Journal of pharmaceutical and biomedical analysis最新文献

{"title":"Recycling of active pharmaceutical ingredients Metformin hydrochloride and Losartan potassium from expired medications and crystal structure of a new Losartan polymorphic form.","authors":"Laura Galindo-Leon, Juan D Galofre-Benitez, Julián Corredor-Gamba, Deissy N Jaramillo, Johan D Lozano, Mario A Macías, Elizabeth Jiménez-Díaz","doi":"10.1016/j.jpba.2025.117090","DOIUrl":"10.1016/j.jpba.2025.117090","url":null,"abstract":"<p><p>The disposal of expired pharmaceutical tablets, particularly those from household sources, is a growing environmental concern due to the accumulation of pharmaceutical contaminants in water ecosystems. Despite FDA recommendations to discard expired medications, studies have shown that many of these pills retain up to 90 % of their original potency long after their expiration date. This opens up the possibility for recycling and reusing the Active Pharmaceutical Ingredients (APIs) contained in these expired pills. This study presents a simple yet effective extraction protocol for recovering APIs from expired tablets of Metformin hydrochloride and Losartan potassium. The method achieved recovery yields of 88 % for Losartan and 37 % for Metformin. Structural characterization via NMR, HPLC, IR, HRMS, and both powder, and single-crystal X-ray diffraction confirmed the integrity and high purity of the recovered APIs compared to chemical standards. Biological assays carried out in L929 cell line revealed that the extracted APIs retained comparable activity to their chemical-standard counterparts, suggesting their potential for reuse in biochemical assays. During the recrystallization process by using PDRX, a new polymorphic form of Losartan potassium was discovered and named \"Form M\". In addition, a twelve-month stability study showed no degradation for either API at storage conditions. These results suggest that expired APIs are chemically and biologically viable for repurposing, as synthetic precursors in chemical research and in biological assays. The protocol is scalable, adaptable to pharmaceutical companies, and applicable in both industrial and research settings, offering a sustainable solution to pharmaceutical waste. By recovering APIs for educational and industrial use, this approach promotes environmental stewardship and resource efficiency in pharmaceutical science.</p>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"266 ","pages":"117090"},"PeriodicalIF":3.1,"publicationDate":"2025-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144816971","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 clinical application of an LC-MS/MS method designed to simultaneously measure seven second-line TB drugs and two metabolites in human lung tissue.","authors":"Katie Kriegler Foster, Anil Pooran, Marthinus van der Merwe, Sandra Castel, Anton Joubert, Edda Zangenberg, Keertan Dheda, Lubbe Wiesner","doi":"10.1016/j.jpba.2025.117093","DOIUrl":"10.1016/j.jpba.2025.117093","url":null,"abstract":"<p><p>We developed and validated a novel bioanalytical method for the simultaneous quantification of levofloxacin, linezolid, moxifloxacin, delamanid, bedaquiline, clofazimine, and pretomanid, along with the metabolites of delamanid (DM-6705) and bedaquiline (N-desmethyl-bedaquiline, M2), in human lung tissue samples. Following homogenization by bead beating and extraction by protein precipitation, the analytes were separated on an Agilent 1260 Infinity II HPLC system using a Poroshell 120 C18 EC (2.1 mm×50 mm, 2.7 µm) column with gradient elution, applying a mobile phase consisting of 0.1 % formic acid in water and 0.1 % formic acid in a mixture of acetonitrile and methanol. Detection and quantification of the analytes and their stable isotope labelled internal standards were performed on a Sciex API 5500 QTrap mass spectrometer using positive electrospray ionization and multiple reaction monitoring. Validation according to the guidelines of the FDA and EMA proved the method to be precise, accurate, and robust with no significant influence of matrix components. The application of the method to the analysis of clinical samples demonstrated the feasibility of quantifying the second-line anti-tuberculosis drugs in human lung tissue and the potential to provide insights into the drug distribution across the infection sites in the lung.</p>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"266 ","pages":"117093"},"PeriodicalIF":3.1,"publicationDate":"2025-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144812162","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":"Rapid detection of plasma exosomal LncRNA CASC9 for HCC using RT-RPA-CRISPR/Cas12a assay.","authors":"Jie Wu, Xi Li, Xiangbao Yin, Junwen Hu, Pengcheng Zhou, Xiaofeng Zhong, Mingming Wu","doi":"10.1016/j.jpba.2025.117085","DOIUrl":"10.1016/j.jpba.2025.117085","url":null,"abstract":"<p><p>Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide. Early detection is essential for improving patient outcomes. Long non-coding RNAs (lncRNAs) in plasma exosomes have emerged as promising non-invasive biomarkers. However, sensitive detection methods remain limited. Plasma exosomes were isolated and validated using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot (WB). RNA sequencing identified CASC9 as the most significantly upregulated exosomal lncRNA in HCC patients. Its diagnostic value was evaluated using real-time quantitative PCR (RT-qPCR) and a novel RT-RPA-CRISPR/Cas12a fluorescence assay. Diagnostic performance was assessed through receiver operating characteristic (ROC) curve analysis and compared with alpha-fetoprotein (AFP). Exosomal CASC9 levels were significantly elevated in HCC patients and correlated with tumor size, stage, and number (P < 0.001). ROC analysis demonstrated that CASC9 had superior diagnostic accuracy (area under the curve [AUC] = 0.822) compared to AFP (AUC = 0.795), with further improvement when combined (AUC = 0.875). The RT-RPA-CRISPR/Cas12a assay achieved a detection limit of 0.1 copies/μL, outperforming RT-qPCR. When combined with RT-qPCR and AFP, the method achieved an AUC of 0.987 against normal controls and 0.975 against benign cases. Plasma exosomal CASC9 is a promising diagnostic biomarker for HCC. The RT-RPA-CRISPR/Cas12a assay offers a rapid, ultra-sensitive, and clinically feasible detection strategy.</p>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"266 ","pages":"117085"},"PeriodicalIF":3.1,"publicationDate":"2025-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144804348","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":"Batchwise data analysis with inter-batch feature alignment in large scale platelet lipidomics study using UHPLC-ESI-QTOF-MS/MS by data-independent SWATH acquisition.","authors":"Kristina Dittrich, Xiaoqing Fu, Adrian Brun, Madhumita Chatterjee, Meinrad Gawaz, Michael Lämmerhofer","doi":"10.1016/j.jpba.2025.117088","DOIUrl":"10.1016/j.jpba.2025.117088","url":null,"abstract":"<p><p>Untargeted lipidomics by ultra-high-performance liquid chromatography (UHPLC) hyphenated with tandem mass spectrometry using data-independent acquisition (DIA) is a technique with increasing popularity for generating new hypotheses in support of clinical research. Its strength is its data comprehensiveness on both MS and MS/MS level. However, especially when applying SWATH acquisition for large-scale analysis, e.g. clinical studies with over 1000 s to 10,000 s of samples, simultaneous processing of acquired data in multiple batches over longer period of time may be challenging due to retention time and mass shifts as well as huge bulk of data, particularly when computer power is limited. This problem can be alleviated by a batchwise data processing strategy by inter-batch feature alignment of separately processed sample batches. After batchwise automated data processing in MS-DIAL, feature lists can be combined by aligning identical features from different batches attributed to similarity in precursor m/z and retention time, with the intention to generate a representative reference peak list for targeted data extraction. The workflow was established with detected features from three batches of platelet lipid extracts of coronary artery disease (CAD) patients (n = 120) and then applied on a clinical cohort with 1057 CAD patients measured in 22 batches. As a result, the lipidome coverage was significantly increased when several batches were used to create the target feature list compared to a single batch and the increase of annotated features levelled off with 7-8 batches. Further, the lipid identification was improved in terms of number of structurally annotated features.</p>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"266 ","pages":"117088"},"PeriodicalIF":3.1,"publicationDate":"2025-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144804347","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":"Analysis of ethylene glycol (EG) and diethylene glycol (DEG) in oral liquid products using gas chromatography in tandem with Orbitrap spectrometer (GC-Orbitrap).","authors":"Chee Leong Kee, Jing Quan Lim, Hui Ling Lim, Seo Hua Lau, Xiaowei Ge, Min Yong Low","doi":"10.1016/j.jpba.2025.117086","DOIUrl":"10.1016/j.jpba.2025.117086","url":null,"abstract":"<p><p>A selective GC-Orbitrap method has been developed for analysing ethylene glycol (EG) and diethylene glycol (DEG) in four different categories of oral liquid products: Western medicines (WM), traditional medicines (TM), health supplements (HS) and Chinese proprietary medicines (CM). These glycols were chemically ionised to their respective deprotonated ions using methane gas in negative mode and analysed by targeted selected ion monitoring (t-SIM), maintaining a mass error window of ±5 ppm. Internal calibration was performed using matrix-assisted standard solutions ranging from 5 to 100 µg/mL, with 1,4-butanediol-2,2,3,3-D₄ serving as the internal standard (IS). The limits of detection and quantification for both glycols (1.0 µg/mL; 3.0 µg/mL) are comparable with those achieved using gas chromatography tandem with triple quadrupole mass spectrometer (GC-MS/MS) at 0.4 µg/mL and 1.0 µg/mL, respectively. Results showed that most samples were free of EG and DEG. The trace levels of EG and/or DEG detected were below the safety limit of 0.1 % w/w. This methodology not only resolves the false positive identification issues previously encountered with gas chromatography-flame ionisation detection (GC-FID) but also demonstrates broader applicability across different sample matrices, such as oral gel, as evidenced in this study.</p>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"266 ","pages":"117086"},"PeriodicalIF":3.1,"publicationDate":"2025-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144816970","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 rapid LC-MS/MS method for the simultaneous quantification of 10 key folate cycle intermediates in human plasma.","authors":"Sihan Wang, Xiaona Li, Yuanyuan Zhang, Xianhua Zhang, Xin Xiong, Changqing Yang, Libo Zhao","doi":"10.1016/j.jpba.2025.117178","DOIUrl":"https://doi.org/10.1016/j.jpba.2025.117178","url":null,"abstract":"<p><p>The folate cycle is essential for regulating metabolic processes. Simultaneous measurement of folate cycle intermediates is crucial for understanding metabolic disruptions in hematopoietic, nervous, renal and cardiovascular diseases. Currently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for folate cycle metabolites showed poor retention for highly polar compounds in reversed-phase separations with long analytical run time (30 min). Herein, we developed a novel LC-MS/MS method using hydrophilic interaction liquid chromatography (HILIC) mode for simultaneous measurement of 10 key folate cycle metabolites in human plasma, including 5 folate intermediates, 4 related amino acids, and a cofactor (VB₁₂), with enhanced chromatographic retention and reduced analysis time (8.5 min) without derivatization. Through further method validation, all analytes demonstrated acceptable linearity (R² > 0.989), precision (intra-day precision: 1.3-11.3 %; inter-day precision: 3.4-14.6 %), recovery (89.5-113.8 %) and reasonable matrix effect (81.6-115.8 %). The results presented that all intermediates were stable for 5 h at 5°C (autosampler), 12 h at -40°C and 24 h at -80°C. Moreover, the method was successfully applied in clinical plasma from critically ill patients, revealing distinct metabolic perturbations in acute kidney injury (AKI) inpatients compared with non-AKI controls (NAKI). Levels of 5-MTHF and Gly were significantly elevated in the AKI group. Correlation analysis revealed that SCr levels were positively correlated with both 5-MTHF (r = 0.26, p = 0.04) and hCys (r = 0.27, p = 0.04) concentrations. The study is promising to evaluate folate nutritional status to mitigate the risks of folate-related diseases, such as megaloblastic anemia and neural tube defects.</p>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"268 ","pages":"117178"},"PeriodicalIF":3.1,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145251710","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":"Integrating offline 2D UHPLC-QTOF-MS/MS-based automated analytical platform and UHPLC-QTRAP®-MS: The chemical profiling of modified Gegen Qinlian Decoction","authors":"Xinyi Shi ,&nbsp;Zhitian Peng ,&nbsp;Chongsheng Peng ,&nbsp;Xudong Tang ,&nbsp;Xiaobo Li","doi":"10.1016/j.jpba.2025.117175","DOIUrl":"10.1016/j.jpba.2025.117175","url":null,"abstract":"<div><div>The modified Gegen Qinlian Decoction (MGQD) is an upgraded formula of Gegen Qinlian decoction (GQD) with two additional herbs, Euphorbiae Humifusae Herba and Zingiberis Rhizoma Preparatum based on traditional Chinese medicine (TCM) clinical practice. To elucidate the impact of these additions on the chemical composition of the decoction, this study conducted a comprehensive chemical profiling of MGQD. Firstly, the major components in MGQD were quantified, including total saponins, flavonoids, phenolic acids, inorganic elements, carbohydrates, proteins, and free amino acids. These components collectively accounted for 95.06 % of MGQD, with flavonoids being the most abundant category at 30.41 %. Subsequently, an integrated identification strategy was employed by establishing an in-house mass spectral library for MGQD chemical components and incorporating compound clustering analysis through molecular networking of MS² data obtained from offline two-dimensional ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (2D UHPLC-QTOF MS/MS). This approach enabled the identification of 477 structurally diverse compounds in MGQD. Finally, a quantitative analytical method was developed using ultra high performance liquid chromatography coupled with triple quadrupole linear ion trap tandem mass spectrometry (UHPLC-QTRAP®-MS/MS) for the simultaneous determination of 35 selected compounds, which collectively constituted 23.16 % of MGQD. Among these, baicalin was the most abundant (89.4 ± 1.7 μg/mg), followed by puerarin (32.9 ± 0.4 μg/mg) and wogonoside (20.6 ± 0.3 μg/mg). By comparing with the chemical components with that of the original GQD, the potential pharmacodynamic substances of MGQD was inferred, laying the foundation for subsequent research into the pharmacological mechanism. This study provides an effective analytical approach for studying the complex chemical profiles of TCM formulas.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117175"},"PeriodicalIF":3.1,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145220807","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":"Simultaneous determination of nucleosides, modified nucleosides and mononucleotides in gastric cancer cells by liquid chromatography coupled with tandem mass spectrometry","authors":"Juan Li ,&nbsp;Piao Zhou ,&nbsp;Manni He ,&nbsp;Mengxue Liu ,&nbsp;Duo Chen ,&nbsp;Hongmin Liu","doi":"10.1016/j.jpba.2025.117177","DOIUrl":"10.1016/j.jpba.2025.117177","url":null,"abstract":"<div><div>Nucleosides and their modified forms serve as fundamental building blocks of nucleic acids and play critical roles in epigenetic regulation, RNA metabolism, and cellular signaling. In this study, we developed and validated a rapid, sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous quantification of 27 endogenous nucleosides—including 12 canonical nucleosides, 11 modified nucleosides, and 4 mononucleotides—in cellular samples. Under optimized chromatographic conditions, good separations and peak shapes for 27 target compounds were achieved within a 13.0-min gradient elution. The overall LOQs were between 0.2 and 25.0 ng/mL. The intra-day precision of the method was less than 8.2 %, and the inter-day precision was less than 14.2 %. The accuracy was in the range of 89.9–112.2 % for all analytes. The developed method was successfully applied to analyze the metabolic profiles of gastric cancer cells with varying differentiation degrees (NCI-N87, SGC-7901, MGC-803). The results revealed distinct nucleotide metabolism alterations linked to tumor progression and identified potential biomarkers for gastric cancer advancement.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117177"},"PeriodicalIF":3.1,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145213051","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":"An integrated strategy for quality control of Bletillae Rhizoma based on Q-marker","authors":"Jiawei Wang ,&nbsp;Jiajia Gong ,&nbsp;Jiakang Liu ,&nbsp;Xudong Mao ,&nbsp;Chunyue Huang ,&nbsp;Chun Lei ,&nbsp;Xiao Hu","doi":"10.1016/j.jpba.2025.117174","DOIUrl":"10.1016/j.jpba.2025.117174","url":null,"abstract":"<div><div>Bletillae Rhizoma, the rhizome of <em>Bletilla striata</em> (Tunb.) Rchb.f. (BS), a common traditional Chinese medicine, is used to achieve haemostasis and has been widely utilized in clinics for more than 2000 years. Owing to a shortage of resources, some unofficial substitutes such as <em>Bletilla ochracea</em> (BO), <em>Bletilla formosana</em> (BF), and <em>Anthogonium gracile</em> (AG) are also available on the market. This study aims to enhance the quality control of BS and distinguish unofficial substitutes. In this study, an integrated strategy based on Q-marker was performed. Multiple techniques, including untargeted plant metabolomics, serum pharmacochemistry, and activity testing, were combined to identify the Q-marker of BS. A simultaneous quantification method based on Q-marker was established using UPLC-DAD. Through untargeted plant metabolomics, 20 components were identified as differential constituents of BS for distinguishing counterfeits. Thirty-one components were identified or tentatively characterised in the BS extract, and 24 of the 31 components were detected in the serum of treated rats based on serum pharmacochemistry analysis. Six available, differential, and absorbed components, bletilloside A, batatasin III, gymnoside III, militarine, shancigusin I, and dactylorhin A, exhibited haemostatic or anti-inflammatory activities and were selected as Q-markers of BS. The contents of the six Q-markers in BS and the counterfeits were assayed, demonstrating clear differences across BS and the counterfeit species except BF. By integrating the above results, six identified components can be considered as Q-markers of BS. These markers, which are absorbed components associated with the traditional functions and biological activities of BS, can be used for the quality control of BS.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117174"},"PeriodicalIF":3.1,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145220714","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":"Comparison of authentic urine N-ethylpentedrone metabolites to predicted in silico and in vitro human hepatocyte metabolism","authors":"Gloria Daziani ,&nbsp;Omayema Taoussi ,&nbsp;Diletta Berardinelli ,&nbsp;Giulia Bambagiotti ,&nbsp;Marilyn A. Huestis ,&nbsp;Francesco P. Busardò ,&nbsp;Jeremy Carlier","doi":"10.1016/j.jpba.2025.117170","DOIUrl":"10.1016/j.jpba.2025.117170","url":null,"abstract":"<div><div>Synthetic cathinones are the second most common class of substances belonging to the category of new psychoactive substances (NPS). <em>N</em>-ethylpentedrone (NEPD) self-administration was reported, making it important to elucidate its metabolic pathway to improve its detection. NEPD is often taken together with other substances; confidently identifying its intake is important for accurate toxicological analysis and understanding its toxicity. NEPD metabolism was studied following incubation with human hepatocytes and analysis by liquid chromatography-high-resolution mass spectrometry paired with Compound Discoverer software (Thermo Fisher Scientific). Prediction of possible metabolites was performed with the online software GLORYx. <em>In vivo</em> NEPD metabolism was evaluated by the analysis of two authentic human urine samples collected after NEPD ingestion. Twenty metabolites were identified by <em>in vitro</em> hepatocyte incubation, with eighteen identified in the human urine samples. The most abundant reaction was <em>N</em>-deethylation (in positive ionization mode), followed by carbonyl reduction, and a combination of these processes. A pathway previously identified for other synthetic cathinone metabolism, 3-CMC, 4-CMC, and 4-BMC, consisting of the combination of carbonyl reduction, oxidative deamination, and glucuronidation was identified, with the important addition of <em>N</em>-deethylation. These metabolites were identified in urine through additional analyses conducted using negative ionization and retrospectively also in hepatocytes, as they would not have been detectable when analysing the sample with positive ionization alone. It was therefore possible to identify probable markers of NEPD intake and confirm the relevance of the newly proposed metabolic pathway.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117170"},"PeriodicalIF":3.1,"publicationDate":"2025-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145212979","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}