Pub Date : 2025-12-08DOI: 10.1016/j.jpba.2025.117308
Xiang Li , Ting Yang , Fan Zhang , Guoxiang Sun , Mingxiao Hou , Yunen Liu
Kangfuxin Liquid (KFX), a complex zoogenic medicine, faces challenges in quality control due to its multi-component nature and batch-to-batch variability. This study presents a novel integrated quality assessment strategy combining Four-wavelength maximization profiling (Four-WMP) HPLC with electrochemical fingerprinting (ECFP) for comprehensive evaluation of KFX. The Four-WMP method integrated chromatographic data from four wavelengths (236, 254, 265, and 280 nm) into a unified fingerprint, significantly enhancing chemical comprehensiveness compared to conventional single-wavelength approaches. Concurrently, ECFP provided unique insights into redox-active properties. Strong correlations were established between chemical fingerprints and antioxidant efficacy through Partial Least Squares (PLS) modeling (R2=0.8672, Q2=0.789), identifying critical quality markers including protocatechuic acid and adenosine. Substantial inter-manufacturer variation was observed, particularly in sodium benzoate content, highlighting significant quality inconsistencies. This multi-parametric framework provides a holistic and robust paradigm for quality control of KFX and similar complex medicines, effectively bridging chemical composition with bioactive relevance.
{"title":"Integrating multi–wavelength HPLC fingerprinting and electrochemical profiling with chemometrics for comprehensive quality assessment of Kangfuxin Liquid","authors":"Xiang Li , Ting Yang , Fan Zhang , Guoxiang Sun , Mingxiao Hou , Yunen Liu","doi":"10.1016/j.jpba.2025.117308","DOIUrl":"10.1016/j.jpba.2025.117308","url":null,"abstract":"<div><div>Kangfuxin Liquid (KFX), a complex zoogenic medicine, faces challenges in quality control due to its multi-component nature and batch-to-batch variability. This study presents a novel integrated quality assessment strategy combining Four-wavelength maximization profiling (Four-WMP) HPLC with electrochemical fingerprinting (ECFP) for comprehensive evaluation of KFX. The Four-WMP method integrated chromatographic data from four wavelengths (236, 254, 265, and 280 nm) into a unified fingerprint, significantly enhancing chemical comprehensiveness compared to conventional single-wavelength approaches. Concurrently, ECFP provided unique insights into redox-active properties. Strong correlations were established between chemical fingerprints and antioxidant efficacy through Partial Least Squares (PLS) modeling (R<sup>2</sup>=0.8672, Q<sup>2</sup>=0.789), identifying critical quality markers including protocatechuic acid and adenosine. Substantial inter-manufacturer variation was observed, particularly in sodium benzoate content, highlighting significant quality inconsistencies. This multi-parametric framework provides a holistic and robust paradigm for quality control of KFX and similar complex medicines, effectively bridging chemical composition with bioactive relevance.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"270 ","pages":"Article 117308"},"PeriodicalIF":3.1,"publicationDate":"2025-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145733479","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 : 2025-12-08DOI: 10.1016/j.jpba.2025.117313
Lingyu Shen , Wenxing Li , Yupeng Chen , Daogang Guan , Aiping Lu , Anqi Xu
SuHeXiang Wan (SHXW) is a renowned traditional Chinese medicine (TCM) prescription for treating stroke, but its active components remain largely unidentified. This study aimed to screen potential active compounds of SHXW and reveal their underlying mechanisms in stroke treatment. Ingredients and compounds in SHXW were obtained from TCM databases and subjected to initial screening based on ADMET and physicochemical properties, followed by target gene prediction for each filtered compound. A comprehensive network of filtered compound–target gene–stroke pathogenic gene was constructed and optimized using a multi-objective optimization algorithm. Seventeen potential active compounds were identified, which primarily influenced neuroactive ligand-receptor interactions, arachidonic acid metabolism, and several signaling pathways including PI3K-Akt, calcium, and cAMP. Using an oxygen-glucose deprivation and reoxygenation (OGD/R) model, cirsiliol was identified as the lead compound, significantly enhancing cell viability and morphology, decreasing apoptosis, and reducing oxidative stress and inflammatory responses. Molecular docking and dynamics simulations revealed that cirsiliol stably binds to the NADPH binding pocket of CBR1 protein. Further experiments demonstrated that cirsiliol decreased 4-hydroxynonenal (4-HNE, a substrate of CBR1) levels. This study provides a methodological framework for screening active compounds in TCM prescriptions. The neuroprotective effects of cirsiliol against ischemic stroke merit further investigation.
{"title":"SuHeXiang Wan in the treatment of stroke: Prediction potentially active metabolites using a combination of in silico analysis and experimental viability assessment","authors":"Lingyu Shen , Wenxing Li , Yupeng Chen , Daogang Guan , Aiping Lu , Anqi Xu","doi":"10.1016/j.jpba.2025.117313","DOIUrl":"10.1016/j.jpba.2025.117313","url":null,"abstract":"<div><div>SuHeXiang Wan (SHXW) is a renowned traditional Chinese medicine (TCM) prescription for treating stroke, but its active components remain largely unidentified. This study aimed to screen potential active compounds of SHXW and reveal their underlying mechanisms in stroke treatment. Ingredients and compounds in SHXW were obtained from TCM databases and subjected to initial screening based on ADMET and physicochemical properties, followed by target gene prediction for each filtered compound. A comprehensive network of filtered compound–target gene–stroke pathogenic gene was constructed and optimized using a multi-objective optimization algorithm. Seventeen potential active compounds were identified, which primarily influenced neuroactive ligand-receptor interactions, arachidonic acid metabolism, and several signaling pathways including PI3K-Akt, calcium, and cAMP. Using an oxygen-glucose deprivation and reoxygenation (OGD/R) model, cirsiliol was identified as the lead compound, significantly enhancing cell viability and morphology, decreasing apoptosis, and reducing oxidative stress and inflammatory responses. Molecular docking and dynamics simulations revealed that cirsiliol stably binds to the NADPH binding pocket of CBR1 protein. Further experiments demonstrated that cirsiliol decreased 4-hydroxynonenal (4-HNE, a substrate of CBR1) levels. This study provides a methodological framework for screening active compounds in TCM prescriptions. The neuroprotective effects of cirsiliol against ischemic stroke merit further investigation.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"270 ","pages":"Article 117313"},"PeriodicalIF":3.1,"publicationDate":"2025-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145733402","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 : 2025-12-08DOI: 10.1016/j.jpba.2025.117310
Yanhua Gao , Shi Qiao , Jun Li , Ruofan An , Hongpin Hou , Lianmei Wang , Zheng Yuan , Dezhi Kong , Wei Yang
Allergy is a common immune response often triggered by foods, medicine, and pollution, significantly affecting human health. Small-molecule metabolites, as the most direct phenotypic manifestations of biological systems, can directly elucidate gene functions and molecular mechanisms. However, the roles and mechanisms of small-molecule metabolites in allergies remain incompletely understood. In this study, we established an integrated strategy combining metabolomics, network pharmacology, and proteomics to identify active metabolites and clarify their regulatory roles in bovine serum albumin (BSA)-induced allergy. 61 differential metabolites associated with allergy were identified and validated through untargeted and targeted metabolomic analyses of plasma. Combining network pharmacology and proteomics, eight activated differential metabolites affect the complement and coagulation cascades pathway through the proteins F10, F2, and PLG in BSA-induced allergy. This mechanism was further corroborated by proteomic validation of 12 related proteins altered in the complement and coagulation cascades pathway. Through correlation and receiver operating characteristic (ROC) analyses, six diagnostic biomarkers for diagnosing BSA allergy were screened, with area under the ROC curve values exceeding 0.94. Metabolites No. 39 (Glutamylphenylalanine) and 43 (hippuraldehyde sulphate) show excellent sensitivity and accuracy for diagnosing BSA-induced allergy and play a key role in modulating the complement and coagulation cascade. This study found that active metabolites could serve as critical factors and diagnostic biomarkers in allergic responses, offering novel insights into the underlying mechanisms of allergy.
{"title":"Integrated multi-omics approaches and network pharmacology analysis to explore the diagnosis and regulatory role of active metabolites in allergy","authors":"Yanhua Gao , Shi Qiao , Jun Li , Ruofan An , Hongpin Hou , Lianmei Wang , Zheng Yuan , Dezhi Kong , Wei Yang","doi":"10.1016/j.jpba.2025.117310","DOIUrl":"10.1016/j.jpba.2025.117310","url":null,"abstract":"<div><div>Allergy is a common immune response often triggered by foods, medicine, and pollution, significantly affecting human health. Small-molecule metabolites, as the most direct phenotypic manifestations of biological systems, can directly elucidate gene functions and molecular mechanisms. However, the roles and mechanisms of small-molecule metabolites in allergies remain incompletely understood. In this study, we established an integrated strategy combining metabolomics, network pharmacology, and proteomics to identify active metabolites and clarify their regulatory roles in bovine serum albumin (BSA)-induced allergy. 61 differential metabolites associated with allergy were identified and validated through untargeted and targeted metabolomic analyses of plasma. Combining network pharmacology and proteomics, eight activated differential metabolites affect the complement and coagulation cascades pathway through the proteins F10, F2, and PLG in BSA-induced allergy. This mechanism was further corroborated by proteomic validation of 12 related proteins altered in the complement and coagulation cascades pathway. Through correlation and receiver operating characteristic (ROC) analyses, six diagnostic biomarkers for diagnosing BSA allergy were screened, with area under the ROC curve values exceeding 0.94. Metabolites No. 39 (Glutamylphenylalanine) and 43 (hippuraldehyde sulphate) show excellent sensitivity and accuracy for diagnosing BSA-induced allergy and play a key role in modulating the complement and coagulation cascade. This study found that active metabolites could serve as critical factors and diagnostic biomarkers in allergic responses, offering novel insights into the underlying mechanisms of allergy.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"270 ","pages":"Article 117310"},"PeriodicalIF":3.1,"publicationDate":"2025-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145733395","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 : 2025-12-08DOI: 10.1016/j.jpba.2025.117312
Nian Yang , Shengyun Dai , Dongxue Wu , Rui Wu , Xiaoru Hu , Xu Zhao , Jian Zheng
Aconitum Traditional Chinese medicine, represented by Chuanwu (Aconiti Radix), Caowu (Aconiti Kusnezoffii Radix), Fuzi (Aconiti Lateralis Radix Praeparata), and their processed products ZhiChuanwu (Aconiti Radix Cocta), ZhiCaowu (Aconiti Kusnezoffii Radix Cocta), and ZhiFuzi, constitute a class of Chinese herbs characterized by a "dual nature of toxicity and efficacy." They possess pharmacological effects such as dispelling wind, eliminating dampness, and dispersing cold to relieve pain. Due to their high toxicity, the processed forms—ZhiChuanwu, ZhiCaowu, and ZhiFuzi—are predominantly used in clinical practice. ZhiFuzi is a key herb for warming Yang. ZhiChuanwu specializes in treating cold-bi syndrome (painful obstruction due to cold), and ZhiCaowu excels in pain relief. Therefore, the correct classification of these toxic Aconite materials is crucial for ensuring their quality control and appropriate clinical application. This study proposes a data fusion and multivariate analysis strategy based on near-infrared spectroscopy (NIR), electronic nose (E-nose), and high-resolution mass spectrometry (HRMS) for classifying these toxic Aconite herbs. Traditional chemometric modeling based on a single data source was found incapable of correctly classifying the three types of herbs. However, chemometrics combined with data fusion strategies enhanced the performance of the classification models. Furthermore, to identify the optimal combination of analytical modeling methods, the performance of different classification algorithms under various data training strategies was compared. PCA-Linear Discriminant Analysis (PCA-LDA) and PLS-DA, when combined with the feature-level fused dataset (NIR-E-nose-MS), yielded the best classification results. Overall, the classification strategy established in this study holds significant potential for classifying toxic Aconite medicinal materials.
{"title":"Data fusion and multivariate analysis based on near infrared spectroscopy, electronic noses, and high resolution mass spectrometry: A synergetic approach to boost performance on the authenticity analysis of toxic herbs for Aconitum","authors":"Nian Yang , Shengyun Dai , Dongxue Wu , Rui Wu , Xiaoru Hu , Xu Zhao , Jian Zheng","doi":"10.1016/j.jpba.2025.117312","DOIUrl":"10.1016/j.jpba.2025.117312","url":null,"abstract":"<div><div>Aconitum Traditional Chinese medicine, represented by Chuanwu (Aconiti Radix), Caowu (Aconiti Kusnezoffii Radix), Fuzi (Aconiti Lateralis Radix Praeparata), and their processed products ZhiChuanwu (Aconiti Radix Cocta), ZhiCaowu (Aconiti Kusnezoffii Radix Cocta), and ZhiFuzi, constitute a class of Chinese herbs characterized by a \"dual nature of toxicity and efficacy.\" They possess pharmacological effects such as dispelling wind, eliminating dampness, and dispersing cold to relieve pain. Due to their high toxicity, the processed forms—ZhiChuanwu, ZhiCaowu, and ZhiFuzi—are predominantly used in clinical practice. ZhiFuzi is a key herb for warming Yang. ZhiChuanwu specializes in treating cold-bi syndrome (painful obstruction due to cold), and ZhiCaowu excels in pain relief. Therefore, the correct classification of these toxic Aconite materials is crucial for ensuring their quality control and appropriate clinical application. This study proposes a data fusion and multivariate analysis strategy based on near-infrared spectroscopy (NIR), electronic nose (E-nose), and high-resolution mass spectrometry (HRMS) for classifying these toxic Aconite herbs. Traditional chemometric modeling based on a single data source was found incapable of correctly classifying the three types of herbs. However, chemometrics combined with data fusion strategies enhanced the performance of the classification models. Furthermore, to identify the optimal combination of analytical modeling methods, the performance of different classification algorithms under various data training strategies was compared. PCA-Linear Discriminant Analysis (PCA-LDA) and PLS-DA, when combined with the feature-level fused dataset (NIR-E-nose-MS), yielded the best classification results. Overall, the classification strategy established in this study holds significant potential for classifying toxic Aconite medicinal materials.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"270 ","pages":"Article 117312"},"PeriodicalIF":3.1,"publicationDate":"2025-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145733478","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 : 2025-12-07DOI: 10.1016/j.jpba.2025.117309
Noah Gould , Kendall Johnson , Anne-Lise Marie , Jared R. Auclair , Alexander R. Ivanov
Monoclonal antibodies (mAbs) are among the most common biotherapeutics, and their clinical applications and global markets have demonstrated increased growth during the last decade, which are expected to grow further due to the shift of therapeutic treatments from general to personalized approaches. As the number and complexity of mAb-based therapeutics grow, there is an urgent need for highly efficient and high-throughput analytical techniques to characterize them and ensure their safety, potency, stability, and efficacy both from a development and regulatory standpoints. A key component in ensuring the methods used for the characterization of antibody-based therapeutics can provide accurate information, with the potential to influence important pipeline decisions, is the availability of diverse commercially available reference standards with well-known and catalogued properties that can serve as benchmarks and validation for the quality assurance of biotherapeutics in development. In this study, we developed and optimized a microfluidic capillary zone electrophoresis electrospray ionization-mass spectrometry (CZE-ESI-MS) method, utilizing the high-mass range MS data acquisition, to characterize three intact mAb reference standards and their proteoforms under near-native conditions using short analysis times (<15 min), low sample amounts (500 picograms), and minimal sample preparation. The developed CZE-MS method identified a number of modifications at the intact mAb level, including relatively high abundance C-terminal lysine clipping (>95 %) and N-linked glycosylation (>99 %), as well as lower abundance modifications, such as deamidation (∼3–7 %) and N-glycan sialylation (<1.5 %). The developed technique is uniquely suited for thorough and reproducible characterization of mAb-based biotherapeutics in industrial settings.
{"title":"Charge variant analysis of intact monoclonal antibody reference standards using microfluidic capillary zone electrophoresis","authors":"Noah Gould , Kendall Johnson , Anne-Lise Marie , Jared R. Auclair , Alexander R. Ivanov","doi":"10.1016/j.jpba.2025.117309","DOIUrl":"10.1016/j.jpba.2025.117309","url":null,"abstract":"<div><div>Monoclonal antibodies (mAbs) are among the most common biotherapeutics, and their clinical applications and global markets have demonstrated increased growth during the last decade, which are expected to grow further due to the shift of therapeutic treatments from general to personalized approaches. As the number and complexity of mAb-based therapeutics grow, there is an urgent need for highly efficient and high-throughput analytical techniques to characterize them and ensure their safety, potency, stability, and efficacy both from a development and regulatory standpoints. A key component in ensuring the methods used for the characterization of antibody-based therapeutics can provide accurate information, with the potential to influence important pipeline decisions, is the availability of diverse commercially available reference standards with well-known and catalogued properties that can serve as benchmarks and validation for the quality assurance of biotherapeutics in development. In this study, we developed and optimized a microfluidic capillary zone electrophoresis electrospray ionization-mass spectrometry (CZE-ESI-MS) method, utilizing the high-mass range MS data acquisition, to characterize three intact mAb reference standards and their proteoforms under near-native conditions using short analysis times (<15 min), low sample amounts (500 picograms), and minimal sample preparation. The developed CZE-MS method identified a number of modifications at the intact mAb level, including relatively high abundance C-terminal lysine clipping (>95 %) and N-linked glycosylation (>99 %), as well as lower abundance modifications, such as deamidation (∼3–7 %) and N-glycan sialylation (<1.5 %). The developed technique is uniquely suited for thorough and reproducible characterization of mAb-based biotherapeutics in industrial settings.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"270 ","pages":"Article 117309"},"PeriodicalIF":3.1,"publicationDate":"2025-12-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145733398","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 : 2025-12-05DOI: 10.1016/j.jpba.2025.117306
Mintao Jian , Jiaqing Xiong , Kui Li
This study investigates the molecular mechanisms by which Xiangpi Shengji Ointment (XPSJO), combined with a heat-clearing and dampness-drying compound, accelerates postoperative wound healing in an anal fistula (AF) rat model, employing integrated proteomic and metabolomic approaches. An AF rat model was established and treated topically with the herbal combination. Wound healing progression was monitored postoperatively. Histopathological assessment (hematoxylin and eosin [H&E] staining), enzyme-linked immunosorbent assay [ELISA] quantification of inflammatory markers (e.g., tumor necrosis factor-alpha [TNF-α], interleukin [IL]-6) and vascular endothelial growth factor [VEGF], and proteomic (liquid chromatography-tandem mass spectrometry [LC-MS/MS]) and metabolomic (gas chromatography-mass spectrometry [GC-MS]) analyses identified critical pathways and biomolecules involved. In vitro, lipopolysaccharide (LPS)-stimulated THP-1 macrophages and human umbilical vein endothelial cells (HUVECs) were co-treated with XPSJO and Coptis chinensis-Phellodendron amurense Rupr. Extract (CGE). Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway modulation was examined via reverse transcription quantitative polymerase chain reaction (RT-qPCR), Western blot, and functional assays (cell counting kit [CCK]-8, flow cytometry, scratch wound, tube formation). Lentiviral NF-κB overexpression validated mechanistic specificity in vitro and in vivo. Results showed that XPSJO combined with the compound accelerated AF wound closuresuppressed NF-κB signaling pathway activation (evidenced by downregulation of pathway-associated proteins in proteomics and reduced p65 phosphorylation), decreased pro-inflammatory cytokine secretion, and enhanced VEGF expression. Critically, it promoted M2 macrophage polarization (increased Arg-1/CD206, decreased iNOS/CD80) and stimulated HUVEC migration/angiogenesis. Multi-omics analysis further confirmed NF-κB, TNF, and IL-17 pathways as central therapeutic targets. The study concludes that XPSJO combined with a heat-clearing and dampness-drying compound promotes AF healing through pharmacological inhibition of the NF-κB pathway, macrophage reprogramming, and tissue remodeling.
{"title":"Integrated proteomics and metabolomics reveal NF-κB pathway modulation by Xiangpi Shengji ointment and Qingre Zaoshi compound in anal fistula wound healing","authors":"Mintao Jian , Jiaqing Xiong , Kui Li","doi":"10.1016/j.jpba.2025.117306","DOIUrl":"10.1016/j.jpba.2025.117306","url":null,"abstract":"<div><div>This study investigates the molecular mechanisms by which Xiangpi Shengji Ointment (XPSJO), combined with a heat-clearing and dampness-drying compound, accelerates postoperative wound healing in an anal fistula (AF) rat model, employing integrated proteomic and metabolomic approaches. An AF rat model was established and treated topically with the herbal combination. Wound healing progression was monitored postoperatively. Histopathological assessment (hematoxylin and eosin [H&E] staining), enzyme-linked immunosorbent assay [ELISA] quantification of inflammatory markers (e.g., tumor necrosis factor-alpha [TNF-α], interleukin [IL]-6) and vascular endothelial growth factor [VEGF], and proteomic (liquid chromatography-tandem mass spectrometry [LC-MS/MS]) and metabolomic (gas chromatography-mass spectrometry [GC-MS]) analyses identified critical pathways and biomolecules involved. <em>In vitro</em>, lipopolysaccharide (LPS)-stimulated THP-1 macrophages and human umbilical vein endothelial cells (HUVECs) were co-treated with XPSJO and <em>Coptis chinensis</em>-<em>Phellodendron amurense</em> Rupr. Extract (CGE). Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway modulation was examined via reverse transcription quantitative polymerase chain reaction (RT-qPCR), Western blot, and functional assays (cell counting kit [CCK]-8, flow cytometry, scratch wound, tube formation). Lentiviral NF-κB overexpression validated mechanistic specificity <em>in vitro</em> and <em>in vivo</em>. Results showed that XPSJO combined with the compound accelerated AF wound closuresuppressed NF-κB signaling pathway activation (evidenced by downregulation of pathway-associated proteins in proteomics and reduced p65 phosphorylation), decreased pro-inflammatory cytokine secretion, and enhanced VEGF expression. Critically, it promoted M2 macrophage polarization (increased Arg-1/CD206, decreased iNOS/CD80) and stimulated HUVEC migration/angiogenesis. Multi-omics analysis further confirmed NF-κB, TNF, and IL-17 pathways as central therapeutic targets. The study concludes that XPSJO combined with a heat-clearing and dampness-drying compound promotes AF healing through pharmacological inhibition of the NF-κB pathway, macrophage reprogramming, and tissue remodeling.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"270 ","pages":"Article 117306"},"PeriodicalIF":3.1,"publicationDate":"2025-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145733403","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 evaluate the impact of biorelevant bicarbonate buffer solutions (BCB) on drug degradation relative to phosphate buffer solutions (PPB). Esomeprazole (ESM) was selected as a model compound. Stability studies were conducted at 37 °C using 10 mM BCB and 31 mM PPB (both at pH 5.5; buffer capacity (β) = 4.0 mM/pH; ionic strength (I) = 0.14 M). Degradation products were quantified at 4 time points by HPLC. No significant difference was observed in the degradation rate constant of ESM between BCB and PPB. However, LC–MS analysis revealed a distinct tendency that two degradation products with m/z values of 330 and 298 were more abundant in BCB than in PPB. The putative structures of these degradation products matched the previously reported compounds. These findings suggest that buffer species can influence degradation pathways even under equivalent β and I. Consequently, the use of biorelevant buffer systems is essential for accurate assessments of degradation products.
本研究旨在评价生物相关碳酸氢盐缓冲液（BCB）相对于磷酸盐缓冲液（PPB）对药物降解的影响。选择埃索美拉唑（ESM）作为模型化合物。稳定性研究在37°C下进行，使用10 mM BCB和31 mM PPB (pH均为5.5，缓冲容量(β) = 4.0 mM/pH；离子强度(I) = 0.14 M)。用高效液相色谱法测定4个时间点的降解产物。BCB和PPB对ESM的降解速率常数无显著差异。但LC-MS分析显示，m/z值为330和298的两种降解产物在BCB中的含量明显高于PPB。这些降解产物的推定结构与先前报道的化合物相匹配。这些发现表明，即使在相同的β和i下，缓冲物种也可以影响降解途径。因此，使用生物相关的缓冲系统对于准确评估降解产物至关重要。
{"title":"Chemical stability of esomeprazole in biorelevant bicarbonate buffer and an exploratory study for specific degradation products","authors":"Ayaka Takasusuki , Takahiro Takayama , Koichi Inoue , Kiyohiko Sugano","doi":"10.1016/j.jpba.2025.117305","DOIUrl":"10.1016/j.jpba.2025.117305","url":null,"abstract":"<div><div>This study aimed to evaluate the impact of biorelevant bicarbonate buffer solutions (BCB) on drug degradation relative to phosphate buffer solutions (PPB). Esomeprazole (ESM) was selected as a model compound. Stability studies were conducted at 37 °C using 10 mM BCB and 31 mM PPB (both at pH 5.5; buffer capacity (β) = 4.0 mM/pH; ionic strength (<em>I</em>) = 0.14 M). Degradation products were quantified at 4 time points by HPLC. No significant difference was observed in the degradation rate constant of ESM between BCB and PPB. However, LC–MS analysis revealed a distinct tendency that two degradation products with <em>m/z</em> values of 330 and 298 were more abundant in BCB than in PPB. The putative structures of these degradation products matched the previously reported compounds. These findings suggest that buffer species can influence degradation pathways even under equivalent β and <em>I</em>. Consequently, the use of biorelevant buffer systems is essential for accurate assessments of degradation products.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"270 ","pages":"Article 117305"},"PeriodicalIF":3.1,"publicationDate":"2025-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145724126","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 : 2025-12-05DOI: 10.1016/j.jpba.2025.117304
Xiao Dong , Jianliang Li , Yixuan Ni , Shuna Bao , Kexin Fan , Jingjing Xu , Qiao Deng , Xu Wang
Glycoproteins play a crucial role in various biological processes, including protein folding, information transmission, nerve conduction, and molecular recognition. In this study, a novel magnetic phenylboronic acid-functionalized covalent organic framework core-shell material (Fe3O4@BBCOF-BA) was successfully synthesized for the highly selective enrichment of glycoproteins from complex biological samples. Systematic optimization of experimental conditions revealed that under weakly alkaline conditions at pH 8.5, the material specifically recognized glycoproteins through boronate ester bonds. The maximum adsorption capacity for ovalbumin (OVA) reached as high as 469.45 mg/g, representing a two-to threefold improvement over traditional magnetic boronate affinity materials. Adsorption kinetics indicated that equilibrium was reached within 120 min, and rapid separation was achieved within 30 s utilizing the superparamagnetic property. Mild elution was performed using an ethanol-formic acid (90:10, v/v) solution, achieving an OVA recovery rate exceeding 90 %. Selectivity experiments confirmed that the material exhibited significantly higher adsorption of glycoproteins (OVA, HRP) than non-glycoproteins (BSA, Lys). Practical application evaluation demonstrated efficient enrichment of trace glycoproteins from 500-fold diluted egg white and complex protein mixtures. SDS-PAGE analysis confirmed low non-specific adsorption, underscoring its strong potential for application in the pretreatment of complex biological samples.
{"title":"Preparation of Novel magnetic boronic acid-affinity material and investigation of adsorption performance toward ovalbumin","authors":"Xiao Dong , Jianliang Li , Yixuan Ni , Shuna Bao , Kexin Fan , Jingjing Xu , Qiao Deng , Xu Wang","doi":"10.1016/j.jpba.2025.117304","DOIUrl":"10.1016/j.jpba.2025.117304","url":null,"abstract":"<div><div>Glycoproteins play a crucial role in various biological processes, including protein folding, information transmission, nerve conduction, and molecular recognition. In this study, a novel magnetic phenylboronic acid-functionalized covalent organic framework core-shell material (Fe<sub>3</sub>O<sub>4</sub>@BBCOF-BA) was successfully synthesized for the highly selective enrichment of glycoproteins from complex biological samples. Systematic optimization of experimental conditions revealed that under weakly alkaline conditions at pH 8.5, the material specifically recognized glycoproteins through boronate ester bonds. The maximum adsorption capacity for ovalbumin (OVA) reached as high as 469.45 mg/g, representing a two-to threefold improvement over traditional magnetic boronate affinity materials. Adsorption kinetics indicated that equilibrium was reached within 120 min, and rapid separation was achieved within 30 s utilizing the superparamagnetic property. Mild elution was performed using an ethanol-formic acid (90:10, v/v) solution, achieving an OVA recovery rate exceeding 90 %. Selectivity experiments confirmed that the material exhibited significantly higher adsorption of glycoproteins (OVA, HRP) than non-glycoproteins (BSA, Lys). Practical application evaluation demonstrated efficient enrichment of trace glycoproteins from 500-fold diluted egg white and complex protein mixtures. SDS-PAGE analysis confirmed low non-specific adsorption, underscoring its strong potential for application in the pretreatment of complex biological samples.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"270 ","pages":"Article 117304"},"PeriodicalIF":3.1,"publicationDate":"2025-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145714569","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 : 2025-12-04DOI: 10.1016/j.jpba.2025.117303
Vildan Sanko , Mustafa Ali Güngör , Filiz Kuralay
The rapid growth of the pharmaceutical industry has highlighted the importance of controlling both the quality control process and appropriate dosing in drug purchasing to a critical level. In addition to this, pharmaceutical pollutants can harm the environment, especially water resources, creating a need for low-cost, sensitive, selective, and portable technologies to monitor personal health and environmental applications. Because monitoring more than one drug simultaneously provides convenience in terms of diagnosis and treatment, it increases the drug monitoring capacity and features. Therefore, various electrochemical methods and micro/nanomaterials integration have been employed to achieve well-separated peaks for drug molecules and enhance sensitivity. However, detecting more than one drug using an electrochemical method remains a challenging research topic. This review discusses electrochemical multi-drug (bio)sensing techniques, focusing on future trends. For this purpose, drugs were categorized as anticancer, anti-inflammatory, antidepressant, antibacterial, antiviral, and antifungal according to the area they act on. Moreover, integration of herbal drugs, which are highlighted by their rising therapeutic importance and intricate phytochemical compositions, has found place in electrochemical sensing. Current electrochemical (bio)sensors that detect multiple drugs for each group were discussed. In addition, mixed-type multi-drugs containing different drug groups were also discussed in a separate section.
{"title":"Electrochemical multi-drug sensors: Current advances, challenges, and future perspectives","authors":"Vildan Sanko , Mustafa Ali Güngör , Filiz Kuralay","doi":"10.1016/j.jpba.2025.117303","DOIUrl":"10.1016/j.jpba.2025.117303","url":null,"abstract":"<div><div>The rapid growth of the pharmaceutical industry has highlighted the importance of controlling both the quality control process and appropriate dosing in drug purchasing to a critical level. In addition to this, pharmaceutical pollutants can harm the environment, especially water resources, creating a need for low-cost, sensitive, selective, and portable technologies to monitor personal health and environmental applications. Because monitoring more than one drug simultaneously provides convenience in terms of diagnosis and treatment, it increases the drug monitoring capacity and features. Therefore, various electrochemical methods and micro/nanomaterials integration have been employed to achieve well-separated peaks for drug molecules and enhance sensitivity. However, detecting more than one drug using an electrochemical method remains a challenging research topic. This review discusses electrochemical multi-drug (bio)sensing techniques, focusing on future trends. For this purpose, drugs were categorized as anticancer, anti-inflammatory, antidepressant, antibacterial, antiviral, and antifungal according to the area they act on. Moreover, integration of herbal drugs, which are highlighted by their rising therapeutic importance and intricate phytochemical compositions, has found place in electrochemical sensing. Current electrochemical (bio)sensors that detect multiple drugs for each group were discussed. In addition, mixed-type multi-drugs containing different drug groups were also discussed in a separate section.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"270 ","pages":"Article 117303"},"PeriodicalIF":3.1,"publicationDate":"2025-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145733400","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}
Metabolite profiling and identification of oligonucleotide-based therapeutics (OBTs) is important in drug discovery and development. Conventional liquid chromatography-high resolution mass spectrometry (LC-HRMS) methods (using ProMass, BioPharma Finder) are effective for targeted analysis of predictable nuclease-derived metabolites but often miss unexpected non-nuclease-mediated ones. This study aimed to develop a universal analytical workflow for comprehensive metabolite profiling of multiple classes OBTs by integrating BioPharma Finder for targeted identification with a Background Subtraction Filter (BSF) for untargeted detection of unexpected biotransformation products. Inclisiran, a GalNAc-conjugated siRNA, was used as a model compound. Samples incubated with rat, monkey, and human liver S9 fractions were analyzed by the LC-HRMS workflow. As a result, a total of 25 (22 predicted +3 unpredicted) inclisiran metabolites were identified across the three species. BioPharma Finder enabled rapid and sensitive identification of 22 predicted metabolites, supported by automated fragment ion assignment. The BSF data processing revealed 22 metabolites not present in control samples, including SS-1GalNAc, SS-2GalNAc, and SS-3GalNAc, which were missed by BioPharma Finder and likely formed via β-N-acetylglucosaminidase-mediated biotransformation. The structures of BSF-detected metabolites were confirmed by comparison with BioPharma Finder-identified metabolites or characterized through manual MS/MS interpretation. Product ion spectra of these metabolites showed weak or absent diagnostic ion (m/z 94.936), a characteristic fragment of phosphorothioate-containing oligonucleotides, suggesting that the product ion filtering of this ion exhibits limited utility in discovering inclisiran metabolites. Overall, the integrated LC-HRMS workflow shows strong potential as a universal platform for biotransformation studies of OBTs.
代谢物分析和鉴定基于寡核苷酸的疗法（OBTs）在药物发现和开发中具有重要意义。传统的液相色谱-高分辨率质谱（LC-HRMS）方法（使用ProMass， BioPharma Finder）对于可预测的核酸酶衍生代谢物的靶向分析是有效的，但经常错过意想不到的非核酸酶介导的代谢物。本研究旨在开发一种通用的分析工作流程，用于多种obt的综合代谢物分析，方法是将用于靶向鉴定的BioPharma Finder与用于非预期生物转化产物非靶向检测的背景减除过滤器（BSF）集成在一起。Inclisiran是一种GalNAc-conjugated siRNA，被用作模型化合物。用大鼠、猴和人肝脏S9部分孵育的样品采用LC-HRMS工作流程进行分析。结果，在三个物种中共鉴定出25个（22个预测的+3个未预测的）inclisiran代谢物。BioPharma Finder能够快速、灵敏地识别22种预测的代谢物，并支持自动片段离子分配。BSF数据处理显示22种代谢物在对照样品中不存在，包括SS-1GalNAc、SS-2GalNAc和SS-3GalNAc，这些代谢物可能是通过β- n -乙酰氨基葡萄糖酶介导的生物转化形成的。bsf检测到的代谢物的结构通过与BioPharma finder鉴定的代谢物进行比较来确认，或者通过手动MS/MS解释来表征。这些代谢物的产物离子谱显示弱或缺失诊断离子（m/z为94.936），这是含硫代磷酸酯寡核苷酸的特征片段，表明该离子的产物离子过滤在发现含硫代磷酸酯代谢物方面的应用有限。总的来说，LC-HRMS集成工作流显示出强大的潜力，作为obt生物转化研究的通用平台。
{"title":"A universal LC-HRMS workflow integrating targeted and untargeted strategies for rapid and comprehensive metabolite profiling of oligonucleotide-based therapeutics","authors":"Yuqing Zhao , Yue Yang , Mingshe Zhu , Chongzhuang Tang","doi":"10.1016/j.jpba.2025.117298","DOIUrl":"10.1016/j.jpba.2025.117298","url":null,"abstract":"<div><div>Metabolite profiling and identification of oligonucleotide-based therapeutics (OBTs) is important in drug discovery and development. Conventional liquid chromatography-high resolution mass spectrometry (LC-HRMS) methods (using ProMass, BioPharma Finder) are effective for targeted analysis of predictable nuclease-derived metabolites but often miss unexpected non-nuclease-mediated ones. This study aimed to develop a universal analytical workflow for comprehensive metabolite profiling of multiple classes OBTs by integrating BioPharma Finder for targeted identification with a Background Subtraction Filter (BSF) for untargeted detection of unexpected biotransformation products. Inclisiran, a GalNAc-conjugated siRNA, was used as a model compound. Samples incubated with rat, monkey, and human liver S9 fractions were analyzed by the LC-HRMS workflow. As a result, a total of 25 (22 predicted +3 unpredicted) inclisiran metabolites were identified across the three species. BioPharma Finder enabled rapid and sensitive identification of 22 predicted metabolites, supported by automated fragment ion assignment. The BSF data processing revealed 22 metabolites not present in control samples, including SS-1GalNAc, SS-2GalNAc, and SS-3GalNAc, which were missed by BioPharma Finder and likely formed via β-<em>N</em>-acetylglucosaminidase-mediated biotransformation. The structures of BSF-detected metabolites were confirmed by comparison with BioPharma Finder-identified metabolites or characterized through manual MS/MS interpretation. Product ion spectra of these metabolites showed weak or absent diagnostic ion (<em>m</em>/<em>z</em> 94.936), a characteristic fragment of phosphorothioate-containing oligonucleotides, suggesting that the product ion filtering of this ion exhibits limited utility in discovering inclisiran metabolites. Overall, the integrated LC-HRMS workflow shows strong potential as a universal platform for biotransformation studies of OBTs.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"270 ","pages":"Article 117298"},"PeriodicalIF":3.1,"publicationDate":"2025-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145714504","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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