Journal of pharmaceutical and biomedical analysis最新文献

{"title":"A fragment-aware model for novel psychoactive substances analysis with uncertainty quantification","authors":"Pengfei Liu ,&nbsp;Jing Guo ,&nbsp;Jun Xie ,&nbsp;Liang Li ,&nbsp;Jun Tao","doi":"10.1016/j.jpba.2025.117132","DOIUrl":"10.1016/j.jpba.2025.117132","url":null,"abstract":"<div><div>The rapid emergence of novel psychoactive substances (NPS) poses significant challenges to forensic toxicology, as traditional detection methods struggle to keep pace with their increasing complexity and diversity. To address this, we propose the NPS Fragment-Aware Chemical Language Model (NPS-FACL), a large language model (LLM) framework that enhances NPS detection by leveraging their inherent chemical substructures. Our approach utilizes fragment-aware tokenization, achieving a 20.33% reduction in token representation complexity, which contributes to a 2.01% increase in F1-score. Furthermore, the model integrates explainable uncertainty quantification, enhancing prediction reliability and providing insights into substructure-driven biases for improved forensic analysis of NPS. This work marks a shift towards explainable AI-assisted decision-making, providing proactive alerts and uncertainty-driven insights for early warning of emerging synthetic drug threats, in contrast to conventional Liquid Chromatography-Mass Spectrometry (LC-MS) detection methods, which focus on molecule detection through extensive sample preparation and lack forward-looking predictive capabilities. Beyond forensic toxicology, the approach supports broader applications in public health surveillance, drug regulation, and harm reduction strategies by enabling rapid and scalable identification of novel substances.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117132"},"PeriodicalIF":3.1,"publicationDate":"2025-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145105793","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":"Features of sample preparation of cell culture samples for metabolomic screening by LC-MS/MS","authors":"Nikita V. Basov ,&nbsp;Ekaterina A. Butikova ,&nbsp;Maria A. Sotnikova ,&nbsp;Ivan A. Razumov ,&nbsp;Yulia S. Sotnikova ,&nbsp;Yuriy V. Patrushev ,&nbsp;Artem D. Rogachev ,&nbsp;Nariman F. Salakhutdinov ,&nbsp;Andrey G. Pokrovsky","doi":"10.1016/j.jpba.2025.117146","DOIUrl":"10.1016/j.jpba.2025.117146","url":null,"abstract":"<div><div>Metabolomic analysis has become an essential tool in the life sciences, providing insights into cellular metabolism. However, preparing cell cultures for metabolomic screening remains challenging, especially with samples containing variable cell numbers. Standardized and reproducible protocols are required to ensure reliable data while maintaining compatibility with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Using melanoma cell lines SK-MEL-28 (human) and B16 (mouse) as models, we developed and optimized a convenient sample preparation protocol for metabolomic screening by HPLC-MS/MS. The study is focused on optimizing key steps, including cell lysis, metabolite extraction, and normalization strategies for accurate semiquantitative analysis. The effects of cell count on metabolomic coverage and detection sensitivity were evaluated using hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) chromatography. The protocol enables efficient detection of several metabolite classes from samples containing as few as 10,000 cells. The optimal cell count for reliable analysis was found to be 400,000 – 500,000 cells, ensuring consistent and reproducible detection within the method’s analytical coverage. Our findings emphasize the importance of cell size and number in metabolomic studies, as larger cells provide improved metabolomic coverage. Moreover, metabolites exhibited varying detection limits, highlighting the need to adjust sample preparation strategies according to metabolite characteristics. The proposed protocol offers a robust and reproducible approach for the metabolomic screening of adherent melanoma cell cultures by HPLC-MS/MS and can be adapted for non-adherent and other cell types. Balancing sensitivity, reproducibility, and feasibility, this method provides a standardized solution for cell metabolomic studies in pharmacometabolomics, cancer research, and related fields.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117146"},"PeriodicalIF":3.1,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145091956","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":"Icariin enhances contextual fear extinction via modulation of platelet activation and ECM-associated signaling in the hippocampus: A multiomics perspective","authors":"Zhixin Fan ,&nbsp;Simeng Yao ,&nbsp;Xiayu Gong ,&nbsp;Hanfang Xu ,&nbsp;Guangfu Chen","doi":"10.1016/j.jpba.2025.117148","DOIUrl":"10.1016/j.jpba.2025.117148","url":null,"abstract":"<div><div>Impaired fear extinction is a hallmark feature of stress- and anxiety-related disorders. Icariin (ICA), a flavonoid derived from <em>Epimedium</em>, exhibits neuroprotective effects in various preclinical models, but its role in fear modulation remains unclear. In this study, we examined the impact of ICA on contextual fear extinction in a rat model subjected to foot shock (FS). Proteomic and metabolomic profiling of the hippocampus was conducted using data-independent acquisition (DIA) proteomics and untargeted metabolomics. ICA treatment significantly enhanced fear extinction in FS-exposed rats. Proteomic analysis identified 175 differentially expressed proteins, with enrichment in platelet activation and extracellular matrix (ECM)-associated pathways. Metabolomic profiling revealed 50 upregulated and 229 downregulated metabolites, highlighting the involvement of the sphingolipid signaling pathway. Among the altered proteins, Myl9, Gp1ba, Parvb, and Prkaca emerged as key candidates implicated in ICA’s effects on fear extinction, with Myl9 and Prkaca levels significantly correlated with freezing behavior. Integrative analysis further linked multiple differential metabolites, including (±)12,13-DiHOME, to Myl9 and Prkaca expression. These findings provide novel insights into the molecular mechanisms underlying ICA’s therapeutic potential for fear-related neuropsychiatric disorders.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117148"},"PeriodicalIF":3.1,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145102662","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":"A UPLC-Q/TOF-MS-based plasma and hippocampal metabolomics analysis was conducted to investigate the impact of Erjing Pills on Alzheimer's Disease rats","authors":"Xu-dong Zhu ,&nbsp;Li-ping Huang ,&nbsp;Na Zhu ,&nbsp;Xi-yang Yang ,&nbsp;Fei-xia Yan ,&nbsp;Min Liu ,&nbsp;Peng Zhang ,&nbsp;Qiu-yun Wu ,&nbsp;Jian Zhou ,&nbsp;Yi Wu","doi":"10.1016/j.jpba.2025.117147","DOIUrl":"10.1016/j.jpba.2025.117147","url":null,"abstract":"<div><div>Alzheimer’s Disease (AD), the most common form of dementia, places a significant burden on individuals and society. In line with the doctrines of Chinese medical practice, kidney deficiency and insufficient marrow cannot nourish the brain, leading to insufficient blood, cerebral dysfunction, and ultimately dementia. Erjing Pills contain wolfberry and Polygonati Rhizoma, which tonify the kidneys and improve the essence. Although pharmacological studies have demonstrated that Erjing Pills can be used to prevent and treat AD, the detailed action mechanism is yet to be determined. To comprehensively reveal the mechanism of action of Erjing Pills in the prevention and treatment of AD, three pharmacodynamic evaluations, the water maze test, both hematoxylin-eosin and immunohistochemical techniques were utilized to assess the effects of Erjing Pills on AD rats. Furthermore, plasma and hippocampus metabolomics were conducted by various statistical analyses combined with UPLC-Q/TOF-MS were employed for a comprehensive and accurate analysis of the in vivo anti-AD effects of Erjing Pills. Erjing Pills enhanced learning and memory capacity decreased hippocampal Aβ deposition expression, and enhanced hippocampal morphology in AD rats. After Erjing Pills treatment, 38 plasma metabolites and 15 hippocampal metabolites of AD rats were regressed to levels similar to those of controls. Moreover, pathways impacted by Erjing Pills on AD included arachidonic acid metabolism, retinol metabolism, glycerophospholipid metabolism, and caffeine metabolism. Additionally, adrenaline, leukotriene B4, retinol, and paraxanthine clearly distinguished the Erjing Pills group from the AD group. These findings significantly enhance our comprehension of the metabolic pathways implicated in AD and shed light on the therapeutic mechanisms of Erjing Pills in alleviating symptoms in rats with AD.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117147"},"PeriodicalIF":3.1,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145102702","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":"Identification of chemical constituents of Smilax glabra Roxb. and comparative study on pharmacokinetics of 12 main bioactive components in normal and hyperuricemia rats by UPLC-Q-Exactive Orbitrap-HRMS method","authors":"Shiyang Li ,&nbsp;Changyu Long ,&nbsp;Mi Li ,&nbsp;Lingyu Tian ,&nbsp;Rongsheng Li ,&nbsp;Jing Guo ,&nbsp;Zhenyu Xuan","doi":"10.1016/j.jpba.2025.117149","DOIUrl":"10.1016/j.jpba.2025.117149","url":null,"abstract":"<div><div><em>Smilax glabra</em> (<em>S. glabra</em>), a dual-purpose medicinal herb, demonstrates anti-hyperuricemic potential, but its active constituents remain unclear. Here, we systematically characterized its chemical profile and compared pharmacokinetics between normal and hyperuricemic (HUA) rats. Using Ultra-Performance Liquid Chromatography coupled with Q-Exactive Orbitrap High-Resolution Mass Spectrometry (UPLC-Q-Exactive Orbitrap-HRMS), we identified 89 compounds, with 12 quantitatively analyzed by a validated UPLC-MS/MS method (precision RSD &lt;15 %, accuracy 85–115 %). The pharmacokinetic analysis demonstrated that most compounds achieved peak plasma concentrations within 0.5–3 h with relatively short elimination half-lives, while comparative assessment revealed significantly increased systemic exposure and prolonged time for seven key constituents (caffeic acid, neoastilbin, astilbin, neoisoastilbin, isoastilbin, engeletin, and quercetin) in HUA rats. The results of this study provide a direction and basis for further revealing the active components of <em>S. glabra</em> in the treatment of HUA.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117149"},"PeriodicalIF":3.1,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145091967","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":"Study of oxidation products of morphine and their reactivity by electrochemistry/(liquid chromatography/)mass spectrometry","authors":"Semyon K. IIyushonok ,&nbsp;Konstantin A. Krasnov ,&nbsp;Kristina A. Rose ,&nbsp;Alexey S. Gladchuk ,&nbsp;Petr P. Beltyukov ,&nbsp;Elizaveta V. Volchkova ,&nbsp;Andrey S. Radilov ,&nbsp;Ekaterina P. Podolskaya ,&nbsp;Vladimir N. Babakov","doi":"10.1016/j.jpba.2025.117141","DOIUrl":"10.1016/j.jpba.2025.117141","url":null,"abstract":"<div><div>Morphine is a potent analgesic that is commonly used for the clinical management of chronic and acute pain. Several studies indicated that some adverse reactions of morphine might be in part related to oxidation processes during phase I metabolism, resulting in generation of reactive metabolites, which could bind to glutathione (GSH) or macromolecules. In this work we utilized electrochemistry/(liquid chromatography/)mass spectrometry for <em>in vitro</em> study of the oxidative transformation of morphine. Morphine was oxidized in a thin layer flow cell equipped with a glassy carbon working electrode controlled by Roxy™ potentiostat. Using a purely instrumental approach, eighteen oxidation products of morphine were identified based on the accurate mass measurements, chromatographic retention time on a reversed-phase column and the fragmentation patterns. Incubation of morphine oxidation products with GSH resulted in formation of five adducts, including isomeric forms. We also demonstrated that morphine oxidation products could also bind to thiol groups of larger molecules, particularly human serum albumin (HSA). Analysis of the tryptic digest of HSA, isolated from human plasma after incubation with morphine oxidative metabolites, confirmed that the protein was modified at the only free cysteine residue (Cys34) with dihydroxylation product of morphinone.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117141"},"PeriodicalIF":3.1,"publicationDate":"2025-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145086469","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 related substances of 6-Aminopenicillanic acid using UPLC-Q-Exactive Orbitrap-MS/MS and NMR","authors":"Jiajie Wu ,&nbsp;Jing Lu ,&nbsp;Ju Liu ,&nbsp;Huimin Yi ,&nbsp;Ben Zhou ,&nbsp;Hua Chang ,&nbsp;Hongjuan Pan","doi":"10.1016/j.jpba.2025.117143","DOIUrl":"10.1016/j.jpba.2025.117143","url":null,"abstract":"<div><div>6-Aminopenicillanic acid (6-APA), a key intermediate in the synthesis of semi-synthetic penicillin antibiotics, was subjected to forced degradation under thermal, alkaline, acidic, oxidative, and photolytic conditions. The resulting degradation products were identified by ultra high-pressure liquid chromatography-quadrupole-Exactive Orbitrap mass/mass spectrometry (UPLC-Q-Exactive Orbitrap-MS/MS) and nuclear magnetic resonance (NMR) techniques. An oxidative forced degradation of 6-APA, designated RS7, was isolated and purified using semi-preparative liquid chromatography (semi-Prep-LC). Structural characterization confirmed RS7 as penicillamine disulfide. A total of 18 related substances were identified, comprising three process-related substances (including 2 pairs of isomers), and 14 degradation products (including 5 pairs of isomers). The degradation pathways of 6-APA were proposed and summarized. For the first time, the oxidative and reductive degradation products of 6-APA were identified, including the new detection of penicillamine disulfide among its forced degradation products.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117143"},"PeriodicalIF":3.1,"publicationDate":"2025-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145102738","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":"A novel method for the determination of dried saliva spots (DSS) based on UHPLC-MS/MS for three α-dicarbonyl compounds: An application in the saliva of diabetes patients","authors":"Guangyi Zhang ,&nbsp;Chengqiang Han ,&nbsp;Yuxuan Li,&nbsp;Xi-Ling Li,&nbsp;Hongmei Jin,&nbsp;Jun Zhe Min","doi":"10.1016/j.jpba.2025.117142","DOIUrl":"10.1016/j.jpba.2025.117142","url":null,"abstract":"<div><div>α-Dicarbonyl compounds (α-DCs) in human saliva are closely associated with diabetes, but their inherent instability presents challenges for sample transportation and detection. This study introduces a novel approach for the simultaneous detection of three α-DCs—3-deoxyglucoside (3-DG), glyoxal (GO), and methylglyoxal (MG)—in dried saliva spots using 4,5-dimethyl-1,2-phenylenediamine (DMPD) derivatization combined with UHPLC-MS/MS. 5.0 mm diameter double-circle qualitative filter paper was selected as the substrate for dried saliva, with optimal extraction conditions involving two 3-minute extractions. Chromatographic separation was performed on a CSH™ C18 column (2.1 × 50 mm, 1.7 μm) via gradient elution, utilizing a mobile phase consisting of 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile. The entire separation process was completed within a 5.0 min timeframe. The linearity of 3-DG and GO in the concentration range of 0.10–12.5 μM, and MG in the range of 0.24–31.25 μM. (R² ≥ 0.9994). Intra- and inter-day precision yielded RSD values ranging from 0.59 % to 5.78 %, with average recoveries between 99.78 % and 108.50 %. The detection limits were 0.85–12.85 fmol. This method was applied to analyze dried saliva samples from 44 healthy volunteers (HVs) and 46 diabetic patients (DPs), revealing significant differences (<em>p</em> &lt; 0.001) in the concentrations of 3-DG, GO, and MG between HVs and DPs. The research outcomes validate the practicality of α-DCs in dried saliva samples as prospective biomarker candidates for diabetes, while introducing an innovative methodology for their utilization in clinical settings.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117142"},"PeriodicalIF":3.1,"publicationDate":"2025-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145045730","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":"Validation and quantitation of six neurotransmitters in rat plasma and brain using liquid chromatography quadrupole-time-of-flight mass spectrometry","authors":"Laerissa Reveil ,&nbsp;Maria Lasaosa ,&nbsp;Amanda Furman ,&nbsp;A.M. Iqbal O’Meara ,&nbsp;Matthew S. Halquist","doi":"10.1016/j.jpba.2025.117145","DOIUrl":"10.1016/j.jpba.2025.117145","url":null,"abstract":"<div><div>Delirium is a cognitive dysfunction observed following sedation of critically injured patients. Neurotransmitters are endogenous chemical messengers that play key roles in a variety of essential nervous system functions, and their dysregulation is believed to contribute to the development of delirium. Monitoring neurotransmitters such as acetylcholine, dopamine, serotonin, gamma-aminobutyric acid, gamma-hydroxybutyric acid, and glutamic acid for biomarkers of delirium could improve our understanding of delirium pathogenesis and provide evidence for more efficacious treatments. However, current methods have limited scopes of analysis, require large sample volumes, or use complicated extraction methods. In this study, a novel, analytical approach was developed using liquid chromatography quadrupole-time-of-flight mass spectrometry to measure neurotransmitters in limited sample. This method was used to measure neurotransmitters in plasma and brain tissue samples taken from rats treated with a benzodiazepine sedative and opioid analgesic, a drug combination commonly prescribed in intensive care units and known to precipitate delirium. The analytical method was validated for selectivity, matrix effect, accuracy and precision, dilution integrity, and stability in rat plasma and partially validated in rat brain. The calibration curve ranged from 10 to 5000 ng/mL across the analytes. Within-run and between-run bias and precision of the LLOQ and QCs in plasma and brain were within 20 % and 15 %, respectively. Analytes were stable at 2 ºC and −80 ºC. Extraction recoveries from a simple protein precipitation ranged from 85 % to 133 %. The method was successfully applied to measure analytes in rat brain homogenates from a delirium model study.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117145"},"PeriodicalIF":3.1,"publicationDate":"2025-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145081033","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":"Determination of temozolomide and its metabolite 5-aminoimidazole-4-carboxamide in plasma using UPLC-MS/MS: Implications for therapeutic drug monitoring with high-grade gliomas","authors":"Ning Zhang ,&nbsp;Shengwei Shen ,&nbsp;Chuanguang Han ,&nbsp;Jinhui Qiu ,&nbsp;Zhiwei Wang ,&nbsp;Chenlin Shen ,&nbsp;Shuai Song ,&nbsp;Huihui Ma","doi":"10.1016/j.jpba.2025.117144","DOIUrl":"10.1016/j.jpba.2025.117144","url":null,"abstract":"<div><div>Temozolomide (TMZ) combined with radiotherapy is the standard regimen for high-grade gliomas (HGG). TMZ exhibits linear pharmacokinetics and consistent blood-brain barrier penetration, metabolizing to equimolar 5-aminoimidazole-4-carboxamide (AIC) and methyl diazonium cation, forming the basis of the Two-Ratio Hypothesis (plasma-to-cerebrospinal fluid TMZ/AIC ratios) for therapeutic drug monitoring. We developed a validated ultra-performance liquid chromatography-tandem mass spectrometry method to quantify TMZ and AIC in HGG patients. Addressing polarity differences, TMZ was enriched by ethyl acetate liquid-liquid extraction while AIC underwent strong cation exchange SPE. This method demonstrated linearity (TMZ: 77.66–19,415 ng/mL, AIC: 5.00–2000 ng/mL; <em>r²</em> &gt; 0.99), precision (intra-batch RSD ≤ 11.85 %, inter-batch RSD ≤10.23 %), accuracy (RE: −7.48 to 4.94 % for TMZ; −5.25 to 5.26 % for AIC), recovery (97.26–102.5 %), and stability compliant with FDA/Chinese Pharmacopoeia guidelines. Analysis of 46 plasma samples (37 patients) revealed higher dose-normalized TMZ peak concentrations (1-h post-dose) in concurrent chemoradiotherapy versus adjuvant chemotherapy (3.54 <em>vs.</em> 2.48 μg/mL, <em>P</em> &lt; 0.05). Females showed 52.4 % greater TMZ exposure than males (3.55 <em>vs.</em> 2.33 μg/mL, <em>P</em> &lt; 0.05), while AIC exhibited no statistically significant gender- or treatment-related variations. Gender-adjusted TMZ-AIC correlation was moderate (<em>r</em>_<em>s</em>= 0.540, <em>P</em> &lt; 0.01). This study p<em>r</em>ovides the first analytical validation of the Two-Ratio Hypothesis and reveals gender-dependent TMZ pharmacokinetics, supporting personalized neuro-oncology dosing.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117144"},"PeriodicalIF":3.1,"publicationDate":"2025-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145045731","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}