Pub Date : 2026-08-01Epub Date: 2026-03-09DOI: 10.1016/j.jpba.2026.117455
Dominique O. Farrera, Max M. Maloney, Christopher S. LaMadrid, Paige Lenzen-Hammerel, Lauren R. Radtke, Nathan J. Cherrington
Following administration of ezetimibe (EZE), levels of the glucuronidated metabolite ezetimibe-glucuronide (EZEG) are altered in patients with hepatic impairment. A rapid and sensitive liquid chromatography–tandem mass spectrometry method (LC-MS/MS) method was developed and validated to quantify EZE and EZEG in human plasma and urine. Samples were prepared using a protein precipitation procedure with methanol containing stable isotope-labeled internal standards (EZE-d4 and EZEG-d4). Separation of the analytes was achieved using acetonitrile–water (0.1% formic acid) as the mobile phase at a flow rate of 0.5 mL/min on a C18 column. The analytes were detected using negative ionization in multiple reaction monitoring (MRM) mode. The mass transition pairs of m/z 408.4→271.0 and m/z 584.5→271.0 were used to quantify EZE and EZEG, respectively. The method was linear over a concentration range of 1.5 ng/mL – 1 µg/mL for EZE and EZEG in the plasma. In the urine, EZE was detectable a linear range of 5 ng/mL – 1 µg/mL and EZEG ranged from 3 ng/mL – 1 µg/mL. The method was validated in accordance with U.S. Food and Drug Administration and European Medicines Agency regulatory standards, including specificity, sensitivity, stability, repeatability and reproducibility. This ensures accuracy and reliability of test results which thereby enhances patient safety and helps support clinical decisions.
{"title":"Detection and quantification of ezetimibe and its major glucuronide in patients with hepatic impairment via liquid chromatography-tandem mass spectrometry","authors":"Dominique O. Farrera, Max M. Maloney, Christopher S. LaMadrid, Paige Lenzen-Hammerel, Lauren R. Radtke, Nathan J. Cherrington","doi":"10.1016/j.jpba.2026.117455","DOIUrl":"10.1016/j.jpba.2026.117455","url":null,"abstract":"<div><div>Following administration of ezetimibe (EZE), levels of the glucuronidated metabolite ezetimibe-glucuronide (EZEG) are altered in patients with hepatic impairment. A rapid and sensitive liquid chromatography–tandem mass spectrometry method (LC-MS/MS) method was developed and validated to quantify EZE and EZEG in human plasma and urine. Samples were prepared using a protein precipitation procedure with methanol containing stable isotope-labeled internal standards (EZE-d<sub>4</sub> and EZEG-d<sub>4</sub>). Separation of the analytes was achieved using acetonitrile–water (0.1% formic acid) as the mobile phase at a flow rate of 0.5 mL/min on a C18 column. The analytes were detected using negative ionization in multiple reaction monitoring (MRM) mode. The mass transition pairs of <em>m/z</em> 408.4→271.0 and <em>m/z</em> 584.5→271.0 were used to quantify EZE and EZEG, respectively. The method was linear over a concentration range of 1.5 ng/mL – 1 µg/mL for EZE and EZEG in the plasma. In the urine, EZE was detectable a linear range of 5 ng/mL – 1 µg/mL and EZEG ranged from 3 ng/mL – 1 µg/mL. The method was validated in accordance with U.S. Food and Drug Administration and European Medicines Agency regulatory standards, including specificity, sensitivity, stability, repeatability and reproducibility. This ensures accuracy and reliability of test results which thereby enhances patient safety and helps support clinical decisions.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"275 ","pages":"Article 117455"},"PeriodicalIF":3.1,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147387784","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 : 2026-08-01Epub Date: 2026-02-26DOI: 10.1016/j.jpba.2026.117417
Magdaléna Vágnerová , Petr Palivec , Monika Mrňavá , Eliška Mizerová , Bronislav Jurásek , Silvie Rimpelová , Jan Sácký , Klára Šíchová , Čestmír Vejmola , Tomáš Páleníček , David Sýkora , Martin Kuchař
N-benzylphenethylamines are a class of new psychoactive substances (NPS) that are increasingly being used as recreational drugs with a wide range of adverse effects, possibly even death. Currently, the appearance of new N-benzylphenethylamines far outweighs the studies of their metabolism. One of such compounds, 25E-NBOH, has been on the market for the last seven years but its pharmacological and toxicological effects have not yet been thoroughly reported. To provide a basis for such studies, we investigated 25E-NBOH metabolism in three different systems: human liver microsomes, Wistar rat urine, and Cunninghamella elegans fungus, which contains enzymes similar to those found in mammals and serves as an environmentally sustainable and ethically favourable alternative to animal-based metabolic models. Untargeted LC-HRMS/MS was used to detect phase I and phase II 25E-NBOH metabolites in all three systems. A total of 56 metabolites were annotated, many of which occurred in multiple isomeric forms. Despite metabolic differences between the systems, several abundant metabolites were found in all of them. The primary metabolic pathways detected were hydroxylation at various positions, O-demethylation, and N-debenzylation, followed by conjugation with glucuronic acid, sulphate, or glucose. To confirm the presence of metabolites, we synthesised and measured ten substances under the same LC-HRMS/MS conditions as the real samples. Seven of these were successfully matched to detected metabolites based on retention time and MS/MS spectra, enabling structural assignment and isomer distinction. Additionally, the identity of 2C-E, a known psychoactive substance, was confirmed as one of the metabolites using a commercial reference standard. Lastly, we report the structures of three main biomarkers, suggested by both this study and prior literature. This study provides the first comprehensive in vitro and in vivo metabolic profile of 25E-NBOH, identifying the structures of specific metabolites using in-house synthesised reference standards and proposing structures for the main biomarkers. These findings establish a solid foundation for future pharmacological and toxicological studies, supporting clinicians in the accurate diagnosis of intoxication cases.
n -苄基苯乙胺是一类新的精神活性物质（NPS），越来越多地被用作娱乐性药物，具有广泛的副作用，甚至可能导致死亡。目前，新n -苄基苯乙胺的出现远远超过了对其代谢的研究。其中一种化合物，25E-NBOH，已经在市场上销售了7年，但其药理和毒理学效应尚未得到彻底的报道。为了提供此类研究的基础，我们研究了25E-NBOH在三种不同系统中的代谢：人肝微粒体、Wistar大鼠尿液和秀丽隐线虫真菌，后者含有与哺乳动物相似的酶，是一种环境可持续和道德上有利的替代动物代谢模型。非靶向LC-HRMS/MS用于检测所有三种系统的I期和II期25E-NBOH代谢物。共有56种代谢物被注释，其中许多以多种异构体形式出现。尽管系统之间存在代谢差异，但在所有系统中都发现了一些丰富的代谢物。检测到的主要代谢途径是不同位置的羟基化、o -去甲基化和n -去苄基化，其次是与葡萄糖醛酸、硫酸盐或葡萄糖偶联。为了确认代谢物的存在，我们在与实际样品相同的LC-HRMS/MS条件下合成并测量了10种物质。根据保留时间和MS/MS光谱，其中7个与检测到的代谢物成功匹配，从而实现了结构分配和异构体区分。此外，使用商业参考标准确认了已知精神活性物质2C-E的身份，作为代谢物之一。最后，我们报告了本研究和先前文献提出的三个主要生物标志物的结构。本研究提供了第一个全面的25E-NBOH体外和体内代谢谱，使用内部合成参考标准确定了特定代谢物的结构，并提出了主要生物标志物的结构。这些发现为未来的药理学和毒理学研究奠定了坚实的基础，支持临床医生准确诊断中毒病例。
{"title":"Metabolic profile of 25E-NBOH in human liver microsomes, rat urine, and fungus Cunninghamella elegans","authors":"Magdaléna Vágnerová , Petr Palivec , Monika Mrňavá , Eliška Mizerová , Bronislav Jurásek , Silvie Rimpelová , Jan Sácký , Klára Šíchová , Čestmír Vejmola , Tomáš Páleníček , David Sýkora , Martin Kuchař","doi":"10.1016/j.jpba.2026.117417","DOIUrl":"10.1016/j.jpba.2026.117417","url":null,"abstract":"<div><div><em>N</em>-benzylphenethylamines are a class of new psychoactive substances (NPS) that are increasingly being used as recreational drugs with a wide range of adverse effects, possibly even death. Currently, the appearance of new <em>N</em>-benzylphenethylamines far outweighs the studies of their metabolism. One of such compounds, 25E-NBOH, has been on the market for the last seven years but its pharmacological and toxicological effects have not yet been thoroughly reported. To provide a basis for such studies, we investigated 25E-NBOH metabolism in three different systems: human liver microsomes, Wistar rat urine, and <em>Cunninghamella elegans</em> fungus, which contains enzymes similar to those found in mammals and serves as an environmentally sustainable and ethically favourable alternative to animal-based metabolic models. Untargeted LC-HRMS/MS was used to detect phase I and phase II 25E-NBOH metabolites in all three systems. A total of 56 metabolites were annotated, many of which occurred in multiple isomeric forms. Despite metabolic differences between the systems, several abundant metabolites were found in all of them. The primary metabolic pathways detected were hydroxylation at various positions, <em>O</em>-demethylation, and <em>N</em>-debenzylation, followed by conjugation with glucuronic acid, sulphate, or glucose. To confirm the presence of metabolites, we synthesised and measured ten substances under the same LC-HRMS/MS conditions as the real samples. Seven of these were successfully matched to detected metabolites based on retention time and MS/MS spectra, enabling structural assignment and isomer distinction. Additionally, the identity of 2C-E, a known psychoactive substance, was confirmed as one of the metabolites using a commercial reference standard. Lastly, we report the structures of three main biomarkers, suggested by both this study and prior literature. This study provides the first comprehensive <em>in vitro</em> and <em>in vivo</em> metabolic profile of 25E-NBOH, identifying the structures of specific metabolites using in-house synthesised reference standards and proposing structures for the main biomarkers. These findings establish a solid foundation for future pharmacological and toxicological studies, supporting clinicians in the accurate diagnosis of intoxication cases.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"275 ","pages":"Article 117417"},"PeriodicalIF":3.1,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147387785","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 : 2026-08-01Epub Date: 2026-03-02DOI: 10.1016/j.jpba.2026.117445
Paweł Wityk , Joanna Raczak-Gutknecht , Margot Biesemans , Beata Krawczyk , Wiktoria Brzezińska , Michał J. Markuszewski
Urinary tract infections (UTIs) and urosepsis necessitate a deeper understanding of host–pathogen interactions at the metabolic level. We use LC-MS and GC-MS techniques to characterize metabolic pathway alterations in patients and Escherichia coli isolates during UTI and urosepsis. Our findings reveal substantial metabolic adaptations in the human host, including increased porphyrin metabolism, suggesting oxidative stress response or tissue damage. Activation of the pentose phosphate pathway (PPP) and tricarboxylic acid cycle (TCA) highlights the host’s heightened immune and energy demands during infection. Additionally, enhanced malate–aspartate shuttle activity suggests a greater reliance on glycolysis for energy production, while increased pyruvaldehyde degradation indicates active detoxification of harmful metabolic byproducts. In E. coli, distinct metabolic shifts depended on the extracellular/intracellular niche and infection stage. Intracellular metabolites of E. coli during urosepsis exhibited upregulated purine and biotin metabolism, reflecting a focus on replication and essential metabolic functions. Conversely, intracellular metabolites of E. coli during UTI displayed increased aspartate metabolism, TCA cycle activity, Warburg effect, fatty acid biosynthesis, and glycine/serine metabolism, indicative of urinary tract adaptation. Extracellular metabolites of E. coli during urosepsis exhibited a broad activation of sugar metabolism, highlighting its ability to exploit diverse nutrient sources in systemic infection. In contrast, extracellular metabolites of E. coli during UTI demonstrated specific metabolic changes, including propanoate metabolism activation and homocysteine dysregulation, reflecting unique urinary tract conditions. These findings provide insights into the metabolic pathways employed by host and pathogen during UTI and urosepsis, uncovering potential metabolic vulnerabilities in E. coli.
{"title":"Untargeted LC-MS-based metabolic fingerprinting of Escherichia coli-associated urinary tract infections and urosepsis: Insights into the urine, serum, and bacterial interactomes","authors":"Paweł Wityk , Joanna Raczak-Gutknecht , Margot Biesemans , Beata Krawczyk , Wiktoria Brzezińska , Michał J. Markuszewski","doi":"10.1016/j.jpba.2026.117445","DOIUrl":"10.1016/j.jpba.2026.117445","url":null,"abstract":"<div><div>Urinary tract infections (UTIs) and urosepsis necessitate a deeper understanding of host–pathogen interactions at the metabolic level. We use LC-MS and GC-MS techniques to characterize metabolic pathway alterations in patients and <em>Escherichia coli</em> isolates during UTI and urosepsis. Our findings reveal substantial metabolic adaptations in the human host, including increased porphyrin metabolism, suggesting oxidative stress response or tissue damage. Activation of the pentose phosphate pathway (PPP) and tricarboxylic acid cycle (TCA) highlights the host’s heightened immune and energy demands during infection. Additionally, enhanced malate–aspartate shuttle activity suggests a greater reliance on glycolysis for energy production, while increased pyruvaldehyde degradation indicates active detoxification of harmful metabolic byproducts. In <em>E. coli</em>, distinct metabolic shifts depended on the extracellular/intracellular niche and infection stage. Intracellular metabolites of <em>E. coli</em> during urosepsis exhibited upregulated purine and biotin metabolism, reflecting a focus on replication and essential metabolic functions. Conversely, intracellular metabolites of <em>E. coli</em> during UTI displayed increased aspartate metabolism, TCA cycle activity, Warburg effect, fatty acid biosynthesis, and glycine/serine metabolism, indicative of urinary tract adaptation. Extracellular metabolites of <em>E. coli</em> during urosepsis exhibited a broad activation of sugar metabolism, highlighting its ability to exploit diverse nutrient sources in systemic infection. In contrast, extracellular metabolites of <em>E. coli</em> during UTI demonstrated specific metabolic changes, including propanoate metabolism activation and homocysteine dysregulation, reflecting unique urinary tract conditions. These findings provide insights into the metabolic pathways employed by host and pathogen during UTI and urosepsis, uncovering potential metabolic vulnerabilities in <em>E. coli</em>.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"275 ","pages":"Article 117445"},"PeriodicalIF":3.1,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147372847","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 : 2026-08-01Epub Date: 2026-03-01DOI: 10.1016/j.jpba.2026.117444
Juan Wang , Zhongda Zeng , Wei Duan , Zhuo Man , Miaomiao Wang , Zhong Yang
Hyperuricemia is the second most prevalent metabolic disorder globally and exhibits pronounced heterogeneity in clinical practice. Only about 10% of individuals with asymptomatic hyperuricemia (HUA) progress to gout, yet the lack of specific biomarkers and precise therapeutic targets hinders accurate prediction of disease progression. This work focuses on investigating the heterogeneity of hyperuricemia, aiming to elucidate potential mechanisms underlying its transition from asymptomatic to symptomatic stages. A total of 107 serum samples were analyzed, including 36 healthy controls (Con), and 30, 32 and 9 patients with HUA, gouty arthritis (gout), and gout complicated by uric acid nephrolithiasis (UAN), respectively. Using an advanced SCIEX ZenoTOF 7600-based untargeted metabolomics platform, 925 metabolites were identified in both positive and negative ionization modes. The assessment of quality control (QC) samples showed high precision in instrumental measurement with RSD values of the ion features less than 30% achieving 91.76% and 89.73% respectively in the two ionization modes. A total of 46, 37, 28, and 26 differential metabolites were identified among the four groups. Among them, four metabolites showed high sensitivity and specificity in the analysis of the first three groups. Five metabolic pathways remained consistently dysregulated throughout disease progression, which indicates their potential as mechanistic drivers. These findings provide novel biological insights for the early diagnosis and precision treatment of gout and its complications, and offer a crucial foundation for understanding the metabolic basis of hyperuricemia heterogeneity.
{"title":"Unraveling the heterogeneity of hyperuricemia using untargeted metabolomics and statistical modeling","authors":"Juan Wang , Zhongda Zeng , Wei Duan , Zhuo Man , Miaomiao Wang , Zhong Yang","doi":"10.1016/j.jpba.2026.117444","DOIUrl":"10.1016/j.jpba.2026.117444","url":null,"abstract":"<div><div>Hyperuricemia is the second most prevalent metabolic disorder globally and exhibits pronounced heterogeneity in clinical practice. Only about 10% of individuals with asymptomatic hyperuricemia (HUA) progress to gout, yet the lack of specific biomarkers and precise therapeutic targets hinders accurate prediction of disease progression. This work focuses on investigating the heterogeneity of hyperuricemia, aiming to elucidate potential mechanisms underlying its transition from asymptomatic to symptomatic stages. A total of 107 serum samples were analyzed, including 36 healthy controls (Con), and 30, 32 and 9 patients with HUA, gouty arthritis (gout), and gout complicated by uric acid nephrolithiasis (UAN), respectively. Using an advanced SCIEX ZenoTOF 7600-based untargeted metabolomics platform, 925 metabolites were identified in both positive and negative ionization modes. The assessment of quality control (QC) samples showed high precision in instrumental measurement with RSD values of the ion features less than 30% achieving 91.76% and 89.73% respectively in the two ionization modes. A total of 46, 37, 28, and 26 differential metabolites were identified among the four groups. Among them, four metabolites showed high sensitivity and specificity in the analysis of the first three groups. Five metabolic pathways remained consistently dysregulated throughout disease progression, which indicates their potential as mechanistic drivers. These findings provide novel biological insights for the early diagnosis and precision treatment of gout and its complications, and offer a crucial foundation for understanding the metabolic basis of hyperuricemia heterogeneity.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"275 ","pages":"Article 117444"},"PeriodicalIF":3.1,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147369643","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}
{"title":"Corrigendum to “Systematic characterization of chemical constituents and in vivo metabolites of Jiuwei Qianghuo Granules by UPLC-Q-TOF-MS/MS” [J. Pharm. Biomed. Anal. 274 (2026) 117443]","authors":"Zihang Xu , Chen Zhou , Mengru Wu , Shenqi Sun , Hongfei Wu , Jingyao Xia , Mengjiao Zhou , Yehan Zhu , Beibei Xiang , Yaqi Yao","doi":"10.1016/j.jpba.2026.117447","DOIUrl":"10.1016/j.jpba.2026.117447","url":null,"abstract":"","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"275 ","pages":"Article 117447"},"PeriodicalIF":3.1,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147377874","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 : 2026-06-15Epub Date: 2026-02-09DOI: 10.1016/j.jpba.2026.117409
Fabian E. Bülow , Adam Matkowski , Alexander Weng
Saponins display medicinal properties such as enhancing the drug delivery of biologicals, and boosting immune responses within vaccinations. As plant derived molecules, their phytochemical analysis is key to ensure pharmaceutical quality. The quantification of saponins is typically carried out by high-performance liquid chromatography (HPLC). The aim of this study was the development of a simple thin-layer chromatography (TLC) based method for the quantification of the sapogenin quillaic acid (QA), the triterpenoid backbone of saponins such as QS21 that are used as immunological adjuvants. Following acidic hydrolysis of the saponins, the corresponding sapogenins were derivatized with 4-hydrazino-7-nitro-2,1,3-benzoxadiazole hydrazine (NBD-H) to form fluorescent hydrazones, increasing both the sensitivity and selectivity of the method. The detection of sapogenins with NBD-H is reported here for the first time. The QA-containing TLC bands were identified by mass spectrometry and their quantification was subsequently performed by densitometry. After validation, the method was applied to two plant species from Caryophyllaceae. To verify the plant’s QA contents determined by TLC, a complementary HPLC method was developed. This study presents a new cost-effective method to quantify QA, enabling laboratories with limited resources to monitor plant cultivation and perform phytopharmaceutical quality control.
{"title":"A novel thin layer chromatography based method for the quantification of quillaic acid saponins","authors":"Fabian E. Bülow , Adam Matkowski , Alexander Weng","doi":"10.1016/j.jpba.2026.117409","DOIUrl":"10.1016/j.jpba.2026.117409","url":null,"abstract":"<div><div>Saponins display medicinal properties such as enhancing the drug delivery of biologicals, and boosting immune responses within vaccinations. As plant derived molecules, their phytochemical analysis is key to ensure pharmaceutical quality. The quantification of saponins is typically carried out by high-performance liquid chromatography (HPLC). The aim of this study was the development of a simple thin-layer chromatography (TLC) based method for the quantification of the sapogenin quillaic acid (QA), the triterpenoid backbone of saponins such as QS21 that are used as immunological adjuvants. Following acidic hydrolysis of the saponins, the corresponding sapogenins were derivatized with 4-hydrazino-7-nitro-2,1,3-benzoxadiazole hydrazine (NBD-H) to form fluorescent hydrazones, increasing both the sensitivity and selectivity of the method. The detection of sapogenins with NBD-H is reported here for the first time. The QA-containing TLC bands were identified by mass spectrometry and their quantification was subsequently performed by densitometry. After validation, the method was applied to two plant species from Caryophyllaceae. To verify the plant’s QA contents determined by TLC, a complementary HPLC method was developed. This study presents a new cost-effective method to quantify QA, enabling laboratories with limited resources to monitor plant cultivation and perform phytopharmaceutical quality control.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117409"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146192203","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 : 2026-06-15Epub Date: 2026-02-10DOI: 10.1016/j.jpba.2026.117410
Jiyu Xu , Mingyao Wang , Zhifei Hou , Jie Kong , Mengge Zhang , Yizhuo Tian , Jiangfeng Liu , Juntao Yang , Huaping Dai
Chronic inhalation of coal dust causes coal workers’ pneumoconiosis (CWP), which is one of the leading occupational diseases. Coal workers’ pneumoconiosis is currently incurable, posing a serious public health threat. Identifying the underlying molecular mechanisms of CWP is critical to overcome this challenge. Nowadays, metabolomics has bridged underlying molecular alterations with disease progression, providing a useful tool for researching the pathogenesis and finding biomarkers. In this study, a comprehensive view of metabolic characterization of serum from CWP patients at all stages was provided using untargeted metabolomic analysis. As a result, when compared to healthy controls, the specific alteration patterns of each stage were observed. The results showed arginine and cortisol could be core metabolites in CWP progression. Moreover, five metabolites that significantly changed when going from “solely chronic coal-dust exposure” to an early stage were screened out as potential biomarkers. The receiver operating characteristic results were 0.691–0.862 (individual) and 0.884–0.907 (combined). These findings will benefit the application of metabolomics to understand the pathological mechanism and identify diagnostic biomarkers for CWP.
{"title":"Serum metabolomics reveal metabolic changes in coal workers’ pneumoconiosis progression","authors":"Jiyu Xu , Mingyao Wang , Zhifei Hou , Jie Kong , Mengge Zhang , Yizhuo Tian , Jiangfeng Liu , Juntao Yang , Huaping Dai","doi":"10.1016/j.jpba.2026.117410","DOIUrl":"10.1016/j.jpba.2026.117410","url":null,"abstract":"<div><div>Chronic inhalation of coal dust causes coal workers’ pneumoconiosis (CWP), which is one of the leading occupational diseases. Coal workers’ pneumoconiosis is currently incurable, posing a serious public health threat. Identifying the underlying molecular mechanisms of CWP is critical to overcome this challenge. Nowadays, metabolomics has bridged underlying molecular alterations with disease progression, providing a useful tool for researching the pathogenesis and finding biomarkers. In this study, a comprehensive view of metabolic characterization of serum from CWP patients at all stages was provided using untargeted metabolomic analysis. As a result, when compared to healthy controls, the specific alteration patterns of each stage were observed. The results showed arginine and cortisol could be core metabolites in CWP progression. Moreover, five metabolites that significantly changed when going from “solely chronic coal-dust exposure” to an early stage were screened out as potential biomarkers. The receiver operating characteristic results were 0.691–0.862 (individual) and 0.884–0.907 (combined). These findings will benefit the application of metabolomics to understand the pathological mechanism and identify diagnostic biomarkers for CWP.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117410"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146192204","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 : 2026-06-15Epub Date: 2026-02-02DOI: 10.1016/j.jpba.2026.117388
Jiexia Shi , Fenfang Deng , Yongxian Li , Juntao Li , Rongfei Peng , Jun Yuan , Lei Tan
Accurate and reliable quantification of antigen components in vaccines is critical in vaccine quality control and evaluation of immunogenic consistency. However, conventional immunoassays often suffer from limited specificity, trace-level antigen concentrations, and indirect quantification. In this study, we demonstrated an ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for the determination of effective antigen components in inactivated SARS-CoV-2 vaccines. Specifically, the vaccine samples were denatured and digested with trypsin to generate tryptic peptides. Then, the signature peptides derived from the nucleocapsid protein and their stable isotope–labeled internal standards were selectively captured and separated using anti-peptide antibody-conjugated magnetic beads. The signature peptides were characterized by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, which confirmed their amino acid sequence and multi-charged ionization states. Quantitative analysis was then performed using UHPLC-MS/MS in positive electrospray ionization mode with multiple reaction monitoring. Chromatographic separation of the signature peptides was achieved on an ACQUITY Premier Peptide BEH C₁₈ column using 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile as the mobile phases. The method was validated and exhibited excellent linearity for the signature peptides over the concentration range of 1–60 μg/L, with correlation coefficients higher than 0.999. The recovery ranged from 75.1 % to 86.3 %, with intra-day precision (RSD) of 0.8–1.0 % and inter-day precision of 1.3–3.6 %. Finally, the method was successfully applied to determine the effective antigen components in inactivated SARS-CoV-2 vaccine samples. The concentrations of the signature peptide ADETQALPQR ranged from 4.95 to 12.95 µg/L across the three vaccine batches analyzed, corresponding to 4.38–11.47 nmol/L of nucleocapsid protein. The results indicated that the method exhibited great promise for the determination of active antigenic proteins in inactivated SARS-CoV-2 vaccine samples and provided an alternative analytical platform for vaccine quality control.
{"title":"Determination of antigen components in inactivated SARS-CoV-2 vaccine using ultra-high-performance liquid chromatography tandem mass spectrometry","authors":"Jiexia Shi , Fenfang Deng , Yongxian Li , Juntao Li , Rongfei Peng , Jun Yuan , Lei Tan","doi":"10.1016/j.jpba.2026.117388","DOIUrl":"10.1016/j.jpba.2026.117388","url":null,"abstract":"<div><div>Accurate and reliable quantification of antigen components in vaccines is critical in vaccine quality control and evaluation of immunogenic consistency. However, conventional immunoassays often suffer from limited specificity, trace-level antigen concentrations, and indirect quantification. In this study, we demonstrated an ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for the determination of effective antigen components in inactivated SARS-CoV-2 vaccines. Specifically, the vaccine samples were denatured and digested with trypsin to generate tryptic peptides. Then, the signature peptides derived from the nucleocapsid protein and their stable isotope–labeled internal standards were selectively captured and separated using anti-peptide antibody-conjugated magnetic beads. The signature peptides were characterized by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, which confirmed their amino acid sequence and multi-charged ionization states. Quantitative analysis was then performed using UHPLC-MS/MS in positive electrospray ionization mode with multiple reaction monitoring. Chromatographic separation of the signature peptides was achieved on an ACQUITY Premier Peptide BEH C₁₈ column using 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile as the mobile phases. The method was validated and exhibited excellent linearity for the signature peptides over the concentration range of 1–60 μg/L, with correlation coefficients higher than 0.999. The recovery ranged from 75.1 % to 86.3 %, with intra-day precision (RSD) of 0.8–1.0 % and inter-day precision of 1.3–3.6 %. Finally, the method was successfully applied to determine the effective antigen components in inactivated SARS-CoV-2 vaccine samples. The concentrations of the signature peptide ADETQALPQR ranged from 4.95 to 12.95 µg/L across the three vaccine batches analyzed, corresponding to 4.38–11.47 nmol/L of nucleocapsid protein. The results indicated that the method exhibited great promise for the determination of active antigenic proteins in inactivated SARS-CoV-2 vaccine samples and provided an alternative analytical platform for vaccine quality control.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117388"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146125450","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 : 2026-06-15Epub Date: 2026-02-09DOI: 10.1016/j.jpba.2026.117408
Yunwang Chen , Long Liu , Yinting Wei , Qing Zhang , Yanping Wang
Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by recurrent episodes of intestinal inflammation and mucosal injury. Qingkui Yuyang decoction (QKY), a clinically validated traditional Chinese medicinal formula, has been widely used in the treatment of UC; however, its pharmacodynamically active constituents and underlying mechanisms of action have not been fully elucidated. In this study, we explored the therapeutic mechanisms of QKY in treating UC by employing a combination of serum pharmacochemistry, network pharmacology, and molecular docking techniques. Initially, using UPLC-Q-Exactive Orbitrap-MS/MS, 28 candidate active compounds in the serum of rats treated with QKY were identified. Subsequently, network pharmacology analysis identified 43 overlapping targets between UC and the active components, and 30 related signaling pathways. Further analysis and molecular docking studies have confirmed that the key active components (Loureirin A, Berberine, Ellagic acid) possess potential for effective therapeutic effects with the core targets (RELA, AKT1). In addition, in vitro experiments demonstrated that QKY significantly downregulated the expression levels of the pro-inflammatory cytokines IL-6 and TNF-α. QKY also markedly reduced the phosphorylation levels of NF-κB p65 and p38 MAPK, as well as the corresponding mRNA expression levels of these signaling molecules. These results suggest that QKY may exert its therapeutic effects on UC by modulating the MAPK and NF-κB signaling pathways, offering a promising strategy for the prevention and treatment of UC.
{"title":"Identification of blood-absorbed components of Qingkui Yuyang decoction and its mechanistic roles in ulcerative colitis based on UPLC-Q-Exactive Orbitrap-MS/MS and network pharmacology","authors":"Yunwang Chen , Long Liu , Yinting Wei , Qing Zhang , Yanping Wang","doi":"10.1016/j.jpba.2026.117408","DOIUrl":"10.1016/j.jpba.2026.117408","url":null,"abstract":"<div><div>Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by recurrent episodes of intestinal inflammation and mucosal injury. Qingkui Yuyang decoction (QKY), a clinically validated traditional Chinese medicinal formula, has been widely used in the treatment of UC; however, its pharmacodynamically active constituents and underlying mechanisms of action have not been fully elucidated. In this study, we explored the therapeutic mechanisms of QKY in treating UC by employing a combination of serum pharmacochemistry, network pharmacology, and molecular docking techniques. Initially, using UPLC-Q-Exactive Orbitrap-MS/MS, 28 candidate active compounds in the serum of rats treated with QKY were identified. Subsequently, network pharmacology analysis identified 43 overlapping targets between UC and the active components, and 30 related signaling pathways. Further analysis and molecular docking studies have confirmed that the key active components (Loureirin A, Berberine, Ellagic acid) possess potential for effective therapeutic effects with the core targets (RELA, AKT1). In addition, in vitro experiments demonstrated that QKY significantly downregulated the expression levels of the pro-inflammatory cytokines IL-6 and TNF-α. QKY also markedly reduced the phosphorylation levels of NF-κB p65 and p38 MAPK, as well as the corresponding mRNA expression levels of these signaling molecules. These results suggest that QKY may exert its therapeutic effects on UC by modulating the MAPK and NF-κB signaling pathways, offering a promising strategy for the prevention and treatment of UC.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117408"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146180310","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 : 2026-06-15Epub Date: 2026-01-28DOI: 10.1016/j.jpba.2026.117383
Jérôme Jonveaux, Marc Faudon, Pauline Heymes, Valentina Lucchini, Maria Fernanda Zuluaga Estrada, Michael Jahn, Mostafa Zarei
Isomerization of aspartic acid (Asp) to isoaspartic acid (isoAsp) within the complementarity-determining regions (CDRs) of monoclonal antibodies (mAbs) can lead to conformational changes that decrease antigen-binding affinity. Although isomerization can significantly alter the chromatographic and electrophoretic profiles, precise localization of this modification requires a mass spectrometry-based approach, such as peptide mapping. In this work, we present a case study that investigates various analytical strategies to identify the root cause of significant changes observed in the chromatographic and electrophoretic profiles of an mAb during formulation development. LC-MS analysis of reduced mAb using high-resolution mass spectrometry, peptide mapping using trypsin digestion, and fraction collection of the newly identified peak followed by trypsin digestion suggested that isomerization occurs within the CDR of the mAb. However, due to the presence of three Asp residues within a single tryptic peptide, this modification could not be precisely localized. To overcome this limitation, we developed a sequential enzymatic digestion strategy, utilizing trypsin followed by Asp-N digestion, which enabled accurate localization and quantification of the isomerization site. The resulting data indicated that the main isoAsp signal originated from isomerization at the DS motif that increased substantially over time in the liquid formulation, while no significant change was observed in the lyophilized formulation. The level of isomerization determined through this sequential digestion method correlated well with the LC-UV quantitation data of the reduced mAb.
{"title":"Sequential digestions enable identification and quantification of rapid aspartic acid isomerization in the CDR of a monoclonal antibody light chain","authors":"Jérôme Jonveaux, Marc Faudon, Pauline Heymes, Valentina Lucchini, Maria Fernanda Zuluaga Estrada, Michael Jahn, Mostafa Zarei","doi":"10.1016/j.jpba.2026.117383","DOIUrl":"10.1016/j.jpba.2026.117383","url":null,"abstract":"<div><div>Isomerization of aspartic acid (Asp) to isoaspartic acid (isoAsp) within the complementarity-determining regions (CDRs) of monoclonal antibodies (mAbs) can lead to conformational changes that decrease antigen-binding affinity. Although isomerization can significantly alter the chromatographic and electrophoretic profiles, precise localization of this modification requires a mass spectrometry-based approach, such as peptide mapping. In this work, we present a case study that investigates various analytical strategies to identify the root cause of significant changes observed in the chromatographic and electrophoretic profiles of an mAb during formulation development. LC-MS analysis of reduced mAb using high-resolution mass spectrometry, peptide mapping using trypsin digestion, and fraction collection of the newly identified peak followed by trypsin digestion suggested that isomerization occurs within the CDR of the mAb. However, due to the presence of three Asp residues within a single tryptic peptide, this modification could not be precisely localized. To overcome this limitation, we developed a sequential enzymatic digestion strategy, utilizing trypsin followed by Asp-N digestion, which enabled accurate localization and quantification of the isomerization site. The resulting data indicated that the main isoAsp signal originated from isomerization at the DS motif that increased substantially over time in the liquid formulation, while no significant change was observed in the lyophilized formulation. The level of isomerization determined through this sequential digestion method correlated well with the LC-UV quantitation data of the reduced mAb.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117383"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146119332","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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