Pub Date : 2026-01-12DOI: 10.1016/j.jpba.2026.117353
Xiaoyan Zhang , Shuang Feng , Ziluo Zhang , Chuya Wang , Yongshan Ai , Yalin Xi , Lei Yin , Meiyun Shi
This study established a robust and selective ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) method to investigate the excretion behavior of polyethylene glycol 1000 (PEG1K) oligomers (N = 13–26) in rats. PEG is widely used in pharmaceutical applications, particularly in drug delivery systems, yet detailed excretion profiles of lower molecular weight PEGs remain insufficiently characterized. The developed method utilized ammonium adducts ([M+NH₄]⁺) as precursor ions in multiple reaction monitoring mode, enabling sensitive and high-throughput quantification of individual oligomers in biological matrices. Following intravenous administration in rats, excretion kinetics were monitored over 48 h. Cumulative recovery analysis revealed that renal excretion was the dominant elimination pathway, accounting for 47.30–68.52 % of the administered dose in urine, while fecal excretion represented only 0.66–3.38 %. These findings provide critical insights into the in vivo disposition of PEG1K, supporting its safety evaluation and rational application in drug formulation. The presented analytical platform offers a reliable tool for polymer pharmacokinetic studies.
{"title":"Unraveling excretion behavior of polyethylene glycol 1000 with 13–26 subunits in rats by UHPLC-MS/MS","authors":"Xiaoyan Zhang , Shuang Feng , Ziluo Zhang , Chuya Wang , Yongshan Ai , Yalin Xi , Lei Yin , Meiyun Shi","doi":"10.1016/j.jpba.2026.117353","DOIUrl":"10.1016/j.jpba.2026.117353","url":null,"abstract":"<div><div>This study established a robust and selective ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) method to investigate the excretion behavior of polyethylene glycol 1000 (PEG1K) oligomers (N = 13–26) in rats. PEG is widely used in pharmaceutical applications, particularly in drug delivery systems, yet detailed excretion profiles of lower molecular weight PEGs remain insufficiently characterized. The developed method utilized ammonium adducts ([M+NH₄]⁺) as precursor ions in multiple reaction monitoring mode, enabling sensitive and high-throughput quantification of individual oligomers in biological matrices. Following intravenous administration in rats, excretion kinetics were monitored over 48 h. Cumulative recovery analysis revealed that renal excretion was the dominant elimination pathway, accounting for 47.30–68.52 % of the administered dose in urine, while fecal excretion represented only 0.66–3.38 %. These findings provide critical insights into the in vivo disposition of PEG1K, supporting its safety evaluation and rational application in drug formulation. The presented analytical platform offers a reliable tool for polymer pharmacokinetic studies.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"272 ","pages":"Article 117353"},"PeriodicalIF":3.1,"publicationDate":"2026-01-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145980444","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}
Extracellular vesicles (EV) are lipid-based nanoparticles naturally released by cells, exhibiting considerable heterogeneity in size, surface charge, and biomolecular composition. Recently, increasing attention has been directed toward the characterization of distinct EV-subpopulations, particularly based on unique surface antigen expression profiles. Therefore, a method for analyzing EV-subpopulations using versatile equipment would be highly valuable. In this study, we developed a labeling method for analyzing specific populations in small EVs (sEVs) distinguished by their levels phosphatidylserine (PS) exposure. For visualization, sEVs were labeled with two fusion proteins (enhanced green fluorescent protein [EGFP] linked to lactadherin [LA] and mCherry-Vn96) comprising a PS-binding protein or sEV-tropic peptide (Vn96) combined with fluorescent proteins. Using ultracentrifugation, bulk sEVs were collected, and a fraction of PS(−) sEVs (PS(+) sEV-depleted fraction) were isolated by depleting PS(+) sEVs from bulk sEVs. In bulk sEVs, the colocalization of EGFP-LA and mCherry-Vn96-derived signals was detected. In contrast, the PS(+) sEV-depleted fraction exhibited reduced EGFP-LA fluorescence signal, with only mCherry-Vn96 fluorescence remaining detectable. In conclusion, our labeling technique facilitates the identification and analysis of sEV-subpopulations using fluorescence microscopy in small sample volumes. This platform can also be adapted for broader applications by incorporating additional protein markers.
{"title":"Development of a simple labeling method using fluorescent protein fusion proteins targeting the membrane lipids of small extracellular vesicles","authors":"Yuki Kobayashi, Yuki Takahashi, Hiroto Otera, Yuriko Higuchi, Yoshinobu Takakura","doi":"10.1016/j.jpba.2026.117356","DOIUrl":"10.1016/j.jpba.2026.117356","url":null,"abstract":"<div><div>Extracellular vesicles (EV) are lipid-based nanoparticles naturally released by cells, exhibiting considerable heterogeneity in size, surface charge, and biomolecular composition. Recently, increasing attention has been directed toward the characterization of distinct EV-subpopulations, particularly based on unique surface antigen expression profiles. Therefore, a method for analyzing EV-subpopulations using versatile equipment would be highly valuable. In this study, we developed a labeling method for analyzing specific populations in small EVs (sEVs) distinguished by their levels phosphatidylserine (PS) exposure. For visualization, sEVs were labeled with two fusion proteins (enhanced green fluorescent protein [EGFP] linked to lactadherin [LA] and mCherry-Vn96) comprising a PS-binding protein or sEV-tropic peptide (Vn96) combined with fluorescent proteins. Using ultracentrifugation, bulk sEVs were collected, and a fraction of PS<sup>(−)</sup> sEVs (PS<sup>(+)</sup> sEV-depleted fraction) were isolated by depleting PS<sup>(+)</sup> sEVs from bulk sEVs. In bulk sEVs, the colocalization of EGFP-LA and mCherry-Vn96-derived signals was detected. In contrast, the PS<sup>(+)</sup> sEV-depleted fraction exhibited reduced EGFP-LA fluorescence signal, with only mCherry-Vn96 fluorescence remaining detectable. In conclusion, our labeling technique facilitates the identification and analysis of sEV-subpopulations using fluorescence microscopy in small sample volumes. This platform can also be adapted for broader applications by incorporating additional protein markers.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"272 ","pages":"Article 117356"},"PeriodicalIF":3.1,"publicationDate":"2026-01-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145981047","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 : 2026-01-12DOI: 10.1016/j.jpba.2026.117360
Wei Li , Yaqian Cheng , Tong Wu , Chenning Zhao , Qingsong Shao , Lanying Pan , Ying Zheng
Dysregulation of glucose and lipid metabolism has emerged as a leading cause of cardiovascular diseases worldwide. The current treatment for these diseases is often a combination of drugs. Despite the preference efficacy of combination therapies, adverse reactions have been frequently reported, such as gastrointestinal bleeding. Anoectochilus roxburghii (Wall.) Lindl., one of the traditional Chinese medicines, is recognized for its hypolipidemic and hypoglycemic effects. However, the exact therapeutic potential of A. roxburghii in glucose and lipid metabolism remains unsolved. By employing in vitro and in vivo experiments, this study evaluated the pharmacological activities of A. roxburghii and identified the primary active compounds involved in glucose and lipid metabolism. The results indicated that A. roxburghii can ameliorate dysregulation in glucose and lipid metabolism by promoting glycogen synthesis, and inhibiting gluconeogenesis and fatty acid oxidation. Using UHPLC-QE-MS, a total of 662 compounds were detected in the aqueous extract of A. roxburghii, and 769 were identified in the ethanol extract. Key ingredients such as morin and kaempferol participated in glucose metabolism, while kinsenoside and naringenin modulating lipid metabolism. Furthermore, A. roxburghii may potentially intervene in the insulin resistance signaling pathway by influencing TP53 protein expression, thereby modulating glucose metabolism process. This research provides evidence for the development and application of A. roxburghii as potential drugs in the treatment of glucose and lipid disordered diseases.
{"title":"Exploring the therapeutic potential of Anoectochilus roxburghii in glucose and lipid metabolism disorder: From phenotypic effects to molecular mechanisms and network pharmacology","authors":"Wei Li , Yaqian Cheng , Tong Wu , Chenning Zhao , Qingsong Shao , Lanying Pan , Ying Zheng","doi":"10.1016/j.jpba.2026.117360","DOIUrl":"10.1016/j.jpba.2026.117360","url":null,"abstract":"<div><div>Dysregulation of glucose and lipid metabolism has emerged as a leading cause of cardiovascular diseases worldwide. The current treatment for these diseases is often a combination of drugs. Despite the preference efficacy of combination therapies, adverse reactions have been frequently reported, such as gastrointestinal bleeding. <em>Anoectochilus roxburghii</em> (Wall.) Lindl<em>.</em>, one of the traditional Chinese medicines, is recognized for its hypolipidemic and hypoglycemic effects. However, the exact therapeutic potential of <em>A. roxburghii</em> in glucose and lipid metabolism remains unsolved. By employing <em>in vitro</em> and <em>in vivo</em> experiments, this study evaluated the pharmacological activities of <em>A. roxburghii</em> and identified the primary active compounds involved in glucose and lipid metabolism. The results indicated that <em>A. roxburghii</em> can ameliorate dysregulation in glucose and lipid metabolism by promoting glycogen synthesis, and inhibiting gluconeogenesis and fatty acid oxidation. Using UHPLC-QE-MS, a total of 662 compounds were detected in the aqueous extract of <em>A. roxburghii</em>, and 769 were identified in the ethanol extract. Key ingredients such as morin and kaempferol participated in glucose metabolism, while kinsenoside and naringenin modulating lipid metabolism. Furthermore, <em>A. roxburghii</em> may potentially intervene in the insulin resistance signaling pathway by influencing TP53 protein expression, thereby modulating glucose metabolism process. This research provides evidence for the development and application of <em>A. roxburghii</em> as potential drugs in the treatment of glucose and lipid disordered diseases.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"272 ","pages":"Article 117360"},"PeriodicalIF":3.1,"publicationDate":"2026-01-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145980443","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 : 2026-01-12DOI: 10.1016/j.jpba.2026.117359
Yuqing Tian , Pei Ma , Liang Wang , Bei Song , Jinghua Yang , Dan Qian , Wei Guo
Morus alba L. (M. alba) leaves, valued for their medicinal and edible properties, possess substantial economic and social importance. Notably, diverse germplasm resources of M. alba exhibit considerable variation in both metabolite composition and pharmacological activity. However, the interrelationships among genetic diversity, metabolites profiles, and pharmacological activity across different M. alba varieties remain insufficiently elucidated. This study aimed to construct simple sequence repeat (SSR) and ultra-high performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS) fingerprints, together with antipyretic clustering diagrams, for 69 different varieties of M. alba leaves, and analyze the correlations among their genetic structures, metabolite profiles, and antipyretic activities. In this study, 69 M. alba leaf samples representing different varieties were collected, and their antipyretic effects were evaluated using a rat pharmacodynamic model. SSR fingerprints were generated with TP-M13 SSR molecular markers to screen core primer pairs for variety identification. Simultaneously, metabolite fingerprints were established using UPLC-Q-TOF/MS with common peaks calibrated and compounds qualitatively identified by mass spectrometry. Finally, multivariate chemometric approaches were applied to integrate SSR data and UPLC-Q-TOF/MS fingerprint profiles, and antipyretic activity results for clustering and correlation analysis. The dominant M. alba varieties, Yantai 792, Zhesang 3, 8036, Fengyuan 1, Laiwugusang, Xiajingusang, Gaoqing 792, Zhesang 1 and Heizhenzhu exhibited the strongest antipyretic activities. In contrast, Guiyou, Danbaisang, and Dashi showed weaker effects and were clustered into a single category in the molecule-metabolite-efficacy clustering diagram, thereby indicating the substantial influence of varieties differences on genetic diversity, metabolite composition, and pharmacological activity. Pearson correlation analysis further confirmed significant correlations among genetic components, metabolites, and pharmacological effects. In addition, a core marker set comprising 7 SSR loci was identified, together with 8 key chemical markers including cryptochlorogenic acid, neochlorogenic acid, isoquercitrin, rutin, nicotiflorin, astragalin, salicylic acid, and umbelliferone. These findings help identify the “molecule-metabolite” dual molecular markers with the bias of antipyretic effects for M. alba leaves varieties, and provide valuable insights for refining the medicinal value of M. alba leaves and promoting the utilization of high-value resources.
{"title":"Integrated dual-molecular marker analysis with SSR and UPLC-Q-TOF/MS fingerprints technology reveal the interrelation of the molecule-metabolite in Morus alba L. leaves","authors":"Yuqing Tian , Pei Ma , Liang Wang , Bei Song , Jinghua Yang , Dan Qian , Wei Guo","doi":"10.1016/j.jpba.2026.117359","DOIUrl":"10.1016/j.jpba.2026.117359","url":null,"abstract":"<div><div><em>Morus alba</em> L. (<em>M. alba</em>) leaves, valued for their medicinal and edible properties, possess substantial economic and social importance. Notably, diverse germplasm resources of <em>M. alba</em> exhibit considerable variation in both metabolite composition and pharmacological activity. However, the interrelationships among genetic diversity, metabolites profiles, and pharmacological activity across different <em>M. alba</em> varieties remain insufficiently elucidated. This study aimed to construct simple sequence repeat (SSR) and ultra-high performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS) fingerprints, together with antipyretic clustering diagrams, for 69 different varieties of <em>M. alba</em> leaves, and analyze the correlations among their genetic structures, metabolite profiles, and antipyretic activities. In this study, 69 <em>M. alba</em> leaf samples representing different varieties were collected, and their antipyretic effects were evaluated using a rat pharmacodynamic model. SSR fingerprints were generated with TP-M13 SSR molecular markers to screen core primer pairs for variety identification. Simultaneously, metabolite fingerprints were established using UPLC-Q-TOF/MS with common peaks calibrated and compounds qualitatively identified by mass spectrometry. Finally, multivariate chemometric approaches were applied to integrate SSR data and UPLC-Q-TOF/MS fingerprint profiles, and antipyretic activity results for clustering and correlation analysis. The dominant <em>M. alba</em> varieties, Yantai 792, Zhesang 3, 8036, Fengyuan 1, Laiwugusang, Xiajingusang, Gaoqing 792, Zhesang 1 and Heizhenzhu exhibited the strongest antipyretic activities. In contrast, Guiyou, Danbaisang, and Dashi showed weaker effects and were clustered into a single category in the molecule-metabolite-efficacy clustering diagram, thereby indicating the substantial influence of varieties differences on genetic diversity, metabolite composition, and pharmacological activity. Pearson correlation analysis further confirmed significant correlations among genetic components, metabolites, and pharmacological effects. In addition, a core marker set comprising 7 SSR loci was identified, together with 8 key chemical markers including cryptochlorogenic acid, neochlorogenic acid, isoquercitrin, rutin, nicotiflorin, astragalin, salicylic acid, and umbelliferone. These findings help identify the “molecule-metabolite” dual molecular markers with the bias of antipyretic effects for <em>M. alba</em> leaves varieties, and provide valuable insights for refining the medicinal value of <em>M. alba</em> leaves and promoting the utilization of high-value resources.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"272 ","pages":"Article 117359"},"PeriodicalIF":3.1,"publicationDate":"2026-01-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145981049","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 : 2026-01-12DOI: 10.1016/j.jpba.2026.117357
Enhui Ji , Yuan Liu , Yifan Tian , Limin Li , Pei Qun , Zhengming Yang , Chaoqin Ren , Yanfei Huang , Yongcang Zhang
Meconopsis integrifolia total flavonoids (MITF) have been identified as the hepatoprotective fraction of M. integrifolia, with its main components being flavonoid glycosides, along with lower levels of phenolic acids and alkaloids. Since flavonoid glycosides are poorly absorbed in vivo, metabolism serves as a crucial pathway for their biotransformation. However, the metabolism and material basis of pharmacological effects of MITF in vivo remain unknown. In this study, ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry was used to investigate the metabolic profiles of MITF. A total of 61 absorbed prototype compounds were identified, including 54 in feces (43 flavonoids, 8 phenolic acids, and 3 alkaloids), 8 in urine (2 flavonoids, 3 phenolic acids, 3 alkaloids), and 6 in plasma (2 flavonoids, 4 phenolic acids). Furthermore, 113 metabolites were characterized, including 64 in feces (38 flavonoids, 11 phenolic acids, 15 alkaloids), 46 in urine (17 flavonoids, 16 phenolic acids, 13 alkaloids), and 14 in plasma (5 flavonoids, 9 phenolic acids). The major metabolic pathways were dehydroxylation, hydroxylation, reduction, dehydrogenation, hydration, dehydration, methylation, sulfation, and glucuronidation. The results indicated that the biotransformation and absorption of flavonoids in vivo were limited. In contrast, phenolic acids and alkaloids were extensively absorbed. This research provides crucial insights into the metabolic fate of MITF in rats, thereby clarifying the pharmacologically active substances derived from M. integrifolia.
Data availability
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
综合Meconopsis integrifolia total flavonoids （MITF）是综合Meconopsis integrifolia的保肝成分，其主要成分为黄酮类苷，酚酸和生物碱含量较低。由于黄酮类苷在体内吸收较差，代谢是其生物转化的重要途径。然而，MITF在体内的代谢和药理作用的物质基础尚不清楚。本研究采用超高效液相色谱联用三重四极杆质谱法研究了MITF的代谢谱。共鉴定出61种吸收原型化合物，其中粪便54种（43种黄酮类化合物、8种酚酸类化合物和3种生物碱）、尿液8种（2种黄酮类化合物、3种酚酸类化合物、3种生物碱）和血浆6种（2种黄酮类化合物、4种酚酸类化合物）。共鉴定出113种代谢物，其中粪便64种（38种黄酮类化合物，11种酚酸，15种生物碱），尿液46种（17种黄酮类化合物，16种酚酸，13种生物碱），血浆14种（5种黄酮类化合物，9种酚酸）。主要代谢途径为去羟基化、羟基化、还原、脱氢、水化、脱水、甲基化、磺化和葡萄糖醛酸化。结果表明，黄酮类化合物在体内的生物转化和吸收是有限的。相反，酚酸和生物碱被广泛吸收。本研究为了解MITF在大鼠体内的代谢命运提供了重要的见解，从而阐明了来自整合芽孢杆菌的药理活性物质。数据可获得性支持本研究结果的数据可根据通讯作者的要求提供。由于隐私或道德限制，这些数据不会公开。
{"title":"Identification of metabolites from the Himalayan herbal medicine Meconopsis integrifolia total flavonoids fraction in rats using UPLC-Q-exactive orbitrap-MS analysis","authors":"Enhui Ji , Yuan Liu , Yifan Tian , Limin Li , Pei Qun , Zhengming Yang , Chaoqin Ren , Yanfei Huang , Yongcang Zhang","doi":"10.1016/j.jpba.2026.117357","DOIUrl":"10.1016/j.jpba.2026.117357","url":null,"abstract":"<div><div><em>Meconopsis integrifolia</em> total flavonoids (MITF) have been identified as the hepatoprotective fraction of <em>M. integrifolia</em>, with its main components being flavonoid glycosides, along with lower levels of phenolic acids and alkaloids. Since flavonoid glycosides are poorly absorbed <em>in vivo</em>, metabolism serves as a crucial pathway for their biotransformation. However, the metabolism and material basis of pharmacological effects of MITF <em>in vivo</em> remain unknown. In this study, ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry was used to investigate the metabolic profiles of MITF. A total of 61 absorbed prototype compounds were identified, including 54 in feces (43 flavonoids, 8 phenolic acids, and 3 alkaloids), 8 in urine (2 flavonoids, 3 phenolic acids, 3 alkaloids), and 6 in plasma (2 flavonoids, 4 phenolic acids). Furthermore, 113 metabolites were characterized, including 64 in feces (38 flavonoids, 11 phenolic acids, 15 alkaloids), 46 in urine (17 flavonoids, 16 phenolic acids, 13 alkaloids), and 14 in plasma (5 flavonoids, 9 phenolic acids). The major metabolic pathways were dehydroxylation, hydroxylation, reduction, dehydrogenation, hydration, dehydration, methylation, sulfation, and glucuronidation. The results indicated that the biotransformation and absorption of flavonoids <em>in vivo</em> were limited. In contrast, phenolic acids and alkaloids were extensively absorbed. This research provides crucial insights into the metabolic fate of MITF in rats, thereby clarifying the pharmacologically active substances derived from <em>M. integrifolia</em>.</div></div><div><h3>Data availability</h3><div>The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"272 ","pages":"Article 117357"},"PeriodicalIF":3.1,"publicationDate":"2026-01-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145980445","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 : 2026-01-09DOI: 10.1016/j.jpba.2026.117337
Marisa H Maria, Nuno R Neng, Thomas Berg
The use of alcohol, legal and illicit substances poses great negative consequences on health and economy worldwide. LC-MS/MS allow simultaneous determination of multiple compounds in biological matrices. The aim of this study was to develop a LC-MS/MS method for the determination of the alcohol biomarker phosphatidylethanol (PEth) - including three homologues (PEth 16:0/18:1, PEth 16:0/18:2, PEth 18:0/18:1) - cocaine and three metabolites, and 8 other drugs in whole blood. Whole blood in K2EDTA tubes was prepared by liquid-liquid extraction using heptane/ethyl acetate/2-propanol (16:64:20, v:v:v). Chromatographic separation was achieved on an Acquity BEH C18 column (50 × 2.1 mm I.D., 1.7 µm particles). Mobile phase was 0.025 % ammonia, pH 10.7 (Solvent A) and methanol (Solvent B). The method was fully validated with isotope-labelled internal standards for 10 compounds. Inter-assay precision and accuracy were within ± 16 % for all analytes at five to seven tested concentrations. Recovery was within 42-79 % for 14 compounds and 11 % for benzoylecgonine. Matrix effects were within ± 25 % for most analytes. Internal standards compensated for matrix effects for compounds that had their own internal standards. A robust, precise, and accurate LC-MS/MS method for the determinations of three PEth homologues and 12 drugs and metabolites was, developed and validated. The method is valuable, especially for detecting polydrug use and alcohol consumption. To the best of our knowledge, this is the first LC-MS/MS method for the simultaneous determination of three PEth homologues and different drugs and metabolites.
{"title":"Simultaneous determination of three phosphatidylethanol homologues, 12 drugs and metabolites in whole blood by LC-MS/MS.","authors":"Marisa H Maria, Nuno R Neng, Thomas Berg","doi":"10.1016/j.jpba.2026.117337","DOIUrl":"https://doi.org/10.1016/j.jpba.2026.117337","url":null,"abstract":"<p><p>The use of alcohol, legal and illicit substances poses great negative consequences on health and economy worldwide. LC-MS/MS allow simultaneous determination of multiple compounds in biological matrices. The aim of this study was to develop a LC-MS/MS method for the determination of the alcohol biomarker phosphatidylethanol (PEth) - including three homologues (PEth 16:0/18:1, PEth 16:0/18:2, PEth 18:0/18:1) - cocaine and three metabolites, and 8 other drugs in whole blood. Whole blood in K2EDTA tubes was prepared by liquid-liquid extraction using heptane/ethyl acetate/2-propanol (16:64:20, v:v:v). Chromatographic separation was achieved on an Acquity BEH C<sub>18</sub> column (50 × 2.1 mm I.D., 1.7 µm particles). Mobile phase was 0.025 % ammonia, pH 10.7 (Solvent A) and methanol (Solvent B). The method was fully validated with isotope-labelled internal standards for 10 compounds. Inter-assay precision and accuracy were within ± 16 % for all analytes at five to seven tested concentrations. Recovery was within 42-79 % for 14 compounds and 11 % for benzoylecgonine. Matrix effects were within ± 25 % for most analytes. Internal standards compensated for matrix effects for compounds that had their own internal standards. A robust, precise, and accurate LC-MS/MS method for the determinations of three PEth homologues and 12 drugs and metabolites was, developed and validated. The method is valuable, especially for detecting polydrug use and alcohol consumption. To the best of our knowledge, this is the first LC-MS/MS method for the simultaneous determination of three PEth homologues and different drugs and metabolites.</p>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"117337"},"PeriodicalIF":3.1,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146180306","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 : 2026-01-09DOI: 10.1016/j.jpba.2026.117349
Ying-Shan Li , Xing-Ting Dong , Gui-Zhong Xin , Li-Fang Liu , Zi-Qi Shi
The precise differentiation of isomers is crucial for comprehensive mass spectrometry-based analysis of complex Chinese medicinal formulas (CMFs). To address this challenge, a standard-free strategy was proposed by integrating chromatographic behavior, mass spectrometric behavior, and quantum chemical calculations. Firstly, structural isomers and certain stereoisomers were differentiated based on characteristic fragment ions and calculated partition coefficient (clogP). For remaining indistinguishable isomers, fragment ions with significant intensity differences were identified, and 3D molecular structures of the precursor ions, product ions, and neutral loss fragments were constructed. The bond dissociation enthalpies (BDE) for critical fragmentation pathways were computed using density functional theory (DFT) and correlated with relative fragment ion abundances to achieve differentiation, following the principle that higher cleavage energies correspond to reduced fragmentation efficiency and lower product ion intensities. The methodology was validated using limited standards and the optimal collision energy (OCE) fitting. Using Qiangli Pipa syrup as a case, this integrated approach enabled the identification of 203 compounds, including 40 isomer groups comprising 27 structural isomers and 13 stereoisomers. This strategy overcomes the limitations of conventional methods that are typically restricted to specific structural classes, demonstrating robust capability for systematic isomer differentiation in CMFs.
{"title":"A standard-free strategy for the differentiation of isomers and deep profiling of chemicalome in Chinese medicinal formulas","authors":"Ying-Shan Li , Xing-Ting Dong , Gui-Zhong Xin , Li-Fang Liu , Zi-Qi Shi","doi":"10.1016/j.jpba.2026.117349","DOIUrl":"10.1016/j.jpba.2026.117349","url":null,"abstract":"<div><div>The precise differentiation of isomers is crucial for comprehensive mass spectrometry-based analysis of complex Chinese medicinal formulas (CMFs). To address this challenge, a standard-free strategy was proposed by integrating chromatographic behavior, mass spectrometric behavior, and quantum chemical calculations. Firstly, structural isomers and certain stereoisomers were differentiated based on characteristic fragment ions and calculated partition coefficient (<em>clogP</em>). For remaining indistinguishable isomers, fragment ions with significant intensity differences were identified, and 3D molecular structures of the precursor ions, product ions, and neutral loss fragments were constructed. The bond dissociation enthalpies (BDE) for critical fragmentation pathways were computed using density functional theory (DFT) and correlated with relative fragment ion abundances to achieve differentiation, following the principle that higher cleavage energies correspond to reduced fragmentation efficiency and lower product ion intensities. The methodology was validated using limited standards and the optimal collision energy (OCE) fitting. Using Qiangli Pipa syrup as a case, this integrated approach enabled the identification of 203 compounds, including 40 isomer groups comprising 27 structural isomers and 13 stereoisomers. This strategy overcomes the limitations of conventional methods that are typically restricted to specific structural classes, demonstrating robust capability for systematic isomer differentiation in CMFs.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"271 ","pages":"Article 117349"},"PeriodicalIF":3.1,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145977943","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}
The amounts of the serine (Ser), threonine (Thr) and allothreonine (aThr) enantiomers were determined in tissues (cerebrum, cerebellum, pancreas, liver and kidney) and physiological fluids (plasma and urine) of rats and mice with deficiency of d-amino acid oxidase (DAO). DAO is an enzyme metabolizing d-amino acids in mammals and has been implicated in the pathophysiology of several diseases via the alteration of d-amino acids. To determine trace levels of the amino acid enantiomers, a three-dimensional (3D) HPLC system composed of reversed-phase, anion-exchange and chiral separations was designed and utilized. Prior to the 3D-HPLC analysis, the analytes were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole to enhance the fluorescence detection sensitivity. By using the 3D-HPLC system, the tissues and physiological fluids of F344-Daoldao rats and B6DAO-/- mice (animals with the DAO deficiency) were analyzed. In both species, d-Ser levels were elevated in the absence of DAO activity except for the cerebrum. The amounts of d-Thr and d-aThr were increased in the cerebellum and kidney with the DAO deficiency while their amounts were almost the same in the other tissues and physiological fluids. These results indicated that the intrinsic d-Ser analogues were metabolized by DAO in mammals and further studies to clarify its physiological significance are expected.
{"title":"Three-dimensional high-performance liquid chromatographic determination of serine, threonine and allothreonine enantiomers in the d-amino acid oxidase deficient mice and rats.","authors":"Mai Oyaide, Takeyuki Akita, Chiharu Ishii, Yukiko Shimizu, Masashi Mita, Ryuichi Konno, Tadashi Okamura, Kenji Hamase","doi":"10.1016/j.jpba.2026.117348","DOIUrl":"https://doi.org/10.1016/j.jpba.2026.117348","url":null,"abstract":"<p><p>The amounts of the serine (Ser), threonine (Thr) and allothreonine (aThr) enantiomers were determined in tissues (cerebrum, cerebellum, pancreas, liver and kidney) and physiological fluids (plasma and urine) of rats and mice with deficiency of d-amino acid oxidase (DAO). DAO is an enzyme metabolizing d-amino acids in mammals and has been implicated in the pathophysiology of several diseases via the alteration of d-amino acids. To determine trace levels of the amino acid enantiomers, a three-dimensional (3D) HPLC system composed of reversed-phase, anion-exchange and chiral separations was designed and utilized. Prior to the 3D-HPLC analysis, the analytes were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole to enhance the fluorescence detection sensitivity. By using the 3D-HPLC system, the tissues and physiological fluids of F344-Dao<sup>ldao</sup> rats and B6DAO<sup>-/-</sup> mice (animals with the DAO deficiency) were analyzed. In both species, d-Ser levels were elevated in the absence of DAO activity except for the cerebrum. The amounts of d-Thr and d-aThr were increased in the cerebellum and kidney with the DAO deficiency while their amounts were almost the same in the other tissues and physiological fluids. These results indicated that the intrinsic d-Ser analogues were metabolized by DAO in mammals and further studies to clarify its physiological significance are expected.</p>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"117348"},"PeriodicalIF":3.1,"publicationDate":"2026-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146142845","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 : 2026-01-08DOI: 10.1016/j.jpba.2026.117350
Dominik Kresnik, Bryan Bajor, Christian Steuer
The rising interest in essential oils (EOs) for their therapeutic, antibacterial, and antifungal properties has led to an increasing demand for high-quality products in both medicinal and industrial sectors. To meet these stringent quality requirements, robust, precise, and efficient analytical methods are essential. Gas chromatography (GC) remains the gold standard for EO analysis due to its sensitivity and resolution. Although numerous methods are available –primarily targeting similar analytes in varying combinations standardization remains a challenge, with protocols differing across ISO guidelines and international pharmacopoeias. In this study, Design of Experiments (DoE) was employed to optimize and harmonize existing GC methods, focusing on sample preparation and chromatographic parameters. A polar GC column (60 m length, 0.25 mm inner diameter, 0.25 µm film thickness) was selected for its ability to effectively separate 87 terpenes, sesquiterpenes, and related compounds commonly found in EOs. The optimized temperature gradient enabled complete separation within a 75-minute runtime, outperforming or matching existing methods in terms of resolution and reproducibility. Streamlined sample preparation protocols led to reduced solvent consumption and minimized sample requirements across all tested EOs. As a proof of concept, the final method was applied to 12 different essential oils, demonstrating comparable analytical performance and confirming its broad applicability and efficiency.
由于精油具有治疗、抗菌和抗真菌的特性，人们对精油的兴趣日益浓厚，这导致医药和工业部门对高质量产品的需求不断增加。为了满足这些严格的质量要求，稳健、精确和高效的分析方法是必不可少的。气相色谱法（GC）因其灵敏度和分辨率而成为EO分析的金标准。尽管有许多方法可用，但主要针对不同组合的类似分析物，标准化仍然是一个挑战，ISO指南和国际药典的协议不同。本研究采用实验设计（Design of Experiments, DoE）对现有的气相色谱方法进行优化和协调，重点关注样品制备和色谱参数。极性气相色谱柱（60 m长，0.25 mm内径，0.25 µm膜厚）能够有效分离EOs中常见的87种萜类、倍半萜类及相关化合物。优化后的温度梯度可以在75分钟内完成分离，在分辨率和重现性方面优于或匹配现有的方法。简化的样品制备方案减少了溶剂消耗，并将所有测试EOs的样品需求降至最低。作为概念验证，最后的方法被应用于12种不同的精油，展示了可比的分析性能，并证实了其广泛的适用性和效率。
{"title":"Ease of analysis through unification: One gas chromatographic method for the chemical profiling of essential oils","authors":"Dominik Kresnik, Bryan Bajor, Christian Steuer","doi":"10.1016/j.jpba.2026.117350","DOIUrl":"10.1016/j.jpba.2026.117350","url":null,"abstract":"<div><div>The rising interest in essential oils (EOs) for their therapeutic, antibacterial, and antifungal properties has led to an increasing demand for high-quality products in both medicinal and industrial sectors. To meet these stringent quality requirements, robust, precise, and efficient analytical methods are essential. Gas chromatography (GC) remains the gold standard for EO analysis due to its sensitivity and resolution. Although numerous methods are available –primarily targeting similar analytes in varying combinations standardization remains a challenge, with protocols differing across ISO guidelines and international pharmacopoeias. In this study, Design of Experiments (DoE) was employed to optimize and harmonize existing GC methods, focusing on sample preparation and chromatographic parameters. A polar GC column (60 m length, 0.25 mm inner diameter, 0.25 µm film thickness) was selected for its ability to effectively separate 87 terpenes, sesquiterpenes, and related compounds commonly found in EOs. The optimized temperature gradient enabled complete separation within a 75-minute runtime, outperforming or matching existing methods in terms of resolution and reproducibility. Streamlined sample preparation protocols led to reduced solvent consumption and minimized sample requirements across all tested EOs. As a proof of concept, the final method was applied to 12 different essential oils, demonstrating comparable analytical performance and confirming its broad applicability and efficiency.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"272 ","pages":"Article 117350"},"PeriodicalIF":3.1,"publicationDate":"2026-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145981048","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 : 2026-01-07DOI: 10.1016/j.jpba.2026.117347
Xin Huang , Jingyu Chen , Xinyu He
Cells of breast cancer (BC) can metastasize to lymph nodes or other organs and is the high leading cause of female cancer deaths. Efforts to improve the performance of early detection and precise treatment are urgent and important for BC management. In clinical application, compared with highly complex classification functions, precise, simple and biologically interpretable algorithms can strengthen the understanding of disease development and facilitate the personalization of therapeutic strategies. In this study, a novel readily interpretable decision rule involving multiple forms of molecular relationship (RI-MFR) was proposed for cancer clinical management applications. In RI-MFR, linear and nonlinear pairwise molecule comparisons were comprehensively analyzed by a joint probability mass function for the identification of top scoring pairs from high dimensional biomolecular data. Based on the selected few molecule pairs, accurate, readily interpretable decision rules were inferred to provide biological insight as to how classification was performed. RI-MFR can effectively eliminate the influence of sample variability caused by individual differences. RI-MFR was successfully employed to analyze changes in metabolic mechanisms during BC development based on genomics datasets and metabolic alterations before and after BC therapy using our metabolomic profiling. The experimental results indicated that genes and metabolites involving in the glycosphingolipid metabolism may be the crucial factors associated with BC development and contribute to the enhanced effectiveness of BC treatment. Compared with other algorithms, RI-MFR had the significantly better classification results with p-values < 0.05, which suggested it is a more useful tool to identify important bioinformation for clinical BC management.
{"title":"A readily interpretable rule involving multiple forms of pairwise molecule comparisons with applications for clinical make-decision of breast cancer management","authors":"Xin Huang , Jingyu Chen , Xinyu He","doi":"10.1016/j.jpba.2026.117347","DOIUrl":"10.1016/j.jpba.2026.117347","url":null,"abstract":"<div><div>Cells of breast cancer (BC) can metastasize to lymph nodes or other organs and is the high leading cause of female cancer deaths. Efforts to improve the performance of early detection and precise treatment are urgent and important for BC management. In clinical application, compared with highly complex classification functions, precise, simple and biologically interpretable algorithms can strengthen the understanding of disease development and facilitate the personalization of therapeutic strategies. In this study, a novel readily interpretable decision rule involving multiple forms of molecular relationship (RI-MFR) was proposed for cancer clinical management applications. In RI-MFR, linear and nonlinear pairwise molecule comparisons were comprehensively analyzed by a joint probability mass function for the identification of top scoring pairs from high dimensional biomolecular data. Based on the selected few molecule pairs, accurate, readily interpretable decision rules were inferred to provide biological insight as to how classification was performed. RI-MFR can effectively eliminate the influence of sample variability caused by individual differences. RI-MFR was successfully employed to analyze changes in metabolic mechanisms during BC development based on genomics datasets and metabolic alterations before and after BC therapy using our metabolomic profiling. The experimental results indicated that genes and metabolites involving in the glycosphingolipid metabolism may be the crucial factors associated with BC development and contribute to the enhanced effectiveness of BC treatment. Compared with other algorithms, RI-MFR had the significantly better classification results with <em>p</em>-values < 0.05, which suggested it is a more useful tool to identify important bioinformation for clinical BC management.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"271 ","pages":"Article 117347"},"PeriodicalIF":3.1,"publicationDate":"2026-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145926877","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}
扫码关注我们
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号