried out using a green analytical method based on rotating disk sorptive extraction. Considering the complexity of wastewater matrices, analytes were determined using gas chromatography coupled to mass spectrometry (GC–MS) in selective ion monitoring mode. A Doehlert experimental design was applied to optimize the extraction process, yielding the following optimal conditions: extraction time of 87 min, sample volume of 16 mL, and stirring speed of 1080 rpm. Hospital wastewater samples from Manizales (Caldas, Colombia) were analyzed, and concentrations of E1, E2, EE2, and E3 were 7.2, 0.1, 6.7, and 33.2 µg L⁻¹, respectively. Limits of detection ranged from 0.06 to 0.40 µg L⁻¹, and limits of quantification from 0.16 to 1.20 µg L⁻¹. Calibration curves for all analytes showed R² values above 0.98. Recovery values obtained through standard addition calibration ranged from 56 % to 90 %, despite significant matrix effects (–67 to 230). The MMT/HAp composite demonstrated comparable performance to the commercial HLB sorbent, confirming its potential for environmental applications.
医院废水处理厂中存在类固醇激素的环境问题突出表明，需要制定有效的监测和去除战略。即使在微量水平，类固醇雌激素也表现出很强的生物活性和生物积累潜力，将其归类为内分泌干扰化合物。本研究提出了蒙脱土/羟基磷灰石（MMT/HAp）复合材料作为一种环保、低成本的提取雌酮（E1）、17β-雌二醇（E2）、17α-炔雌二醇（EE2）和雌三醇（E3）的替代材料。采用旋转圆盘吸附萃取绿色分析方法制备样品。考虑到废水基质的复杂性，在选择性离子监测模式下，采用气相色谱-质谱联用（GC-MS）对分析物进行测定。采用Doehlert实验设计优化提取工艺，得到最佳提取条件：提取时间87 min，进样量16 mL，搅拌速度1080 rpm。医院废水样本马尼萨莱斯(卡尔达斯、哥伦比亚)进行了分析和浓度的E1, E2, EE2,和E3是7.2,0.1,6.7,和33.2µg L⁻¹,分别。检测范围为0.06 - 0.40µg L -毒血症，定量范围为0.16 - 1.20µg L -毒血症。所有分析物的校正曲线R²值均在0.98以上。尽管有显著的基质效应（-67至230），但通过标准添加校准获得的回收率从56%到90%不等。MMT/HAp复合材料表现出与商用HLB吸附剂相当的性能，证实了其在环境应用方面的潜力。
{"title":"Development of a green analytical method based on a Montmorillonite/Hydroxyapatite composite coupled with rotating disk sorptive extraction and GC–MS for monitoring steroid hormones in hospital wastewater","authors":"Maby Moll Martínez-Garzón , José Martin Posada-Lotero , Milton Hernando Rosero-Moreano , Elisabeth Restrepo-Parra , Francy Nelly Jiménez-García","doi":"10.1016/j.chroma.2026.466722","DOIUrl":"10.1016/j.chroma.2026.466722","url":null,"abstract":"<div><div>Environmental concern regarding the presence of steroid hormones in hospital wastewater treatment plants underscores the need for effective monitoring and removal strategies. Even at trace levels, steroid estrogens exhibit strong biological activity and bioaccumulation potential, classifying them as endocrine-disrupting compounds. In this study, a Montmorillonite/Hydroxyapatite (MMT/HAp) composite was proposed as an environmentally friendly and low-cost alternative for the extraction of estrone (E1), 17β-estradiol (E2), 17α-ethinylestradiol (EE2), and estriol (E3). Sample preparation was carried out using a green analytical method based on rotating disk sorptive extraction. Considering the complexity of wastewater matrices, analytes were determined using gas chromatography coupled to mass spectrometry (GC–MS) in selective ion monitoring mode. A Doehlert experimental design was applied to optimize the extraction process, yielding the following optimal conditions: extraction time of 87 min, sample volume of 16 mL, and stirring speed of 1080 rpm. Hospital wastewater samples from Manizales (Caldas, Colombia) were analyzed, and concentrations of E1, E2, EE2, and E3 were 7.2, 0.1, 6.7, and 33.2 µg L⁻¹, respectively. Limits of detection ranged from 0.06 to 0.40 µg L⁻¹, and limits of quantification from 0.16 to 1.20 µg L⁻¹. Calibration curves for all analytes showed R² values above 0.98. Recovery values obtained through standard addition calibration ranged from 56 % to 90 %, despite significant matrix effects (–67 to 230). The MMT/HAp composite demonstrated comparable performance to the commercial HLB sorbent, confirming its potential for environmental applications.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1769 ","pages":"Article 466722"},"PeriodicalIF":4.0,"publicationDate":"2026-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146045902","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 : 2026-01-21DOI: 10.1016/j.chroma.2026.466721
Pooja Mishra , Jing Ma , Huan Xie , Fang Li , Robert Y.L. Tsai , Dong Liang
DNA methylation and hydroxymethylation are important epigenetic modifications that play key roles in cancer development and aging processes by regulating gene expression and genome stability. Traditionally, bisulfite conversion-based or antibody-based enzyme-linked immunosorbent assays are used to find DNA methylation. These tests are non specific, tedious, and not able to differentiate the difference between methylation and hydroxymethylation. To address these issues, we developed a sensitive, reproducible, and specific LC–MS/MS method for simultaneous quantification of two major DNA methylation products, 5-methyl-2′-deoxycytidine (5-mdC) and 5-hydroxymethyl-2′-deoxycytidine (5-hmdC), as well as 2′-deoxycytidine (2-dC), using corresponding stable isotope-labeled internal standards: 5-methyl-2′-deoxycytidine-d₃, 5-(hydroxymethyl)-2′-deoxycytidine-d₃, and 2′-deoxycytidine-¹³C,¹⁵N₂. We purified DNA samples from mouse liver tissue, broke them down with enzymes, filtered them, added internal standards, and then run them through a SCIEX 6500+ Triple Quad LC–MS/MS system with an Atlantis T3 C18 column under a binary gradient. The method showed great chromatographic separation and specificity, with MRM transitions of m/z 228.154 to 112.1 for 2-dC, 242.143 to 126.2 for 5-mdC, and 258.135 to 142.1 for 5-hmdC.Peak area ratio of analyte to internal standard exhibited linearity across calibration ranges of 5–5000 ng /mL for 2-dC, 0.5–500 ng/ mL for 5-mdC, and 0.05–10 ng/mL for 5-hmdC (R² > 0.999), using 2 µL injection and a total runtime of 9 min. The 5-hmdC level in female mouse liver significantly increased with aging from two to sixteen months old (0.0958 % to 0.1984 %; P<0.001), whereas 5-mdC remained unchanged (3.47 % to 3.56 %; n.s.). These data confirm the accurate and reproducible quantification of DNA methylation and hydroxymethylation in tissue samples using the developed LC-MS/MS assay and indicate a broad application to cell culture and clinical biomarker studies.
{"title":"Simultaneous quantification of cytidine, methylcytidine, and hydroxymethylcytidine by isotope-dilution LC–MS/MS with application to mouse liver samples","authors":"Pooja Mishra , Jing Ma , Huan Xie , Fang Li , Robert Y.L. Tsai , Dong Liang","doi":"10.1016/j.chroma.2026.466721","DOIUrl":"10.1016/j.chroma.2026.466721","url":null,"abstract":"<div><div>DNA methylation and hydroxymethylation are important epigenetic modifications that play key roles in cancer development and aging processes by regulating gene expression and genome stability. Traditionally, bisulfite conversion-based or antibody-based enzyme-linked immunosorbent assays are used to find DNA methylation. These tests are non specific, tedious, and not able to differentiate the difference between methylation and hydroxymethylation. To address these issues, we developed a sensitive, reproducible, and specific LC–MS/MS method for simultaneous quantification of two major DNA methylation products, 5-methyl-2′-deoxycytidine (5-mdC) and 5-hydroxymethyl-2′-deoxycytidine (5-hmdC), as well as 2′-deoxycytidine (2-dC), using corresponding stable isotope-labeled internal standards: 5-methyl-2′-deoxycytidine-d₃, 5-(hydroxymethyl)-2′-deoxycytidine-d₃, and 2′-deoxycytidine-¹³C,¹⁵N₂. We purified DNA samples from mouse liver tissue, broke them down with enzymes, filtered them, added internal standards, and then run them through a SCIEX 6500+ Triple Quad LC–MS/MS system with an Atlantis T3 C18 column under a binary gradient. The method showed great chromatographic separation and specificity, with MRM transitions of m/z 228.154 to 112.1 for 2-dC, 242.143 to 126.2 for 5-mdC, and 258.135 to 142.1 for 5-hmdC.Peak area ratio of analyte to internal standard exhibited linearity across calibration ranges of 5–5000 ng /mL for 2-dC, 0.5–500 ng/ mL for 5-mdC, and 0.05–10 ng/mL for 5-hmdC (R² > 0.999), using 2 µL injection and a total runtime of 9 min. The 5-hmdC level in female mouse liver significantly increased with aging from two to sixteen months old (0.0958 % to 0.1984 %; <em>P</em> <em><</em> <em>0.001</em>), whereas 5-mdC remained unchanged (3.47 % to 3.56 %; n.s.). These data confirm the accurate and reproducible quantification of DNA methylation and hydroxymethylation in tissue samples using the developed LC-MS/MS assay and indicate a broad application to cell culture and clinical biomarker studies.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1769 ","pages":"Article 466721"},"PeriodicalIF":4.0,"publicationDate":"2026-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146045931","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Two anionic β-cyclodextrin-based nanohybrid monolithic columns were synthesized and applied in CEC enantioseparation with UV detection. The first monolithic column was fabricated via a "one-pot" approach using glycidyl methacrylate-functionalized sulfated β-cyclodextrin (GMA-S-β-CD), sodium 3-mercaptopropanesulfonate, and alkoxysilanes. The second column was prepared similarly using glutamate-modified β-cyclodextrin (GMA-Glu-β-CD) as the chiral stationary phase. Compared to the Glu-β-CD monolith, the S-β-CD column exhibited significantly higher separation efficiency, achieving baseline separation for 19 out of 27 chiral drugs-including antibiotics, antihistamines, azoles, and other therapeutic agents-whereas the Glu-β-CD monolith resolved only 14. Repeatability was excellent, with relative standard deviations (RSDs) below 4.84% for run-to-run, day-to-day, and column-to-column analyses. Furthermore, quantum mechanics-molecular mechanics (QM-MM) simulations were employed to elucidate the differences in interaction strengths between enantiomers and cyclodextrins within the inclusion complexes.
{"title":"Preparation of two new anionic β-cyclodextrin-based hybrid monolithic chiral stationary phases for CEC enantioseparations of drugs and molecular modeling of enantiomer-chiral selector interactions.","authors":"Feixue Zhou, Qiufang Bai, Jingwen Ma, Wanru Wang, Yuran Shi, Yan Feng, Xiao Zhai, Feng Yang, Xin Wang, Yaxuan Song, Ye Sun, Linwei Li, Guangjun Fan, Linlin Fang","doi":"10.1016/j.chroma.2026.466724","DOIUrl":"https://doi.org/10.1016/j.chroma.2026.466724","url":null,"abstract":"<p><p>Two anionic β-cyclodextrin-based nanohybrid monolithic columns were synthesized and applied in CEC enantioseparation with UV detection. The first monolithic column was fabricated via a \"one-pot\" approach using glycidyl methacrylate-functionalized sulfated β-cyclodextrin (GMA-S-β-CD), sodium 3-mercaptopropanesulfonate, and alkoxysilanes. The second column was prepared similarly using glutamate-modified β-cyclodextrin (GMA-Glu-β-CD) as the chiral stationary phase. Compared to the Glu-β-CD monolith, the S-β-CD column exhibited significantly higher separation efficiency, achieving baseline separation for 19 out of 27 chiral drugs-including antibiotics, antihistamines, azoles, and other therapeutic agents-whereas the Glu-β-CD monolith resolved only 14. Repeatability was excellent, with relative standard deviations (RSDs) below 4.84% for run-to-run, day-to-day, and column-to-column analyses. Furthermore, quantum mechanics-molecular mechanics (QM-MM) simulations were employed to elucidate the differences in interaction strengths between enantiomers and cyclodextrins within the inclusion complexes.</p>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1770 ","pages":"466724"},"PeriodicalIF":4.0,"publicationDate":"2026-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146117259","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 : 2026-01-21DOI: 10.1016/j.chroma.2026.466725
Clara M.A. Eichler , Hannah Calder , Bharat Chandramouli , Matthew Curtis , Heidi Hayes , Benjamin Kim , Ruth Marfil-Vega , Cristina Matos Mejías , Laura Miles , Alan Owens , Jack Stuff , Kurt Thaxton , Jochen Vandenberg , Nicola Watson , David Wevill , Jackie A. Whitecavage , Xiaoyu Liu
Fluorotelomer alcohols (FTOHs) are a group of volatile and semi-volatile per- and polyfluoroalkyl substances (PFAS) commonly found in indoor air and contribute to PFAS inhalation exposure. Improving our understanding of the contribution of FTOHs to human exposure to PFAS indoors is of high interest. Consensus standard test methods play an essential role in environmental risk assessment and management. We present the outcome of an international interlaboratory study (ILS) conducted with nine laboratories to evaluate the precision of ASTM International Standard Test Method D8591. The ILS was organized by the United States Environmental Protection Agency. The test method specifies the analysis of four FTOHs (4:2, 6:2, 8:2 and 10:2 FTOH) collected on PFAS-specific thermal desorption tubes by gas chromatography coupled with tandem mass spectrometry. During the ILS, participating laboratories were instructed to use the test method to analyze three samples (A, B, and C), each three times. Each sample contained the target FTOHs at defined concentrations unknown to the laboratories. The results from seven laboratories show that the relative reproducibility standard deviation (RSDR) of the method ranges from 14% to 26% and the relative repeatability standard deviation (RSDr) ranges from 4.6% to 11%, with RSDs decreasing with decreasing volatility of the FTOHs. Bias ranged from -13% to 6.0% and was generally larger and negative for less volatile FTOHs. The test method in conjunction with the precision statistics from this ILS will provide a reliable, defendable method that can be used in the context of studying PFAS sources, transport, and human exposure.
{"title":"Measuring fluorotelomer alcohols by thermal desorption-gas chromatography-tandem mass spectrometry: Interlaboratory study results","authors":"Clara M.A. Eichler , Hannah Calder , Bharat Chandramouli , Matthew Curtis , Heidi Hayes , Benjamin Kim , Ruth Marfil-Vega , Cristina Matos Mejías , Laura Miles , Alan Owens , Jack Stuff , Kurt Thaxton , Jochen Vandenberg , Nicola Watson , David Wevill , Jackie A. Whitecavage , Xiaoyu Liu","doi":"10.1016/j.chroma.2026.466725","DOIUrl":"10.1016/j.chroma.2026.466725","url":null,"abstract":"<div><div>Fluorotelomer alcohols (FTOHs) are a group of volatile and semi-volatile per- and polyfluoroalkyl substances (PFAS) commonly found in indoor air and contribute to PFAS inhalation exposure. Improving our understanding of the contribution of FTOHs to human exposure to PFAS indoors is of high interest. Consensus standard test methods play an essential role in environmental risk assessment and management. We present the outcome of an international interlaboratory study (ILS) conducted with nine laboratories to evaluate the precision of ASTM International Standard Test Method D8591. The ILS was organized by the United States Environmental Protection Agency. The test method specifies the analysis of four FTOHs (4:2, 6:2, 8:2 and 10:2 FTOH) collected on PFAS-specific thermal desorption tubes by gas chromatography coupled with tandem mass spectrometry. During the ILS, participating laboratories were instructed to use the test method to analyze three samples (A, B, and C), each three times. Each sample contained the target FTOHs at defined concentrations unknown to the laboratories. The results from seven laboratories show that the relative reproducibility standard deviation (<em>RSD<sub>R</sub></em>) of the method ranges from 14% to 26% and the relative repeatability standard deviation (<em>RSD<sub>r</sub></em>) ranges from 4.6% to 11%, with RSDs decreasing with decreasing volatility of the FTOHs. Bias ranged from -13% to 6.0% and was generally larger and negative for less volatile FTOHs. The test method in conjunction with the precision statistics from this ILS will provide a reliable, defendable method that can be used in the context of studying PFAS sources, transport, and human exposure.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1769 ","pages":"Article 466725"},"PeriodicalIF":4.0,"publicationDate":"2026-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146023326","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 : 2026-01-21DOI: 10.1016/j.chroma.2026.466727
Ahmed Serag , Manal E. Alosaimi , Maram H Abduljabbar , Adnan Alharbi , Faisal Alsenani , Farooq M. Almutairi , Muneef M. Aldhafeeri , Atiah H. Almalki
A sustainable salting-out assisted liquid-liquid extraction coupled with liquid chromatography-tandem mass spectrometry (SALLE-LC-MS/MS) method was developed and validated for amisulpride quantification in human plasma according to ICH M10 guidelines. Box-Behnken experimental design systematically optimized SALLE parameters through evaluation of sample pH (5.0–10.0), acetonitrile volume (500–1500 μL), salt concentration (2.0–6.0 mol/L), and centrifugation time (3.0–10.0 min). The developed polynomial model demonstrated strong predictive capability with subsequent numerical optimization identifying optimal conditions of pH 8.5, acetonitrile 1200 μL, ammonium acetate 4.5 mol/L, and 3-minute centrifugation. These conditions achieved 97.6% extraction recovery for amisulpride with minimal deviation from predicted values. Following optimization, chromatographic separation was achieved using a C18 column with isocratic elution (acetonitrile:water with 0.1% formic acid, 70:30 v/v) and 4-minute analysis time, with multiple reaction monitoring employing transitions m/z370.1→242.1 for amisulpride and m/z376.2→165.1 for haloperidol internal standard. Comprehensive validation subsequently demonstrated linearity across 2–1500 ng/mL with acceptable accuracy, precision, and stability under clinical storage conditions. Matrix effects ranged from 88–105% with low variability, while extraction recovery exceeded 98% across all quality control levels. Clinical application in healthy volunteers (n = 5) following 200 mg oral amisulpride administration successfully characterized key pharmacokinetic parameters including Cmax (506 ng/mL), tmax (3.8 h), and t1/2 (13.3 h) over 48 h. Finally, multi-metric sustainability assessment using CaFRI (75/100), BAGI (77.5/100), and RGB12 (83.1/100 whiteness) frameworks confirmed achievement of white analytical chemistry through balanced analytical performance, environmental friendliness, and practical implementation feasibility. The method offers significant advantages including minimal waste generation, reduced solvent consumption, and enhanced throughput while maintaining regulatory compliance for amisulpride therapeutic drug monitoring applications.
{"title":"Box-Behnken optimized salting-out assisted liquid-liquid extraction coupled with LC-MS/MS for sustainable amisulpride quantification in human plasma","authors":"Ahmed Serag , Manal E. Alosaimi , Maram H Abduljabbar , Adnan Alharbi , Faisal Alsenani , Farooq M. Almutairi , Muneef M. Aldhafeeri , Atiah H. Almalki","doi":"10.1016/j.chroma.2026.466727","DOIUrl":"10.1016/j.chroma.2026.466727","url":null,"abstract":"<div><div>A sustainable salting-out assisted liquid-liquid extraction coupled with liquid chromatography-tandem mass spectrometry (SALLE-LC-MS/MS) method was developed and validated for amisulpride quantification in human plasma according to ICH M10 guidelines. Box-Behnken experimental design systematically optimized SALLE parameters through evaluation of sample pH (5.0–10.0), acetonitrile volume (500–1500 μL), salt concentration (2.0–6.0 mol/L), and centrifugation time (3.0–10.0 min). The developed polynomial model demonstrated strong predictive capability with subsequent numerical optimization identifying optimal conditions of pH 8.5, acetonitrile 1200 μL, ammonium acetate 4.5 mol/L, and 3-minute centrifugation. These conditions achieved 97.6% extraction recovery for amisulpride with minimal deviation from predicted values. Following optimization, chromatographic separation was achieved using a C18 column with isocratic elution (acetonitrile:water with 0.1% formic acid, 70:30 v/v) and 4-minute analysis time, with multiple reaction monitoring employing transitions <em>m/z</em>370.1→242.1 for amisulpride and <em>m/z</em>376.2→165.1 for haloperidol internal standard. Comprehensive validation subsequently demonstrated linearity across 2–1500 ng/mL with acceptable accuracy, precision, and stability under clinical storage conditions. Matrix effects ranged from 88–105% with low variability, while extraction recovery exceeded 98% across all quality control levels. Clinical application in healthy volunteers (<em>n</em> = 5) following 200 mg oral amisulpride administration successfully characterized key pharmacokinetic parameters including C<sub>max</sub> (506 ng/mL), t<sub>max</sub> (3.8 h), and t<sub>1/2</sub> (13.3 h) over 48 h. Finally, multi-metric sustainability assessment using CaFRI (75/100), BAGI (77.5/100), and RGB12 (83.1/100 whiteness) frameworks confirmed achievement of white analytical chemistry through balanced analytical performance, environmental friendliness, and practical implementation feasibility. The method offers significant advantages including minimal waste generation, reduced solvent consumption, and enhanced throughput while maintaining regulatory compliance for amisulpride therapeutic drug monitoring applications.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1770 ","pages":"Article 466727"},"PeriodicalIF":4.0,"publicationDate":"2026-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146090511","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 : 2026-01-20DOI: 10.1016/j.chroma.2026.466717
Nataliia Leonova , Olga Konovalova , Peter Tarábek
Inclusion of emerging contaminants into water quality monitoring programs can serve as an additional layer for protecting water resources against deterioration. Based on prior non-target screening using LC–QTOF, a direct injection liquid chromatography–electrospray ionization-tandem mass spectrometry with multiple reaction monitoring method was developed for the determination of 23 micropollutants in natural waters. The combination of methodological simplicity, flexibility, environmental compatibility, and demonstrated performance under real-world monitoring conditions represents a significant advancement over existing chromatographic strategies for the determination of relevant and emerging pollutants. The method provides high sensitivity and reliability, enabling the simultaneous quantification of target analytes (pesticides, pharmaceuticals and products of their transformation) spanning a wide polarity range (XLogP3 0.3–6.1) and extending the applicability of multianalytes chromatographic analysis to complex environmental matrices. The limits of detection and quantification and linearity were evaluated in deionized water, groundwater, and surface water, while accuracy in terms of recovery and precision were determined in surface water. Pesticides provided satisfactory recoveries ranging from 83 to 120%, with quantification limits between 0.02 µg L−1 and 0.18 µg L−1, whereas pharmaceuticals displayed recoveries of 83–119%, with quantification limits of 0.03–0.20 µg L−1 in different types of water. Using the developed method, 599 water samples were analyzed between 2023 and 2025. The most frequently detected compounds at concentrations above the limit of quantification were terbumeton-desethyl, prometon and benzenesulfonamide in groundwater; fluconazole and sulfamethoxazole in surface water; and propazine-2‑hydroxy, terbuthylazine-2‑hydroxy and carbamazepine in both types of water samples.
{"title":"Development of an LC–ESI-MS/MS method for the determination of contaminants of emerging concern – towards extending quality surveillance of water resources","authors":"Nataliia Leonova , Olga Konovalova , Peter Tarábek","doi":"10.1016/j.chroma.2026.466717","DOIUrl":"10.1016/j.chroma.2026.466717","url":null,"abstract":"<div><div>Inclusion of emerging contaminants into water quality monitoring programs can serve as an additional layer for protecting water resources against deterioration. Based on prior non-target screening using LC–QTOF, a direct injection liquid chromatography–electrospray ionization-tandem mass spectrometry with multiple reaction monitoring method was developed for the determination of 23 micropollutants in natural waters. The combination of methodological simplicity, flexibility, environmental compatibility, and demonstrated performance under real-world monitoring conditions represents a significant advancement over existing chromatographic strategies for the determination of relevant and emerging pollutants. The method provides high sensitivity and reliability, enabling the simultaneous quantification of target analytes (pesticides, pharmaceuticals and products of their transformation) spanning a wide polarity range (XLogP3 0.3–6.1) and extending the applicability of multianalytes chromatographic analysis to complex environmental matrices. The limits of detection and quantification and linearity were evaluated in deionized water, groundwater, and surface water, while accuracy in terms of recovery and precision were determined in surface water. Pesticides provided satisfactory recoveries ranging from 83 to 120%, with quantification limits between 0.02 µg L<sup>−1</sup> and 0.18 µg L<sup>−1</sup>, whereas pharmaceuticals displayed recoveries of 83–119%, with quantification limits of 0.03–0.20 µg L<sup>−1</sup> in different types of water. Using the developed method, 599 water samples were analyzed between 2023 and 2025. The most frequently detected compounds at concentrations above the limit of quantification were terbumeton-desethyl, prometon and benzenesulfonamide in groundwater; fluconazole and sulfamethoxazole in surface water; and propazine-2‑hydroxy, terbuthylazine-2‑hydroxy and carbamazepine in both types of water samples.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1768 ","pages":"Article 466717"},"PeriodicalIF":4.0,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146034352","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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