Journal of pharmaceutical and biomedical analysis最新文献

{"title":"Microflow LC-MS/MS reveals platform-specific post-translational modification signatures in recombinant adeno-associated virus capsids linked to enhanced potency and stability: A quality control framework for gene therapy","authors":"Jing Jin ,&nbsp;Wentao Wang ,&nbsp;Tie Gao ,&nbsp;Yangguang Dai ,&nbsp;Lei Tao ,&nbsp;Qiang Ma ,&nbsp;Lijun Wang ,&nbsp;Xiu Li ,&nbsp;Hongxu Chen ,&nbsp;Yong Zhou","doi":"10.1016/j.jpba.2025.117112","DOIUrl":"10.1016/j.jpba.2025.117112","url":null,"abstract":"<div><div>Recombinant adeno-associated viruses (rAAVs) are pivotal gene therapy vectors due to their safety and stable transduction, yet comprehensive characterization of capsid post-translational modifications (PTMs)—critical for potency, immunogenicity, and manufacturing consistency—remains limited across production platforms. This study employs microflow LC-MS/MS coupled with electron-activated dissociation (EAD) to analyze PTMs in clinically relevant rAAV5 and rAAV9 serotypes produced via mammalian (HEK293) and insect (Sf9) cells, with parallel cellular-level evaluation of vector potency and infectivity, conducted under matched purity and capsid thermal stability conditions to isolate PTM-specific effects. Intact mass analysis revealed conserved N-terminal acetylation in VP1/VP3 across both platforms, while PTM profiling identified six distinct modification types, including deamidation, oxidation, and phosphorylation, with Sf9-derived vectors exhibiting 14 % more PTMs than HEK293-produced counterparts. Despite comparable purity and thermostability, HEK293-derived vectors demonstrated superior in vitro potency (1.9-fold higher eGFP expression) and lower physical-to-infectious particle ratios (P:I, 1.8–3.2-fold reduction), linking PTM patterns to enhanced infectivity. EAD fragmentation mapped isoaspartate (IsoAsp) formation to specific asparagine residues, implicating deamidation-driven instability, while analysis of four Sf9-produced rAAV9 batches revealed ≤ 5 % lot-to-lot variability in PTM site counts. Preliminary data identified low-variance PTM sites (e.g., N57, N452; coefficient of variation, CV ≤ 15 %) and IsoAsp levels (CV ≤ 10 %) as potential stability markers for batch consistency monitoring, though their definitive utility as critical quality attributes requires further validation. These findings establish serotype- and platform-specific PTM landscapes under controlled biophysical parameters, providing actionable insights for optimizing production systems and establishing PTM-driven quality control in gene therapy.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"266 ","pages":"Article 117112"},"PeriodicalIF":3.1,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144886809","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":"Characterisation of small RNA-based therapeutics and their process impurities by fast and sensitive liquid chromatography high resolution mass spectrometry","authors":"Silvia Millán-Martín ,&nbsp;Felipe Guapo ,&nbsp;Sara Carillo ,&nbsp;Ulrik H. Mistarz ,&nbsp;Ken Cook ,&nbsp;Jonathan Bones","doi":"10.1016/j.jpba.2025.117097","DOIUrl":"10.1016/j.jpba.2025.117097","url":null,"abstract":"<div><div>Oligonucleotides offer a powerful class of new therapeutic modalities, which requires the support of robust and sensitive analytical methods. The primary structure of RNA-based therapeutic drugs is considered a critical quality attribute by regulatory agencies, and must be empirically confirmed to ensure quality, safety and efficacy, together with the analysis of the 5′ and 3′ termini, and any site-specific modifications. This study highlights the use of amine-based ion pair reversed-phase LC coupled to a high-resolution MS and MS/MS method (HRMS) for the characterisation of small RNA-based molecules, single stranded antisense oligonucleotides (ss-ASOs), designed with different chemical modifications from second and third generation categories, including backbone modifications, sugar modifications at the 2’ position and base modifications. The study evaluates the applicability of an alternative ion pairing reagent, with moderate hydrophobicity, and the use of a mild ESI source, with the aim to reduce the formation of ion pair-adducts and in-source induced impurities. The developed method allows for sequence verification and confident identification of low abundant impurities with high sensitivity and high mass accuracy at the intact and sequencing level. Intact analysis allowed for the detection and quantification of full-length products with different sizes and purity values, and the detection of multiple impurities and degradants, even without being fully chromatographically resolved, with high sensitivity. Most common impurity found in analysed RNA-based ss-ASOs was the presence of the phosphodiester (PO) conversion, with fractional abundances from 9.2 % to 1.2 %, followed by failure sequence shortmers.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"268 ","pages":"Article 117097"},"PeriodicalIF":3.1,"publicationDate":"2025-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144880301","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":"Dysregulated tryptophan metabolism and gut microbiota in chronic hypoxia-induced pulmonary hypertension rats","authors":"Jinhao Shuai ,&nbsp;Sheng He ,&nbsp;Yang Wang ,&nbsp;Junjie Zhen ,&nbsp;Duonan Yu ,&nbsp;Mengying Lv","doi":"10.1016/j.jpba.2025.117111","DOIUrl":"10.1016/j.jpba.2025.117111","url":null,"abstract":"<div><div>Pulmonary hypertension (PH) is a life-threatening disease, mainly caused by pulmonary arterial inflammation and remodeling, which resulted in elevated pulmonary arterial pressure (PAP) and ultimate rightsided heart failure. In the present study, Wistar rats were exposed to hypoxia for four weeks to induce PH. Multiomics approaches integrating 16S rRNA sequencing, untargeted and targeted metabolomics were utilized to identify alterations in gut microbiota and host metabolism associated with hypoxia-induced pulmonary hypertension. Rats exposed to hypoxia for four weeks exhibited the increased right ventricular systolic pressure (RVSP), pulmonary vascular remodeling, right ventricular hypertrophy, gut microbiota dysbiosis and disturbed serum metabolism. Tryptophan-pathway targeted metabolomics showed that chronic hypoxia exposure induced an increase of tryptophan, xanthurenic acid and 3-indoleacetic acid levels and a decrease of kynurenine, nicotinamide and kynurenic acid levels. Our research would offer promising targets for early diagnosis and treatment of PH.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"266 ","pages":"Article 117111"},"PeriodicalIF":3.1,"publicationDate":"2025-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144852472","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":"Strategies for the electrochemical determination of drugs in biofluids: The role of sample preparation in sensitivity and selectivity enhancement","authors":"Marina Bascón-Humanes,&nbsp;M. Laura Soriano,&nbsp;Rafael Lucena,&nbsp;Soledad Cárdenas","doi":"10.1016/j.jpba.2025.117107","DOIUrl":"10.1016/j.jpba.2025.117107","url":null,"abstract":"<div><div>The presence of drugs in different matrices is a growing concern that must be addressed to ensure prompt and accurate responses. They constitute a heterogeneous group of compounds and their low concentrations, as well as the potential presence of metabolites, make analytical determination more complex. In this context, sample preparation is a valuable tool for achieving the sensitivity and selectivity required for drug determination. This review discusses the role of this analytical process in combination with electrochemical detection for determining drugs in biofluids, compiling the scarce early examples that emerged before 2020 and critically evaluating the advances reported until 2025. The modification of electrodes and the introduction of extraction techniques have been critically discussed. Integrating sample preparation with electrochemical detection is identified as one of the most powerful approaches.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"266 ","pages":"Article 117107"},"PeriodicalIF":3.1,"publicationDate":"2025-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144878593","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":"MicroRNAs as emerging biomarkers for chronic wound monitoring: Analytical methods for the point-of-care detection","authors":"Diego Alvarez-Rafael ,&nbsp;Alfredo de la Escosura-Muñiz","doi":"10.1016/j.jpba.2025.117110","DOIUrl":"10.1016/j.jpba.2025.117110","url":null,"abstract":"<div><div>Chronic wounds represent a significant clinical challenge due to their prolonged healing time and susceptibility to infection and inflammation. Recent advances in molecular diagnostics have identified microRNAs as promising biomarkers for real-time monitoring of wound status, given their regulatory roles in the three phases of the healing process: inflammation, proliferation, and tissue remodeling. This review explores the potential of microRNAs as diagnostic tools for chronic wound management, with special focus on analytical strategies suitable for point-of-care (POC) detection like paper-based lateral flow assays or electrochemical techniques. We critically assess the advantages and limitations of current technologies regarding sensitivity, specificity, integration, and usability in decentralized healthcare settings.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"266 ","pages":"Article 117110"},"PeriodicalIF":3.1,"publicationDate":"2025-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144841628","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":"Strategies for accurate quantitation of free payloads in antibody-drug-conjugates: application for a payload with a labile lactone group","authors":"Kasie Fang,&nbsp;Minjoo Jung,&nbsp;Timothy Sikorski,&nbsp;Hermes Licea-Perez","doi":"10.1016/j.jpba.2025.117105","DOIUrl":"10.1016/j.jpba.2025.117105","url":null,"abstract":"<div><div>Quantifying free payloads in biological matrices is essential for understanding Antibody-Drug Conjugates (ADC) off-target toxicity and safety. The bioanalysis of payloads is challenging due to the need to measure trace amount amidst abundant ADC, with minor ADC degradation potentially causing substantial payload overestimation. Successful assays require careful evaluation of payload structures and effective management of ADC-related interferences. This study identifies challenges for lactone-containing free payloads, mitigates their impact on bioanalysis, and develops a validated methodology for accurate measurement of these payloads in human serum. Lactone hydrolysis and its formation from carboxylate were evaluated at various pH values in buffer solutions and serum. The lactone was completely hydrolyzed at pH 8.5 (25°C) within 25 min; it took several hours at pH 7 and was stable at pH ≤ 6. Lactone regeneration from carboxylate was rapid at pH 3 (within 5 min) and slower at pH ≥ 4. In human serum, lactone hydrolysis was relatively fast (approximately 2 h at 37°C), suggesting the carboxylate form predominates in circulation. Stability experiments showed lactone hydrolysis in serum is reversible, eliminating the need for sample treatment at clinical sites. These insights were applied in the design of a method based on protein precipitation and solid-phase extraction to quantify total payload exposure (50–10,000 pg/mL) in serum in the presence of ADC (250 µg/mL). A double liquid-liquid extraction was employed to purify the ADC before use to prevent interferences in the selectivity and stability assessment. The assay was validated according to M10 guidance and used to support clinical studies.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"266 ","pages":"Article 117105"},"PeriodicalIF":3.1,"publicationDate":"2025-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144827638","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":"Development and validation of a reporter gene assay for the bioactivity determination of anti-TSLP monoclonal antibodies","authors":"Shuting Hou ,&nbsp;Bojia Yang ,&nbsp;Lan Wang ,&nbsp;Chuanfei Yu ,&nbsp;Junzhi Wang","doi":"10.1016/j.jpba.2025.117106","DOIUrl":"10.1016/j.jpba.2025.117106","url":null,"abstract":"<div><div>Thymic stromal lymphopoietin (TSLP) promotes Th2-mediated inflammation via dendritic cell activation and STAT5 signaling and plays critical roles in inflammatory disorders and allergic diseases. While tezepelumab stands as the first and only approved anti-TSLP monoclonal antibody (mAb) with over 10 anti-TSLP mAbs in clinical development, no validated anti-TSLP mAb bioassay was reported yet. Bioactivity determination is essential for ensuring mAb quality. To bridge this gap, we generated a novel HuT78-STAT5-luc reporter cell line through lentiviral transduction of STAT5 response element-driven luciferase into HuT78 cells. This stable cell line expressed luciferase in a TSLP dose-responsive manner. After systematic optimization of cell density, incubation time, TSLP concentration and mAb concentration, we established a reporter gene assay (RGA) with four-parameter regression compliance for the anti-TSLP mAb bioactivity determination. The RGA underwent full validation according to the International Council for Harmonization (ICH) Q2(R2) guideline, namely specificity, linearity, accuracy, precision and robustness. In conclusion, we established a robust and user-friendly RGA that can be applied for the quality control of anti-TSLP mAbs.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117106"},"PeriodicalIF":3.1,"publicationDate":"2025-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144917605","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":"Multi-residue screening method of 195 drugs of abuse and metabolites in whole blood using LC-MS/MS and application to real blood samples","authors":"Sangeun Lee ,&nbsp;Jihyun Lee ,&nbsp;Hangji Ok ,&nbsp;Eunbin Ryu ,&nbsp;Wonhee Koo ,&nbsp;Yoon Hee Lee ,&nbsp;Jeasung Pyo ,&nbsp;Na Young Lim ,&nbsp;Chan Hyeok Kwon ,&nbsp;Su Jeong Park ,&nbsp;Kikyung Jung ,&nbsp;Yoonsook Eom ,&nbsp;Younghoon Chon ,&nbsp;Min-Kyu Song ,&nbsp;Youngmin Hong ,&nbsp;Eunyoung Han","doi":"10.1016/j.jpba.2025.117096","DOIUrl":"10.1016/j.jpba.2025.117096","url":null,"abstract":"<div><div>Polydrug use is a growing concern in the forensic field due to its high risk of overdose and its significant impact on physical and psychological health. Hence, the development of methods for simultaneously analyzing multiple drugs in blood has become increasingly essential recently. The aim of this study is to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous detection of 195 drugs of abuse (DOAs), including amphetamines, opiates, cathinones, phencyclidines, synthetic cannabinoids, cocaine, and metabolites, in 100 μL of blood. Blood samples were extracted using liquid-liquid extraction (LLE) with acetonitrile containing 0.1 % trifluoroacetic acid, and multiple reaction monitoring (MRM) was employed for the detection of 195 drugs and metabolites in a single run. The method was validated over a concentration range of 1–100 ng/mL for all target compounds, showing good linearity with a coefficient of determination (R²) greater than 0.99 for 193 compounds. The limits of quantification (LOQs) of target analytes ranged 0.1–10 ng/mL. The method was successfully applied to 20 real blood samples, detecting 22 drugs of abuse and their metabolites, with concentrations ranging from 0.2 to 401.4 ng/mL. This developed method is expected to be highly applicable in the forensic or clinical field for the rapid detection of polydrug use.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"266 ","pages":"Article 117096"},"PeriodicalIF":3.1,"publicationDate":"2025-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144841629","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":"Characterization of the glycosylation profile of erythropoiesis-stimulating agents (ESAs) and impact on potency","authors":"Julie TerWee ,&nbsp;Beth McCoy ,&nbsp;Bryan Bernat ,&nbsp;Kaila Wilson-Landy ,&nbsp;Sam Billingham ,&nbsp;Krishana Gulla ,&nbsp;Catherine Srebalus Barnes","doi":"10.1016/j.jpba.2025.117094","DOIUrl":"10.1016/j.jpba.2025.117094","url":null,"abstract":"<div><div>To evaluate the impact of glycosylation of Chinese Hamster Ovary (CHO) cell produced erythropoiesis-stimulating agents (ESAs) on <em>in vivo</em> efficacy, epoetin glycoforms were fractionated and characterized. A comprehensive series of biochemical, <em>in vitro</em> bio-functional analyses and <em>in vivo</em> potency assays were conducted to better understand the relationship of structure to function of epoetin glycoforms. The hyper-glycosylated ESA darbepoetin alfa was also assessed to understand the range of <em>in vivo</em> potency response. Results demonstrate a strong link between sialylation or antennary structure, but not between N-acetyllactosamine repeat number and <em>in vivo</em> potency. Results for darbepoetin as compared to epoetin also confirm an inverse relationship between <em>in vivo</em> potency and potency measured using <em>in vitro</em> methods.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"266 ","pages":"Article 117094"},"PeriodicalIF":3.1,"publicationDate":"2025-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144829016","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":"Non-targeted metabolite characterization in microsomal assay using liquid chromatography coupled to high-resolution mass spectrometry: Application to carisoprodol","authors":"Elena Ferri ,&nbsp;Cristian Caprari ,&nbsp;Maria Angela Vandelli ,&nbsp;Loretta L. Del Mercato ,&nbsp;Cinzia Citti ,&nbsp;Giuseppe Cannazza","doi":"10.1016/j.jpba.2025.117091","DOIUrl":"10.1016/j.jpba.2025.117091","url":null,"abstract":"<div><div>Understanding the metabolic fate of pharmaceutical compounds is critical for assessing drug safety and efficacy. A combination of advanced analytical techniques and <em>in vitro</em> models allows for detailed investigation of biotransformation processes. This study presents an integrated workflow using carisoprodol as a case study to demonstrate the application of modern analytical strategies for metabolic profiling. An analytical platform based on liquid chromatography–high-resolution mass spectrometry (LC-HRMS) was employed, operating in both MS¹ and MS² modes to investigate fragmentation behaviour and identify metabolites. Chromatographic separation was performed using a core-shell C₁₈ column under gradient elution. <em>In vitro</em> metabolic stability studies were conducted using rat liver microsomes, and a deuterated analogue was also tested to assist in structural elucidation of hydroxylated metabolites. Additionally, <em>in silico</em> metabolite prediction tools were applied and compared with experimental results. The compound showed slow metabolic degradation (<em>t₁/₂</em> = 233.72 ± 3.09 min) and low intrinsic clearance (<em>CL</em>_{<em>int, in vitro</em>} = 5.930 ± 0.078 µL/min/mg). LC-HRMS enabled identification of meprobamate and a hydroxylated derivative as major metabolites. MS/MS analysis of the deuterated metabolite excluded hydroxylation on the <em>n</em>-pentyl chain as reported in the literature, indicating alternative modification sites. <em>In silico</em> predictions correctly identified meprobamate but misassigned hydroxylation positions for the other metabolite. This study highlights the effectiveness of a multi-technique analytical approach for elucidating drug metabolism. The integration of LC-HRMS, isotopic labelling, and computational tools provides a comprehensive platform for metabolic characterization, while emphasizing the necessity of experimental validation in refining <em>in silico</em> predictions.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"266 ","pages":"Article 117091"},"PeriodicalIF":3.1,"publicationDate":"2025-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144852471","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}