Pub Date : 2025-12-26DOI: 10.1016/j.jchromb.2025.124906
Huiling Zhou , Simin Dong , Ao Pan , Huan Xu , Xin Tang , Xixi Wang , Linshen Xie , Yongxin Li
Wilson disease (WD) is an inherited disorder of copper metabolism with early diagnostic challenges. Urinary proteomics shows promise for identifying WD-related biomarkers, but current strategies neglect the detection of low-abundance proteins critical for preclinical diagnosis. Urine samples from 53 newly diagnosed WD patients and 47 matched controls were analyzed using an optimized proteomics strategy integrating data-independent acquisition (DIA) with parallel reaction monitoring (PRM). Functional enrichment analysis, hierarchical clustering and other multidimensional analyses were employed to delve into low-abundance protein biomarkers. Recursive feature elimination (RFE) and support vector machine (SVM) were applied to identify the candidate biomarkers, and the performance of the diagnostic model was measured by the receiver operating characteristic (ROC) curve. Furthermore, the potential biomarkers were validated by ELISA in an independent validation cohort. The optimized DIA-based untargeted proteomics identified 2263 urine proteins, including 447 differentially expressed proteins (68 upregulated and 379 downregulated). After LC-PRM-MS verification, 46 new candidate biomarkers for WD were identified and 11 were included in the final model. The SVM model performed the best in classifying WD and healthy control, and the areas under the ROC curve in the training set and the test set were 0.95 and 0.94 respectively. Four proteins were validated by ELISA in an independent cohort, with expression levels consistent with the proteomics data. The proposed DIA-PRM proteomics analysis detect more low-abundance urinary proteins, and a urinary protein biomarker panel with high accuracy for non-invasive diagnosis in WD patients was identified.
{"title":"Comprehensive urinary proteomics using DIA and PRM for low-abundance protein profiling of Wilson disease","authors":"Huiling Zhou , Simin Dong , Ao Pan , Huan Xu , Xin Tang , Xixi Wang , Linshen Xie , Yongxin Li","doi":"10.1016/j.jchromb.2025.124906","DOIUrl":"10.1016/j.jchromb.2025.124906","url":null,"abstract":"<div><div>Wilson disease (WD) is an inherited disorder of copper metabolism with early diagnostic challenges. Urinary proteomics shows promise for identifying WD-related biomarkers, but current strategies neglect the detection of low-abundance proteins critical for preclinical diagnosis. Urine samples from 53 newly diagnosed WD patients and 47 matched controls were analyzed using an optimized proteomics strategy integrating data-independent acquisition (DIA) with parallel reaction monitoring (PRM). Functional enrichment analysis, hierarchical clustering and other multidimensional analyses were employed to delve into low-abundance protein biomarkers. Recursive feature elimination (RFE) and support vector machine (SVM) were applied to identify the candidate biomarkers, and the performance of the diagnostic model was measured by the receiver operating characteristic (ROC) curve. Furthermore, the potential biomarkers were validated by ELISA in an independent validation cohort. The optimized DIA-based untargeted proteomics identified 2263 urine proteins, including 447 differentially expressed proteins (68 upregulated and 379 downregulated). After LC-PRM-MS verification, 46 new candidate biomarkers for WD were identified and 11 were included in the final model. The SVM model performed the best in classifying WD and healthy control, and the areas under the ROC curve in the training set and the test set were 0.95 and 0.94 respectively. Four proteins were validated by ELISA in an independent cohort, with expression levels consistent with the proteomics data. The proposed DIA-PRM proteomics analysis detect more low-abundance urinary proteins, and a urinary protein biomarker panel with high accuracy for non-invasive diagnosis in WD patients was identified.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124906"},"PeriodicalIF":2.8,"publicationDate":"2025-12-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145852138","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-24DOI: 10.1016/j.jchromb.2025.124899
Yao Liu , Huanyi Ren , Qing Bin , Kai Hu , Hongya Zhu , Qing Cheng , Mengqing Zhang , Chao Jiang , Jiahao Feng
<div><h3>Background</h3><div>Acute liver injury (ALI) represents a serious clinical condition characterized by rapid progression toward hepatic functional decompensation, potentially culminating in liver failure. Despite proposed therapeutic interventions, effective management strategies remain limited. Total flavonoids from Apocynum venetum leaves (TFAP), recognized for their antioxidant and anti-inflammatory properties, have demonstrated multi-organ protective effects in prior studies. However, the precise mechanisms underlying TFAP's role in attenuating ALI pathogenesis remain poorly elucidated.</div></div><div><h3>Methods</h3><div>Utilizing the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and analysis platform, in conjunction with the drug mapping server, the active constituents of TFAP and their therapeutic targets for acute liver injury were identified. Specific targets pertinent to the treatment of acute liver injury were screened and compiled via the GEO database. Molecular docking techniques were employed to elucidate the interactions between the active compounds and their respective targets. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to provide a comprehensive analysis of the targets associated with acute liver injury. To validate the network pharmacology findings, CCl4-induced acute liver injury cell and animal models were established. Cell viability was assessed using the Cell Counting Kit-8 (CCK8) solution, while flow cytometry, RT-qPCR, and Western blot assays were conducted to evaluate indicators related to cell apoptosis and the Nrf2 signaling pathway. Biochemical assays were employed to quantify the enzymatic activities of aspartate transaminase (AST), alanine transaminase (ALT), and superoxide dismutase (SOD), as well as the concentration of malondialdehyde (MDA) in both cellular and plasma samples. Hematoxylin-Eosin (HE) staining was conducted to examine the pathological alterations in hepatic tissues of mice across different experimental groups. Additionally, immunohistochemical methods were utilized to assess the expression levels of the proteins Nrf2, Keap1, HO-1, and GCLC in liver tissues.</div></div><div><h3>Results</h3><div>A comprehensive transcriptome analysis combined with network pharmacology has indicated that TFAP may exert therapeutic effects on alcoholic hepatitis (ALD) by modulating the AR and BCHE signaling pathways. In vitro studies demonstrated that TFAP extended the survival of hepatocytes subjected to injury, significantly decreased the levels of AST, ALT, LDH, ROS, and MDA, and enhanced SOD activity in CCl₄-induced LO2 cells. Furthermore, treatment with these flavonoids resulted in a reduction of AR protein expression while increasing the expression of BCHE and Nrf2 proteins. In vivo experiments revealed that, in comparison to the blank control group, the model group exhibited significantly elevated levels of li
{"title":"Hepatoprotection of Apocynum venetum flavonoids: Targeting oxidative stress via coordinated NRF2/BCHE induction and AR inhibition","authors":"Yao Liu , Huanyi Ren , Qing Bin , Kai Hu , Hongya Zhu , Qing Cheng , Mengqing Zhang , Chao Jiang , Jiahao Feng","doi":"10.1016/j.jchromb.2025.124899","DOIUrl":"10.1016/j.jchromb.2025.124899","url":null,"abstract":"<div><h3>Background</h3><div>Acute liver injury (ALI) represents a serious clinical condition characterized by rapid progression toward hepatic functional decompensation, potentially culminating in liver failure. Despite proposed therapeutic interventions, effective management strategies remain limited. Total flavonoids from Apocynum venetum leaves (TFAP), recognized for their antioxidant and anti-inflammatory properties, have demonstrated multi-organ protective effects in prior studies. However, the precise mechanisms underlying TFAP's role in attenuating ALI pathogenesis remain poorly elucidated.</div></div><div><h3>Methods</h3><div>Utilizing the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and analysis platform, in conjunction with the drug mapping server, the active constituents of TFAP and their therapeutic targets for acute liver injury were identified. Specific targets pertinent to the treatment of acute liver injury were screened and compiled via the GEO database. Molecular docking techniques were employed to elucidate the interactions between the active compounds and their respective targets. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to provide a comprehensive analysis of the targets associated with acute liver injury. To validate the network pharmacology findings, CCl4-induced acute liver injury cell and animal models were established. Cell viability was assessed using the Cell Counting Kit-8 (CCK8) solution, while flow cytometry, RT-qPCR, and Western blot assays were conducted to evaluate indicators related to cell apoptosis and the Nrf2 signaling pathway. Biochemical assays were employed to quantify the enzymatic activities of aspartate transaminase (AST), alanine transaminase (ALT), and superoxide dismutase (SOD), as well as the concentration of malondialdehyde (MDA) in both cellular and plasma samples. Hematoxylin-Eosin (HE) staining was conducted to examine the pathological alterations in hepatic tissues of mice across different experimental groups. Additionally, immunohistochemical methods were utilized to assess the expression levels of the proteins Nrf2, Keap1, HO-1, and GCLC in liver tissues.</div></div><div><h3>Results</h3><div>A comprehensive transcriptome analysis combined with network pharmacology has indicated that TFAP may exert therapeutic effects on alcoholic hepatitis (ALD) by modulating the AR and BCHE signaling pathways. In vitro studies demonstrated that TFAP extended the survival of hepatocytes subjected to injury, significantly decreased the levels of AST, ALT, LDH, ROS, and MDA, and enhanced SOD activity in CCl₄-induced LO2 cells. Furthermore, treatment with these flavonoids resulted in a reduction of AR protein expression while increasing the expression of BCHE and Nrf2 proteins. In vivo experiments revealed that, in comparison to the blank control group, the model group exhibited significantly elevated levels of li","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124899"},"PeriodicalIF":2.8,"publicationDate":"2025-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145837741","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-24DOI: 10.1016/j.jchromb.2025.124904
Merna M. Sakr , Reham S. Ibrahim , Amira M. Beltagy , Reham S. Darwish
Sprouting is a widely practiced food processing method, particularly in Western countries. Chickpeas (Cicer arietinum) have been traditionally used in various cuisines worldwide for centuries. Their versatility and nutritional value make them a staple ingredient in many traditional dishes. This study investigated the impact of sprouting chickpea seeds on their phytochemical profile and anticancer properties. Chickpea seeds were germinated under light and dark conditions for eight days, with samples collected on intervals. Extracts were analyzed using UPLC-MS/MS and tested for cytotoxic activity against two different breast cancer cell lines, namely; MDA-MB-231 (triple-negative breast cancer) and HeLa (uterine cervical cancer). UPLC-MS/MS detected 117 metabolites, primarily flavonoids, phenolic acids, and amino acids. Germination affected phytochemical composition and sprout length, with longer sprouts in darkness. Metabolite abundance peaked on day eight, while cytotoxicity was highest on day six. An OPLS model linked cytotoxic effects on HeLa and MDA-MB-231 cells to key metabolites, identified via coefficient plots. Caffeoylquinic acid, naringenin, and biochanin B were major contributors against MDA-MB-231, while hydroxybenzoic acid hexoside, orobol, and biochanin A hexoside were prominent in HeLa cells. Molecular docking of the top three metabolites with IL2 and ABCB1 genes, revealed as common genes between bioactive compounds' genes and cancer genes, showed hydroxybenzoic acid hexoside and naringenin as top binders, aligning with OPLS findings. Although the results demonstrate promising in vitro anticancer potential, further in vivo and mechanistic studies are required to confirm efficacy, safety, and bioavailability before establishing chickpea sprouts as a functional food or a chemo-preventive agent.
{"title":"From seed to bioactive: unveiling sprouting-driven changes in chickpeas via targeted metabolomics and in silico modelling","authors":"Merna M. Sakr , Reham S. Ibrahim , Amira M. Beltagy , Reham S. Darwish","doi":"10.1016/j.jchromb.2025.124904","DOIUrl":"10.1016/j.jchromb.2025.124904","url":null,"abstract":"<div><div>Sprouting is a widely practiced food processing method, particularly in Western countries. Chickpeas (<em>Cicer arietinum</em>) have been traditionally used in various cuisines worldwide for centuries. Their versatility and nutritional value make them a staple ingredient in many traditional dishes. This study investigated the impact of sprouting chickpea seeds on their phytochemical profile and anticancer properties. Chickpea seeds were germinated under light and dark conditions for eight days, with samples collected on intervals. Extracts were analyzed using UPLC-MS/MS and tested for cytotoxic activity against two different breast cancer cell lines, namely; MDA-MB-231 (triple-negative breast cancer) and HeLa (uterine cervical cancer). UPLC-MS/MS detected 117 metabolites, primarily flavonoids, phenolic acids, and amino acids. Germination affected phytochemical composition and sprout length, with longer sprouts in darkness. Metabolite abundance peaked on day eight, while cytotoxicity was highest on day six. An OPLS model linked cytotoxic effects on HeLa and MDA-MB-231 cells to key metabolites, identified <em>via</em> coefficient plots. Caffeoylquinic acid, naringenin, and biochanin B were major contributors against MDA-MB-231, while hydroxybenzoic acid hexoside, orobol, and biochanin A hexoside were prominent in HeLa cells. Molecular docking of the top three metabolites with IL2 and ABCB1 genes, revealed as common genes between bioactive compounds' genes and cancer genes, showed hydroxybenzoic acid hexoside and naringenin as top binders, aligning with OPLS findings. Although the results demonstrate promising <em>in vitro</em> anticancer potential, further <em>in vivo</em> and mechanistic studies are required to confirm efficacy, safety, and bioavailability before establishing chickpea sprouts as a functional food or a chemo-preventive agent.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124904"},"PeriodicalIF":2.8,"publicationDate":"2025-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145880597","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
This study aimed to comprehensively investigate the antidepressant mechanisms of Bupleurum polysaccharide (BP) through the microbiota-gut-brain axis, employing an integrated multi-omics approach. Using a chronic unpredictable mild stress (CUMS) rat model of depression, we evaluated BP's effects on depressive-like behaviors and analyzed its regulatory mechanisms on metabolites and gut microbiota through combined metabolomics and metagenomics. Structural characterization revealed that Bupleurum polysaccharide SPAP-1 is an acidic homogeneous polysaccharide with a molecular weight of approximately 100 kDa, primarily composed of glucose, mannose, rhamnose, and other monosaccharides. Pharmacodynamic assessments demonstrated that BP significantly ameliorated CUMS-induced depressive behaviors, including weight loss, reduced food intake, anhedonia, and behavioral despair (P < 0.05). Metabolomic analysis identified 19 differential metabolites, with BP reversing 11 of them, primarily involved in phenylalanine and tryptophan metabolism pathways. Western blot analysis confirmed BP's regulatory effects on key enzymes Got1 and Lta4h. Metagenomic results showed that BP remarkably reshaped gut microbiota structure, restored microbial diversity, optimized the Firmicutes/Bacteroidetes ratio, enriched beneficial genera (Agathobacter, Phocaeicola), and inhibited pathogenic genera (Ruminococcus). Crucially, integrated multi-omics analysis revealed significant microbiota-metabolite correlations, demonstrating that BP-promoted beneficial bacteria positively correlated with neurotransmitter precursors, while BP-inhibited pathogenic bacteria associated with pro-inflammatory mediators. Mediation analysis further established the “microbiota → metabolite → behavior” causal chain, with Ruminococcus → LTB4 → despair behavior accounting for 42.3 % of the mediation effect. In conclusion, Bupleurum polysaccharide ameliorates depressive-like behaviors through multi-target regulation of the metabolite-microbiota interaction network, highlighting its potential as an antidepressant agent or functional food and providing a novel research paradigm for understanding the multi-target characteristics of traditional Chinese medicine polysaccharides.
{"title":"Bupleurum polysaccharide improves CUMS-induced depressive behavior in rats by regulating the “microbiota-gut-brain Axis”: a mechanism study based on metabolomics and metagenomics","authors":"Hongcai Zhang, Shuxiang Zhang , Xuan Li, Wenran Wang, Haixue Kuang","doi":"10.1016/j.jchromb.2025.124905","DOIUrl":"10.1016/j.jchromb.2025.124905","url":null,"abstract":"<div><div>This study aimed to comprehensively investigate the antidepressant mechanisms of Bupleurum polysaccharide (BP) through the microbiota-gut-brain axis, employing an integrated multi-omics approach. Using a chronic unpredictable mild stress (CUMS) rat model of depression, we evaluated BP's effects on depressive-like behaviors and analyzed its regulatory mechanisms on metabolites and gut microbiota through combined metabolomics and metagenomics. Structural characterization revealed that Bupleurum polysaccharide SPAP-1 is an acidic homogeneous polysaccharide with a molecular weight of approximately 100 kDa, primarily composed of glucose, mannose, rhamnose, and other monosaccharides. Pharmacodynamic assessments demonstrated that BP significantly ameliorated CUMS-induced depressive behaviors, including weight loss, reduced food intake, anhedonia, and behavioral despair (<em>P</em> < 0.05). Metabolomic analysis identified 19 differential metabolites, with BP reversing 11 of them, primarily involved in phenylalanine and tryptophan metabolism pathways. Western blot analysis confirmed BP's regulatory effects on key enzymes Got1 and Lta4h. Metagenomic results showed that BP remarkably reshaped gut microbiota structure, restored microbial diversity, optimized the Firmicutes/Bacteroidetes ratio, enriched beneficial genera (Agathobacter, Phocaeicola), and inhibited pathogenic genera (Ruminococcus). Crucially, integrated multi-omics analysis revealed significant microbiota-metabolite correlations, demonstrating that BP-promoted beneficial bacteria positively correlated with neurotransmitter precursors, while BP-inhibited pathogenic bacteria associated with pro-inflammatory mediators. Mediation analysis further established the “microbiota → metabolite → behavior” causal chain, with Ruminococcus → LTB4 → despair behavior accounting for 42.3 % of the mediation effect. In conclusion, Bupleurum polysaccharide ameliorates depressive-like behaviors through multi-target regulation of the metabolite-microbiota interaction network, highlighting its potential as an antidepressant agent or functional food and providing a novel research paradigm for understanding the multi-target characteristics of traditional Chinese medicine polysaccharides.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124905"},"PeriodicalIF":2.8,"publicationDate":"2025-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145880738","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-23DOI: 10.1016/j.jchromb.2025.124909
Guoli Wu , Chen Li , Yue Zhang , Tingli Qu
Fluoroquinolones are a class of widely used antibiotics, most of which contain chiral centers. Significant differences exist between their enantiomers in terms of antimicrobial activity, pharmacokinetics, and toxicity. In this study, glucosamine chiral-functionalized carbon quantum dots (GA-CQDs) were successfully synthesized and employed as a pseudostationary phase. By optimizing the chromatographic conditions, an efficient and novel chiral separation platform based on capillary electrophoresis (CE) was established for the enantioseparation of six fluoroquinolones. Compared to the CE system using free glucosamine solely as the chiral selector, the CE system employing GA-CQDs as a pseudostationary phase demonstrated a substantial improvement in chiral resolution (Rs) for all six model analytes (Ofloxacin: 1.56 → 9.21; Pazufloxacin: 0 → 8.53; Prulifloxacin: 1.38 → 7.58; Lomefloxacin: 1.01 → 2.84; Gemifloxacin: 0 → 1.25; Balofloxacin: 0 → 0.88). The incorporation of the pseudostationary phase significantly enhanced the chiral separation capability. This proposed strategy is characterized by its operational simplicity and low cost, thereby offering a valuable conceptual framework and a highly promising approach for developing chiral separation methods for fluoroquinolone antibiotics. This study represents the first report on employing GA-CQDs as a chiral selective medium to construct a capillary electrophoresis system, which achieved satisfactory enantioselectivity. Furthermore, the chiral recognition mechanism of glucosamine was preliminarily investigated through molecular modeling simulations, with the binding energy providing insights into the separation process.
{"title":"Chiral separation of fluoroquinolones by capillary electrophoresis using glucosamine-functionalized carbon quantum dots as a Pseudostationary phase","authors":"Guoli Wu , Chen Li , Yue Zhang , Tingli Qu","doi":"10.1016/j.jchromb.2025.124909","DOIUrl":"10.1016/j.jchromb.2025.124909","url":null,"abstract":"<div><div>Fluoroquinolones are a class of widely used antibiotics, most of which contain chiral centers. Significant differences exist between their enantiomers in terms of antimicrobial activity, pharmacokinetics, and toxicity. In this study, glucosamine chiral-functionalized carbon quantum dots (GA-CQDs) were successfully synthesized and employed as a pseudostationary phase. By optimizing the chromatographic conditions, an efficient and novel chiral separation platform based on capillary electrophoresis (CE) was established for the enantioseparation of six fluoroquinolones. Compared to the CE system using free glucosamine solely as the chiral selector, the CE system employing GA-CQDs as a pseudostationary phase demonstrated a substantial improvement in chiral resolution (Rs) for all six model analytes (Ofloxacin: 1.56 → 9.21; Pazufloxacin: 0 → 8.53; Prulifloxacin: 1.38 → 7.58; Lomefloxacin: 1.01 → 2.84; Gemifloxacin: 0 → 1.25; Balofloxacin: 0 → 0.88). The incorporation of the pseudostationary phase significantly enhanced the chiral separation capability. This proposed strategy is characterized by its operational simplicity and low cost, thereby offering a valuable conceptual framework and a highly promising approach for developing chiral separation methods for fluoroquinolone antibiotics. This study represents the first report on employing GA-CQDs as a chiral selective medium to construct a capillary electrophoresis system, which achieved satisfactory enantioselectivity. Furthermore, the chiral recognition mechanism of glucosamine was preliminarily investigated through molecular modeling simulations, with the binding energy providing insights into the separation process.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124909"},"PeriodicalIF":2.8,"publicationDate":"2025-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145867098","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-22DOI: 10.1016/j.jchromb.2025.124907
Yuexin Yang , Chunyan Zhu , Qiannan Xu , Bahriman Xarpidin , Dandan Zhang , Hairong Zhang , Xi Luo , Wenjing Tian , HaiFeng Chen , Caisheng Wu
Hyperlipidemia is a major risk factor for cardiovascular diseases and fatty liver. Its incidence has increased in recent years, becoming a major public health concern that poses a serious threat to human health. Tongmaijiangzhi Capsule (TMJZC), a classical pure traditional Chinese medicine (TCM) formulation, has been widely applied in the clinical management of hyperlipidemia with confirmed efficacy and favorable safety. However, its in vivo exposure profile and pharmacokinetic characteristics in humans remain insufficiently characterized, limiting its rational clinical use. In this study, a human exposure-driven analytical strategy was employed, integrating high-resolution mass spectrometry (HRMS), untargeted intelligent data mining, and multi-component quantitative analysis to systematically elucidate the pharmacokinetic behavior and potential bioactive constituents of TMJZC in humans. Using a self-developed untargeted HRMS data-processing platform, a total of 215 TMJZC-related compounds (including 74 prototype constituents and 141 metabolites) were successfully identified in human plasma and urine. Based on human exposure abundance, quality-control components of the herbal medicine, and their circulating forms in vivo, selected seven potential active compounds for further investigation (Cryptochlorogenic acid, Chlorogenic acid, Kaempferol 3-O-β-sophoroside, Hyperoside, Nuciferine, Ginsenoside Rg1, and Notoginsenoside R1). Subsequently, a sensitive and selective multi-component quantification method was subsequently established to systematically characterize their metabolic profiles in humans. Meanwhile, sex-related differences in pharmacokinetics were observed, with the geometric mean ratios (GMRs, female/male) of Nuciferine, Ginsenoside Rg1 and Hyperoside showing substantially higher Cmax (1134.13 %, 447.65 %, 261.53 %,) and AUC0–∞ (580.47 %, 422.63 %, 290.51 %) in females, respectively. In addition, pharmacological evaluation using hyperlipidemic hepatocyte models (AML12 and HepG2 cells) demonstrated that Hyperoside, Nuciferine, Notoginsenoside R1, and Ginsenoside Rg1 significantly reduced intracellulartriglyceride accumulation. Overall, this study provides the first comprehensive human exposure and pharmacokinetic characterization of TMJZC, highlighting representative circulating constituents and sex-related metabolic differences, and offers a robust analytical basis for quality evaluation, rational clinical application, and further mechanistic studies of this traditional Chinese medicine formulation.
{"title":"Systematic characterization of in vivo exposure and pharmacokinetic features of Tongmaijiangzhi capsule in humans using integrated HRMS and intelligent data processing","authors":"Yuexin Yang , Chunyan Zhu , Qiannan Xu , Bahriman Xarpidin , Dandan Zhang , Hairong Zhang , Xi Luo , Wenjing Tian , HaiFeng Chen , Caisheng Wu","doi":"10.1016/j.jchromb.2025.124907","DOIUrl":"10.1016/j.jchromb.2025.124907","url":null,"abstract":"<div><div>Hyperlipidemia is a major risk factor for cardiovascular diseases and fatty liver. Its incidence has increased in recent years, becoming a major public health concern that poses a serious threat to human health. Tongmaijiangzhi Capsule (TMJZC), a classical pure traditional Chinese medicine (TCM) formulation, has been widely applied in the clinical management of hyperlipidemia with confirmed efficacy and favorable safety. However, its <em>in vivo</em> exposure profile and pharmacokinetic characteristics in humans remain insufficiently characterized, limiting its rational clinical use. In this study, a human exposure-driven analytical strategy was employed, integrating high-resolution mass spectrometry (HRMS), untargeted intelligent data mining, and multi-component quantitative analysis to systematically elucidate the pharmacokinetic behavior and potential bioactive constituents of TMJZC in humans. Using a self-developed untargeted HRMS data-processing platform, a total of 215 TMJZC-related compounds (including 74 prototype constituents and 141 metabolites) were successfully identified in human plasma and urine. Based on human exposure abundance, quality-control components of the herbal medicine, and their circulating forms <em>in vivo</em>, selected seven potential active compounds for further investigation (Cryptochlorogenic acid, Chlorogenic acid, Kaempferol 3-<em>O-β</em>-sophoroside, Hyperoside, Nuciferine, Ginsenoside Rg1, and Notoginsenoside R1). Subsequently, a sensitive and selective multi-component quantification method was subsequently established to systematically characterize their metabolic profiles in humans. Meanwhile, sex-related differences in pharmacokinetics were observed, with the geometric mean ratios (GMRs, female/male) of Nuciferine, Ginsenoside Rg1 and Hyperoside showing substantially higher Cmax (1134.13 %, 447.65 %, 261.53 %,) and AUC<sub>0–∞</sub> (580.47 %, 422.63 %, 290.51 %) in females, respectively. In addition, pharmacological evaluation using hyperlipidemic hepatocyte models (AML12 and HepG2 cells) demonstrated that Hyperoside, Nuciferine, Notoginsenoside R1, and Ginsenoside Rg1 significantly reduced intracellulartriglyceride accumulation. Overall, this study provides the first comprehensive human exposure and pharmacokinetic characterization of TMJZC, highlighting representative circulating constituents and sex-related metabolic differences, and offers a robust analytical basis for quality evaluation, rational clinical application, and further mechanistic studies of this traditional Chinese medicine formulation.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124907"},"PeriodicalIF":2.8,"publicationDate":"2025-12-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145837740","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
This study presents and validates a simple, sensitive, and specific method for the determination of lincomycin in liver, bovine muscle tissue, milk, poultry meat, eggs, and honey. The method employs a liquid-liquid extraction sample preparation technique, eliminating the need for solid-phase extraction cartridges. This approach offers a cost-effective and rapid alternative, which is essential for routine analysis. The validation procedure encompassed selectivity, linearity, limit of detection, limit of quantification, recovery, repeatability, reproducibility, and decision limit. The method demonstrated excellent linearity with correlation coefficients ranging from 0.9977 to 0.9999 across all matrices. Limit of detection values ranged from 0.04 to 2 μg/kg, while limit of quantification values ranged from 0.13 to 6.6 μg/kg. Recovery rates were between 70 % and 124 % and decision limits ranged from 0.15 to 543 μg/kg. Out of the 180 real samples analyzed, 31 were detected as positive. Continuous monitoring studies should be conducted regularly to determine the presence of veterinary drug residues in food of animal origin, poultry meat, and honey. These studies will help identify the sources of these residues and facilitate the implementation of secure preventive and remedial strategies.
{"title":"Lincomycin residues in liver, bovine muscle tissue, milk, poultry meat, eggs, and honey: Method development and validation","authors":"Omar Khaled , Lamia Ryad , Mostafa Nagi , Fawzy Eissa","doi":"10.1016/j.jchromb.2025.124902","DOIUrl":"10.1016/j.jchromb.2025.124902","url":null,"abstract":"<div><div>This study presents and validates a simple, sensitive, and specific method for the determination of lincomycin in liver, bovine muscle tissue, milk, poultry meat, eggs, and honey. The method employs a liquid-liquid extraction sample preparation technique, eliminating the need for solid-phase extraction cartridges. This approach offers a cost-effective and rapid alternative, which is essential for routine analysis. The validation procedure encompassed selectivity, linearity, limit of detection, limit of quantification, recovery, repeatability, reproducibility, and decision limit. The method demonstrated excellent linearity with correlation coefficients ranging from 0.9977 to 0.9999 across all matrices. Limit of detection values ranged from 0.04 to 2 μg/kg, while limit of quantification values ranged from 0.13 to 6.6 μg/kg. Recovery rates were between 70 % and 124 % and decision limits ranged from 0.15 to 543 μg/kg. Out of the 180 real samples analyzed, 31 were detected as positive. Continuous monitoring studies should be conducted regularly to determine the presence of veterinary drug residues in food of animal origin, poultry meat, and honey. These studies will help identify the sources of these residues and facilitate the implementation of secure preventive and remedial strategies.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124902"},"PeriodicalIF":2.8,"publicationDate":"2025-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145812582","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-18DOI: 10.1016/j.jchromb.2025.124895
Yinshuo Xu , Yuanjie Shan , Yuta Nishio , Kenichiro Todoroki , Toshimasa Toyo'oka , Toufeng Jin , Jun Zhe Min
With the development of untargeted metabolomics, studies have shown that alterations in the serum levels of specific amino acids are strongly correlated with the development of early-stage colorectal cancer (CRC). However, the relationship between optically active free amino acids in human nails and the risk of CRC remains unclear. In this study, a highly sensitive UHPLC-MS/MS chiral separation method based on (S)-(−)-DBD-Pro-COCl derivatization was established for the simultaneous detection of DL-Asp, DL-Kyn, DL-α-hydroxybutyric acid (DL-α-HB), and Cystamine in the fingernails of patients with CRC. The derivatization reagents were incubated with DL-amino acids, DL-α-HB and Cyst at 60 °C for 15 min to form diastereomers. Subsequently, an Imtakt cadenza CD-C18 column (3.0 × 150 mm, 3.0 μm) was used for the separation of diastereomers under gradient elution of 10 mM HCOONH4 in H2O as mobile phase A and 0.1 % HCOOH in CH3CN as mobile phase B. The degrees of separation (Rs) ranged from 1.76 to 3.21. R2 was >0.9995, indicating good linearity; the limit of detection (LOD) was 25–100 fmol; the interday and intraday precision of DL-Asp and DL-α-HB detection were 0.85 %–4.91 %; and the average recovery ranged from 81.12 % to 110.2 %. However, during the determination process of fingernail samples, only DL-Asp and DL-α-HB were successfully detected, while DL-Kyn and Cyst were not detected. Therefore, this novel method was used to quantify DL-Asp and DL-α-HB in fingernail samples from 32 healthy volunteers (HVs) and 16 patients with CRC (CPs). The results revealed significant differences in D-Asp (p < 0.05), L-Asp (p < 0.01), and L-α-HB (p < 0.05) levels between male HVs and CPs and in D-α-HB (p < 0.05) levels between female HVs and CPs. In addition, the D/L-α-HB ratio (p < 0.01) was significantly different between male HVs and CPs. These findings validate the use of DL-amino acids, DL-α-HB and Cyst from human fingernails as potential diagnostic biomarkers for CRC, providing a basis for the clinical application of human fingernail samples for the noninvasive diagnosis of CRC.
{"title":"Simultaneous chiral analysis of DL-asp and DL-α-HB with derivatization in human fingernail by UHPLC-MS/MS: an application in colorectal cancer","authors":"Yinshuo Xu , Yuanjie Shan , Yuta Nishio , Kenichiro Todoroki , Toshimasa Toyo'oka , Toufeng Jin , Jun Zhe Min","doi":"10.1016/j.jchromb.2025.124895","DOIUrl":"10.1016/j.jchromb.2025.124895","url":null,"abstract":"<div><div>With the development of untargeted metabolomics, studies have shown that alterations in the serum levels of specific amino acids are strongly correlated with the development of early-stage colorectal cancer (CRC). However, the relationship between optically active free amino acids in human nails and the risk of CRC remains unclear. In this study, a highly sensitive UHPLC-MS/MS chiral separation method based on (<em>S</em>)-(−)-DBD-Pro-COCl derivatization was established for the simultaneous detection of DL-Asp, DL-Kyn, DL-α-hydroxybutyric acid (DL-α-HB), and Cystamine in the fingernails of patients with CRC. The derivatization reagents were incubated with DL-amino acids, DL-α-HB and Cyst at 60 °C for 15 min to form diastereomers. Subsequently, an Imtakt cadenza CD-C<sub>18</sub> column (3.0 × 150 mm, 3.0 μm) was used for the separation of diastereomers under gradient elution of 10 mM HCOONH<sub>4</sub> in H<sub>2</sub>O as mobile phase A and 0.1 % HCOOH in CH<sub>3</sub>CN as mobile phase B. The degrees of separation (Rs) ranged from 1.76 to 3.21. R<sup>2</sup> was >0.9995, indicating good linearity; the limit of detection (LOD) was 25–100 fmol; the interday and intraday precision of DL-Asp and DL-α-HB detection were 0.85 %–4.91 %; and the average recovery ranged from 81.12 % to 110.2 %. However, during the determination process of fingernail samples, only DL-Asp and DL-α-HB were successfully detected, while DL-Kyn and Cyst were not detected. Therefore, this novel method was used to quantify DL-Asp and DL-α-HB in fingernail samples from 32 healthy volunteers (HVs) and 16 patients with CRC (CPs). The results revealed significant differences in D-Asp (<em>p</em> < 0.05), L-Asp (<em>p</em> < 0.01), and L-α-HB (<em>p</em> < 0.05) levels between male HVs and CPs and in D-α-HB (<em>p</em> < 0.05) levels between female HVs and CPs. In addition, the D/L-α-HB ratio (<em>p</em> < 0.01) was significantly different between male HVs and CPs. These findings validate the use of DL-amino acids, DL-α-HB and Cyst from human fingernails as potential diagnostic biomarkers for CRC, providing a basis for the clinical application of human fingernail samples for the noninvasive diagnosis of CRC.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124895"},"PeriodicalIF":2.8,"publicationDate":"2025-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145822581","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-17DOI: 10.1016/j.jchromb.2025.124900
Devendra Kumar , Manish Sharma , Neerja Trivedi
Therapeutic oligonucleotides have emerged as a transformative drug class, yet their physicochemical complexity poses unique analytical challenges in bioanalysis. Liquid chromatography mass spectrometry (LC-MS) has become a powerful platform for their quantification, offering high specificity and structural insight. However, accurate measurement requires addressing challenges such as nonspecific binding, matrix effects, nuclease degradation, and ion-pairing interferences from sample preparation to LC-MS analysis. This review provides a practical roadmap for establishing robust LC-MS workflows for oligonucleotides bioanalysis, emphasizing optimized sample preparation, column and mobile phase selection, ionization control, and fragmentation tuning. Key strategies to minimize analytical artifacts, improve recovery, and ensure regulatory compliance are discussed in the context of current FDA or EMA bioanalytical validation guidelines. Collectively, this work outlines the critical considerations and systematic optimizations needed to achieve reliable, reproducible, and sensitive quantification of therapeutic oligonucleotides in complex biological matrices, supporting their successful clinical translation by informing pharmacokinetics, therapeutic potential, and safety profiles.
{"title":"A Roadmap guide on bioanalysis challenges and practical solutions for accurate quantification of oligonucleotide-based novel therapeutic modalities using LC-MS","authors":"Devendra Kumar , Manish Sharma , Neerja Trivedi","doi":"10.1016/j.jchromb.2025.124900","DOIUrl":"10.1016/j.jchromb.2025.124900","url":null,"abstract":"<div><div>Therapeutic oligonucleotides have emerged as a transformative drug class, yet their physicochemical complexity poses unique analytical challenges in bioanalysis. Liquid chromatography mass spectrometry (LC-MS) has become a powerful platform for their quantification, offering high specificity and structural insight. However, accurate measurement requires addressing challenges such as nonspecific binding, matrix effects, nuclease degradation, and ion-pairing interferences from sample preparation to LC-MS analysis. This review provides a practical roadmap for establishing robust LC-MS workflows for oligonucleotides bioanalysis, emphasizing optimized sample preparation, column and mobile phase selection, ionization control, and fragmentation tuning. Key strategies to minimize analytical artifacts, improve recovery, and ensure regulatory compliance are discussed in the context of current FDA or EMA bioanalytical validation guidelines. Collectively, this work outlines the critical considerations and systematic optimizations needed to achieve reliable, reproducible, and sensitive quantification of therapeutic oligonucleotides in complex biological matrices, supporting their successful clinical translation by informing pharmacokinetics, therapeutic potential, and safety profiles.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124900"},"PeriodicalIF":2.8,"publicationDate":"2025-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145788163","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-17DOI: 10.1016/j.jchromb.2025.124903
Hongjun Luo , Hui Li , Yongyin Zhou , Wenhong Luo , Zhexuan Lin
Global analysis of phospholipid methylation is crucial for understanding the biological roles of phospholipids in mammalian membranes. This study developed and validated a robust liquid chromatography-mass spectrometry (LC-MS) method for the simultaneous determination of ethanolamine (EA) and choline (CL) released from the hydrolysis of phosphatidylethanolamine (PE) and phosphatidylcholine (PC), respectively. The extraction, hydrolysis, and detection procedures were optimized. The compounds were separated on an HILIC column with isocratic elution and detected using a mass spectrometer with electrospray ionization in positive ion mode. The selected ions monitored were m/z 104.1 for CL, m/z 113.1 for d9-CL (internal standard for CL), m/z 62.1 for EA, and m/z 66.1 for d4-EA (internal standard for EA). The method showed satisfactory linearity (r2 > 0.999), precision (intra- and inter-day RSDs ≤6.3 %), and accuracy (−2.1–6.8 %). The limits of detection (LOD) were 0.30 μM for EA and 0.02 μM for CL. This method was applied to human erythrocytes and several tumor cell lines (Hela, EC109, Saos-2), revealing significant differences in the EA/CL ratio (reflecting the PE/PC ratio) among different cell types. This study indicated that the global phospholipid methylation status is cell-type specific and established the approach as a valuable tool for investigating membrane lipid composition in health and disease.
{"title":"A liquid chromatography-mass spectrometry method for profiling global phospholipid methylation via Headgroup analysis revealisng cell-type specificity","authors":"Hongjun Luo , Hui Li , Yongyin Zhou , Wenhong Luo , Zhexuan Lin","doi":"10.1016/j.jchromb.2025.124903","DOIUrl":"10.1016/j.jchromb.2025.124903","url":null,"abstract":"<div><div>Global analysis of phospholipid methylation is crucial for understanding the biological roles of phospholipids in mammalian membranes. This study developed and validated a robust liquid chromatography-mass spectrometry (LC-MS) method for the simultaneous determination of ethanolamine (EA) and choline (CL) released from the hydrolysis of phosphatidylethanolamine (PE) and phosphatidylcholine (PC), respectively. The extraction, hydrolysis, and detection procedures were optimized. The compounds were separated on an HILIC column with isocratic elution and detected using a mass spectrometer with electrospray ionization in positive ion mode. The selected ions monitored were <em>m</em>/<em>z</em> 104.1 for CL, m/z 113.1 for d9-CL (internal standard for CL), m/z 62.1 for EA, and m/z 66.1 for d4-EA (internal standard for EA). The method showed satisfactory linearity (r<sup>2</sup> > 0.999), precision (intra- and inter-day RSDs ≤6.3 %), and accuracy (−2.1–6.8 %). The limits of detection (LOD) were 0.30 μM for EA and 0.02 μM for CL. This method was applied to human erythrocytes and several tumor cell lines (Hela, EC109, Saos-2), revealing significant differences in the EA/CL ratio (reflecting the PE/PC ratio) among different cell types. This study indicated that the global phospholipid methylation status is cell-type specific and established the approach as a valuable tool for investigating membrane lipid composition in health and disease.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124903"},"PeriodicalIF":2.8,"publicationDate":"2025-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145822648","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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