Pub Date : 2026-06-15Epub Date: 2026-02-12DOI: 10.1016/j.jpba.2026.117406
Jiaxi Fang , Luping Wang , Di Zhang , Yan Liang , Shouxin Li , Jingkui Tian , Qiang He , Juan Jin , Wei Zhu
{"title":"Corrigendum to “Integrative analysis of transcriptome and metabolome provide new insights into mechanisms of capilliposide A against cisplatin-induced nephrotoxicity” [J. Pharm. Biomed. Anal. 238 (2024), 115814]","authors":"Jiaxi Fang , Luping Wang , Di Zhang , Yan Liang , Shouxin Li , Jingkui Tian , Qiang He , Juan Jin , Wei Zhu","doi":"10.1016/j.jpba.2026.117406","DOIUrl":"10.1016/j.jpba.2026.117406","url":null,"abstract":"","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117406"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146192142","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: 2026-01-30DOI: 10.1016/j.jpba.2026.117386
Michalina McDermott , Zachary Sargeant , Christopher E. Karlsen , Feng Li , R. Andrew Shalliker , Jake A. Cravino
The demand for rapid and reliable analytical methods in the pharmaceutical industry continues to grow, with Ultra-/High-Performance Liquid Chromatography (U/HPLC) remaining the gold standard for impurity profiling, quantification of active pharmaceutical ingredients, and degradation product analysis. However, traditional HPLC methods are often constrained by pressure limitations at higher flow rates, which can hinder analytical throughput. While recent advancements in column technology have improved performance, they typically exacerbate pressure-related challenges. In this study, we evaluate a novel column technology designed to address these limitations by enabling high-resolution separations at reduced pressures and increased flow rates. Our findings demonstrate that the column, when operated in Radial Flow Stream Splitting (RFS) mode, maintains quantitative accuracy and repeatability while achieving up to a 120 % improvement in separation efficiency and a 30 % reduction in backpressure compared to conventional operation. By way of assaying over-the-counter medication, we have found no difference in the quantitative reliability of the assay when in RFS vs stock mode, despite reducing the analysis time by up to 40 %.
{"title":"Cost-effective routine pharmaceutical testing using radial flow stream splitting HPLC columns: Quantitative analysis and performance metrics in the analysis of over-the-counter drugs","authors":"Michalina McDermott , Zachary Sargeant , Christopher E. Karlsen , Feng Li , R. Andrew Shalliker , Jake A. Cravino","doi":"10.1016/j.jpba.2026.117386","DOIUrl":"10.1016/j.jpba.2026.117386","url":null,"abstract":"<div><div>The demand for rapid and reliable analytical methods in the pharmaceutical industry continues to grow, with Ultra-/High-Performance Liquid Chromatography (U/HPLC) remaining the gold standard for impurity profiling, quantification of active pharmaceutical ingredients, and degradation product analysis. However, traditional HPLC methods are often constrained by pressure limitations at higher flow rates, which can hinder analytical throughput. While recent advancements in column technology have improved performance, they typically exacerbate pressure-related challenges. In this study, we evaluate a novel column technology designed to address these limitations by enabling high-resolution separations at reduced pressures and increased flow rates. Our findings demonstrate that the column, when operated in Radial Flow Stream Splitting (RFS) mode, maintains quantitative accuracy and repeatability while achieving up to a 120 % improvement in separation efficiency and a 30 % reduction in backpressure compared to conventional operation. By way of assaying over-the-counter medication, we have found no difference in the quantitative reliability of the assay when in RFS vs stock mode, despite reducing the analysis time by up to 40 %.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117386"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146119296","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: 2026-02-04DOI: 10.1016/j.jpba.2026.117391
Yi Jin , Yaning Li , Haoran Yang , Huiying Yang , Yaqing Guo , Xianfu Wu
Sevoflurane is an inhalation anesthetic, which is widely favored by anesthesiologists in modern clinical practice. It is reported that sevoflurane readily degrades under certain conditions. Forced degradation studies are essential for elucidating its degradation profile under stressed conditions, as recommended by the ICH guidelines. This study systematically assessed the impact of temperature, light, oxidation, and hydrolysis on sevoflurane stability through forced degradation studies. The results indicated that temperature was identified as the primary trigger for degradation, leading to the formation of four degradation products (DPs) (including the pharmacopeial-known Impurity C and three novel sevoflurane degradants H, I, and J) at 60℃ and 95℃. Light, oxidation, and hydrolysis had no significant effect under the tested conditions. The structures of all DPs were confirmed using nuclear magnetic resonance spectroscopy (NMR). Capitalizing on the inherent quantitative proficiency of this technique, we subsequently employed quantitative proton nuclear magnetic resonance spectroscopy (qHNMR) to determine the relative contents of these four DPs, which can further elucidate the degradation profile of sevoflurane. In silico toxicity and metabolic behavior of them were assessed by Derek Nexus and Meteor Nexus software, respectively. DPs H-J exhibited potential skin irritation/corrosion and sensitization effects across species, potentially attributable to alkyl aldehyde functional groups. All of our efforts are expected to provide guidance for the quality control and optimal storage of sevoflurane.
{"title":"Temperature as the primary risk factor for sevoflurane degradation: Identification and toxicological risk assessment of novel degradants by nuclear magnetic resonance (NMR) spectroscopy","authors":"Yi Jin , Yaning Li , Haoran Yang , Huiying Yang , Yaqing Guo , Xianfu Wu","doi":"10.1016/j.jpba.2026.117391","DOIUrl":"10.1016/j.jpba.2026.117391","url":null,"abstract":"<div><div>Sevoflurane is an inhalation anesthetic, which is widely favored by anesthesiologists in modern clinical practice. It is reported that sevoflurane readily degrades under certain conditions. Forced degradation studies are essential for elucidating its degradation profile under stressed conditions, as recommended by the ICH guidelines. This study systematically assessed the impact of temperature, light, oxidation, and hydrolysis on sevoflurane stability through forced degradation studies. The results indicated that temperature was identified as the primary trigger for degradation, leading to the formation of four degradation products (DPs) (including the pharmacopeial-known Impurity C and three novel sevoflurane degradants H, I, and J) at 60℃ and 95℃. Light, oxidation, and hydrolysis had no significant effect under the tested conditions. The structures of all DPs were confirmed using nuclear magnetic resonance spectroscopy (NMR). Capitalizing on the inherent quantitative proficiency of this technique, we subsequently employed quantitative proton nuclear magnetic resonance spectroscopy (qHNMR) to determine the relative contents of these four DPs, which can further elucidate the degradation profile of sevoflurane. <em>In silico</em> toxicity and metabolic behavior of them were assessed by Derek Nexus and Meteor Nexus software, respectively. DPs H-J exhibited potential skin irritation/corrosion and sensitization effects across species, potentially attributable to alkyl aldehyde functional groups. All of our efforts are expected to provide guidance for the quality control and optimal storage of sevoflurane.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117391"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146157363","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: 2026-01-09DOI: 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":"10.1016/j.jpba.2026.117337","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117337"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","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}
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":"10.1016/j.jpba.2026.117348","url":null,"abstract":"<div><div>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 <span>d</span>-amino acid oxidase (DAO). DAO is an enzyme metabolizing <span>d</span>-amino acids in mammals and has been implicated in the pathophysiology of several diseases <em>via</em> the alteration of <span>d</span>-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-<em>Dao</em><sup><em>ldao</em></sup> rats and B6DAO<sup>-/-</sup> mice (animals with the DAO deficiency) were analyzed. In both species, <span>d</span>-Ser levels were elevated in the absence of DAO activity except for the cerebrum. The amounts of <span>d</span>-Thr and <span>d</span>-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 <span>d</span>-Ser analogues were metabolized by DAO in mammals and further studies to clarify its physiological significance are expected.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117348"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","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 : 2026-06-15Epub Date: 2026-02-04DOI: 10.1016/j.jpba.2026.117395
{"title":"In memoriam of Professor Sándor Görög","authors":"","doi":"10.1016/j.jpba.2026.117395","DOIUrl":"10.1016/j.jpba.2026.117395","url":null,"abstract":"","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117395"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146157390","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.2026.117390
Hanjing Chen , Jiali Li , Fei Yuan , Gongxin He , Hao Wu , Hua Yan , Hongrong Xu , Chao Liu , Lei Sheng , Xuening Li
CG-0255, a thiol prodrug of clopidogrel’s active metabolite H4 (CG-0236), is a novel thienopyridine P2Y12 receptor antagonist under initial clinical development for the treatment of acute coronary syndromes. Unlike clopidogrel, CG-0255 is converted to the active thiol metabolite H4 (CG-0236) in a single hydrolytic step. Compared with clopidogrel, CG-0255 exhibits more efficient and consistent H4 formation in humans, which can be quantified in plasma following either intravenous or oral administration. In this study, we developed and validated a sensitive, rapid, and robust UHPLC–MS/MS method for the simultaneous quantification of CG-0255 and its derivatized active metabolite (MP-H4, CG-0261) in human plasma. After solid-phase extraction from 94.5 μL of plasma, analytes and isotope-labeled internal standards were separated on an ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) using isocratic elution with 0.1 % formic acid in water and acetonitrile (57:43, v/v) at a flow rate of 0.5 mL/min, followed by a 3.5 min column washing and re-equilibration, giving a total analytical run time of 7 min. Baseline separation of CG-0255, CG-0261, and their respective isomers was achieved. Detection was performed using positive electrospray ionization in multiple reaction monitoring mode on a Q-Trap 6500+ mass spectrometer. Calibration curves were linear over 0.05–25 ng/mL for both analytes, corresponding to 0.0353–17.65 ng/mL for H4 (CG-0236). Intra- and inter-day precision and accuracy were within ±15 % at all quality-control levels. The validated assay was successfully applied to two phase I clinical studies conducted at our center, characterizing the pharmacokinetics of CG-0255 following single-dose intravenous and multiple-dose oral administration. This UHPLC–MS/MS method provides a reliable platform for the quantitative evaluation of CG-0255 and its active metabolite in human plasma, and is well suited to support further global clinical development.
{"title":"Development and validation of a sensitive and rapid UHPLC-MS/MS method for the simultaneous quantification of CG-0255 and its active metabolite in human plasma and its application to Phase I studies","authors":"Hanjing Chen , Jiali Li , Fei Yuan , Gongxin He , Hao Wu , Hua Yan , Hongrong Xu , Chao Liu , Lei Sheng , Xuening Li","doi":"10.1016/j.jpba.2026.117390","DOIUrl":"10.1016/j.jpba.2026.117390","url":null,"abstract":"<div><div>CG-0255, a thiol prodrug of clopidogrel’s active metabolite H4 (CG-0236), is a novel thienopyridine P2Y12 receptor antagonist under initial clinical development for the treatment of acute coronary syndromes. Unlike clopidogrel, CG-0255 is converted to the active thiol metabolite H4 (CG-0236) in a single hydrolytic step. Compared with clopidogrel, CG-0255 exhibits more efficient and consistent H4 formation in humans, which can be quantified in plasma following either intravenous or oral administration. In this study, we developed and validated a sensitive, rapid, and robust UHPLC–MS/MS method for the simultaneous quantification of CG-0255 and its derivatized active metabolite (MP-H4, CG-0261) in human plasma. After solid-phase extraction from 94.5 μL of plasma, analytes and isotope-labeled internal standards were separated on an ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) using isocratic elution with 0.1 % formic acid in water and acetonitrile (57:43, v/v) at a flow rate of 0.5 mL/min, followed by a 3.5 min column washing and re-equilibration, giving a total analytical run time of 7 min. Baseline separation of CG-0255, CG-0261, and their respective isomers was achieved. Detection was performed using positive electrospray ionization in multiple reaction monitoring mode on a Q-Trap 6500<sup>+</sup> mass spectrometer. Calibration curves were linear over 0.05–25 ng/mL for both analytes, corresponding to 0.0353–17.65 ng/mL for H4 (CG-0236). Intra- and inter-day precision and accuracy were within ±15 % at all quality-control levels. The validated assay was successfully applied to two phase I clinical studies conducted at our center, characterizing the pharmacokinetics of CG-0255 following single-dose intravenous and multiple-dose oral administration. This UHPLC–MS/MS method provides a reliable platform for the quantitative evaluation of CG-0255 and its active metabolite in human plasma, and is well suited to support further global clinical development.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117390"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146132099","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: 2026-01-30DOI: 10.1016/j.jpba.2026.117379
Rachel Smith , Colin Guy , Rosie Upton , Sam Clawson , Henry Fisher , Mohammad Adam Nasar , David Firth , Allan Watkinson
A streamlined early phase formulation development workflow has been developed for monoclonal antibodies (mAbs) to provide a more efficient process, driving down costs and reducing timelines, without compromising the quality and hence patient safety. The proposed novel workflow combines liquid chromatography-mass spectrometry multi-attribute method (LC-MS MAM) and sensitive ligand binding using surface plasmon resonance (SPR). By linking the two methodologies it is possible to obtain a comprehensive understanding of a mAb’s critical quality attributes (CQAs) and provide a structure/function correlation. As LC-MS MAM cannot address all aspects of degradation, high throughput methods for the analysis of high molecular weight material (HMWM), and conformational and colloidal stability, were also evaluated. The workflow comprises an initial forced degradation study, to verify stability-indication and identify potential degradation routes. Secondly, optimal pH, based on conformational and colloidal stability, is determined. Finally, stabilising excipients are evaluated by design of experiment (DoE). We have verified this workflow using pembrolizumab. In an initial forced degradation study, LC-MS MAM and PD-1 ligand binding could identify the CQAs. Met105 oxidation, located in the CDR3 region, was identified as the major CQA. DoE demonstrated that 25 mM methionine inhibited Met105 oxidation and stabilised PD-1 binding. With this streamlined process, we were able to improve the stability of the protein by formulating in 20 mM histidine, 25 mM methionine, 0.02 % PS80 and 300 mM sucrose, at pH 5.5. The described workflow has the potential to decrease the demand for precious early development material as well as reduce costs and shorten timelines.
{"title":"A streamlined workflow for early phase formulation development of monoclonal antibodies comprising multi-attribute method and ligand binding assay","authors":"Rachel Smith , Colin Guy , Rosie Upton , Sam Clawson , Henry Fisher , Mohammad Adam Nasar , David Firth , Allan Watkinson","doi":"10.1016/j.jpba.2026.117379","DOIUrl":"10.1016/j.jpba.2026.117379","url":null,"abstract":"<div><div>A streamlined early phase formulation development workflow has been developed for monoclonal antibodies (mAbs) to provide a more efficient process, driving down costs and reducing timelines, without compromising the quality and hence patient safety. The proposed novel workflow combines liquid chromatography-mass spectrometry multi-attribute method (LC-MS MAM) and sensitive ligand binding using surface plasmon resonance (SPR). By linking the two methodologies it is possible to obtain a comprehensive understanding of a mAb’s critical quality attributes (CQAs) and provide a structure/function correlation. As LC-MS MAM cannot address all aspects of degradation, high throughput methods for the analysis of high molecular weight material (HMWM), and conformational and colloidal stability, were also evaluated. The workflow comprises an initial forced degradation study, to verify stability-indication and identify potential degradation routes. Secondly, optimal pH, based on conformational and colloidal stability, is determined. Finally, stabilising excipients are evaluated by design of experiment (DoE). We have verified this workflow using pembrolizumab. In an initial forced degradation study, LC-MS MAM and PD-1 ligand binding could identify the CQAs. Met105 oxidation, located in the CDR3 region, was identified as the major CQA. DoE demonstrated that 25 mM methionine inhibited Met105 oxidation and stabilised PD-1 binding. With this streamlined process, we were able to improve the stability of the protein by formulating in 20 mM histidine, 25 mM methionine, 0.02 % PS80 and 300 mM sucrose, at pH 5.5. The described workflow has the potential to decrease the demand for precious early development material as well as reduce costs and shorten timelines.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117379"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146132126","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: 2026-02-01DOI: 10.1016/j.jpba.2026.117387
Yinuo Wu , Xiaoli Li , Haixia Feng , Yifan Chen , Wenwen Wu , Siqin Wang
Insomnia is increasingly recognized as a disorder with complex metabolic underpinnings. We investigated the changes in fecal metabolite levels of 5-hydroxyindoleacetic acid (5-HIAA), octopamine (OA), oleoylethanolamide (OEA), and elaidic acid (EA) in patients with sleep disorders, as well as their correlations with insomnia severity. Sixty participants were divided into two groups, with thirty patients with sleep disorders hospitalized in the Department of Neurology, Zhongda Hospital, Southeast University (October 2024–March 2025) and 30 healthy controls recruited during the same period. Fecal samples were collected from all participants, and metabolite levels were analyzed via untargeted metabolomics analysis using liquid chromatography-mass spectrometry (LC-MS). The Chinese version of the Insomnia Severity Index (C-ISI) was employed to evaluate insomnia severity, and the correlations between insomnia severity and these four metabolites were subjected to statistical analysis. Both univariate and multivariate analyses revealed significant metabolic differences between groups. The experimental group showed significantly lower levels of 5-HIAA (FC = 0.947, P = 0.020) and OA (FC = 0.953, P < 0.001), but higher OEA (FC = 1.101, P < 0.001) and EA (FC = 1.026, P < 0.001). C-ISI scores correlated negatively with 5-HIAA (r = -0.380, P = 0.003) and OA (r = -0.448, P < 0.001), and positively with OEA (r = 0.500, P < 0.001) and EA (r = 0.408, P = 0.001). These fecal metabolites associate with insomnia severity and may serve as potential biomarkers for understanding its pathophysiology and developing interventions.
人们越来越认识到失眠是一种复杂代谢基础的疾病。我们研究了睡眠障碍患者粪便代谢物5-羟基吲哚乙酸（5-HIAA）、章鱼胺（OA）、油基乙醇酰胺（OEA）和elaidic酸（EA）水平的变化及其与失眠严重程度的相关性。60名受试者分为两组，选取东南大学中大医院神经内科住院的睡眠障碍患者30例（2024年10月- 2025年3月）和同期招募的健康对照30例。收集所有参与者的粪便样本，并通过液相色谱-质谱（LC-MS）非靶向代谢组学分析分析代谢物水平。采用中文版失眠症严重程度指数（C-ISI）评估失眠严重程度，并对失眠严重程度与上述四种代谢物的相关性进行统计分析。单因素和多因素分析均显示各组之间的代谢差异显著。实验组5-HIAA （FC = 0.947, P = 0.020）和OA （FC = 0.953, P . 0.05）水平显著低于对照组
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Pub Date : 2026-06-15Epub Date: 2026-01-30DOI: 10.1016/j.jpba.2026.117384
Mohamed A. Gab-Allah , Hyojin Hwang , Mingyu Kim , Ngoc-Trinh Tran , Bong Jik Kim , Minyoung Kim , Jin Hee Han , Yehree Kim , Byung Yoon Choi , Jeongkwon Kim
Accurate detection of point mutations in mitochondrial DNA (mtDNA) is crucial for diagnosing various mitochondrial disorders. In this study, we developed an ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC–HRMS/MS) method for the direct, label-free identification and localization of single-nucleotide mutations using synthetic 20- and 49-mer oligonucleotides as model fragments representing the pathogenic mtDNA point mutation (mt.3243 A>G). Three mobile phase systems, including ammonium bicarbonate (ABC), triethylamine/hexafluoroisopropanol (TEA/HFIP), and tributylamine/HFIP (TBA/HFIP), were systematically evaluated to assess their effects on oligonucleotide retention behavior and duplex stability under denaturing and non-denaturing conditions. The ABC buffer provided optimal performance for maintaining partial duplex integrity, while TEA/HFIP offered superior ionization efficiency for single-stranded analysis. Deconvoluted mass spectra revealed accurate monoisotopic mass differences between wild-type and mutant oligonucleotides, including ∼ + 16 Da for the sense strand (A>G), ∼ –15 Da for the antisense strand (T > C), and ∼ + 1 Da for the duplex, enabling confident mutation discrimination at the intact molecular level. High-resolution MS achieved excellent mass accuracy within ±3 ppm, and high-energy collision dissociation (HCD) MS/MS enabled sequence-specific fragmentation that localized the mutation site with high confidence when compared with theoretical fragments. Overall, this study establishes a reliable analytical framework for mutation detection in oligonucleotide models and highlights the potential of UHPLC–HRMS/MS as a complementary tool for targeted mtDNA fragment analysis.
{"title":"Detection and localization of single-nucleotide mutations in synthetic oligonucleotides by ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry","authors":"Mohamed A. Gab-Allah , Hyojin Hwang , Mingyu Kim , Ngoc-Trinh Tran , Bong Jik Kim , Minyoung Kim , Jin Hee Han , Yehree Kim , Byung Yoon Choi , Jeongkwon Kim","doi":"10.1016/j.jpba.2026.117384","DOIUrl":"10.1016/j.jpba.2026.117384","url":null,"abstract":"<div><div>Accurate detection of point mutations in mitochondrial DNA (mtDNA) is crucial for diagnosing various mitochondrial disorders. In this study, we developed an ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC–HRMS/MS) method for the direct, label-free identification and localization of single-nucleotide mutations using synthetic 20- and 49-mer oligonucleotides as model fragments representing the pathogenic mtDNA point mutation (mt.3243 A>G). Three mobile phase systems, including ammonium bicarbonate (ABC), triethylamine/hexafluoroisopropanol (TEA/HFIP), and tributylamine/HFIP (TBA/HFIP), were systematically evaluated to assess their effects on oligonucleotide retention behavior and duplex stability under denaturing and non-denaturing conditions. The ABC buffer provided optimal performance for maintaining partial duplex integrity, while TEA/HFIP offered superior ionization efficiency for single-stranded analysis. Deconvoluted mass spectra revealed accurate monoisotopic mass differences between wild-type and mutant oligonucleotides, including ∼ + 16 Da for the sense strand (A>G), ∼ –15 Da for the antisense strand (T > C), and ∼ + 1 Da for the duplex, enabling confident mutation discrimination at the intact molecular level. High-resolution MS achieved excellent mass accuracy within ±3 ppm, and high-energy collision dissociation (HCD) MS/MS enabled sequence-specific fragmentation that localized the mutation site with high confidence when compared with theoretical fragments. Overall, this study establishes a reliable analytical framework for mutation detection in oligonucleotide models and highlights the potential of UHPLC–HRMS/MS as a complementary tool for targeted mtDNA fragment analysis.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117384"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146119301","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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