Journal of pharmaceutical and biomedical analysis最新文献

{"title":"Integrated metabolomics and gut microbiota analysis to explore the protective effects of Gushudan on postmenopausal osteoporosis rats via gut-bone axis","authors":"Qinghua Liang ,&nbsp;Hailing Du ,&nbsp;Yajing Wang ,&nbsp;Ying Lai ,&nbsp;Mengxin Ren ,&nbsp;Xiuyan Wei ,&nbsp;Zhili Xiong","doi":"10.1016/j.jpba.2025.116942","DOIUrl":"10.1016/j.jpba.2025.116942","url":null,"abstract":"<div><div>Postmenopausal osteoporosis (PMOP) was caused by significant deviations in gut microbiota and metabolites. Gushudan (GSD), a small traditional Chinese medicine formula, exerted therapeutic effects including kidney-nourishing and bone-strengthening properties. The therapeutic mechanism of GSD in alleviating kidney-yang deficiency syndrome and secondary osteoporosis was systematically investigated through metabolomics and network pharmacology. However, the mechanisms and impact on gut microbiota through which GSD mitigated PMOP remained to be elucidated. In this study, fecal metabolomics was integrated with gut microbiota analysis to comprehensively investigate modification in intestinal flora and metabolic profiles in PMOP rat models from the gut-bone axis framework. Therefore, the GC-MS-based method integrating non-targeted and targeted metabolomics was established to analyze fecal metabolites. The comprehensive analysis of gut microbial communities was performed using 16S rRNA on fecal samples. In the result, 20 potential biomarkers were successfully identified in the non-targeted metabolomics analysis. Subsequently, 12 metabolites related to amino acid metabolism, energy metabolism and bile acid biosynthesis were quantitatively. Then, ten gut microorganisms with significant changes were discovered through 16S rRNA. Furthermore, alterations in fecal metabolites demonstrated a significant correlation with dysbiosis within the gut microorganisms such as <em>[Ruminococcus]_torques_group</em>, <em>Elusimicrobium</em>, <em>Intestinimonas</em> and <em>Papillibacter</em>. GSD effectively modulated abnormal levels of metabolites such as glycine, lactic acid, succinic acid, cholesterol and deoxycholic acid. Specifically, GSD improved the abundance of <em>[Ruminococcus]_torques_group</em>, <em>Elusimicrobium</em>, <em>Intestinimonas</em>. In conclusion, the gut-bone axis was validated as a novel framework, and gut microbiota modulation was further identified as a promising therapeutic target for the prevention of PMOP.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116942"},"PeriodicalIF":3.1,"publicationDate":"2025-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143912808","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 of a new L-fucose purity certified reference material","authors":"Weizhu Chen ,&nbsp;Hua Fang ,&nbsp;Wenhui Jin ,&nbsp;Hui Chen ,&nbsp;Xiaoyuan Huang ,&nbsp;Zhichao Lin ,&nbsp;Zhuan Hong ,&nbsp;Guozong Shi ,&nbsp;Yiping Zhang","doi":"10.1016/j.jpba.2025.116922","DOIUrl":"10.1016/j.jpba.2025.116922","url":null,"abstract":"<div><div>Certified Reference Materials (CRMs) with high accuracy and metrological traceability play an important role in calibrating equipment and validating analytical methods. L- fucose is a ubiquitous natural a six-carbon sugar that has many promising biological physiological functions. It can be used in medicine, food, healthcare products, cosmetics, and other fields. To accurately determine the content of L-fucose in various samples, a reliable CRM needs to be developed. In this study, an new L-fucose purity CRM was developed based on the ISO principle. The study included structural analysis, characterization, homogeneity test, stability evaluation, and uncertainties estimation. The mass balance (MB) and quantitative proton nuclear magnetic resonance (<em>q</em> ¹H NMR) methods were used for characterizing the CRM. Using the MB method, main component, moisture, and nonvolatile and volatile impurities were measured. the PC of CRM was identified by performing high-performance liquid chromatography coupled with ultraviolet detection (HPLC-UV) using the pre-column derivatization procedure. L-fucose purity CRM was found to be stable for 18 months under ambient temperature conditions, whereas, 30 days at 50 °C. It was adequately homogeneous. At a 95 % confidence level, its certified value was 99.5 ± 1.2 % (<em>k</em> = 2).</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116922"},"PeriodicalIF":3.1,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143928373","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, differentiation, and adulteration detection of peppermint essential oil: An NMR approach","authors":"Jianping Zhao ,&nbsp;Mei Wang ,&nbsp;Joseph Lee ,&nbsp;Zulfiqar Ali ,&nbsp;Ikhlas A. Khan","doi":"10.1016/j.jpba.2025.116941","DOIUrl":"10.1016/j.jpba.2025.116941","url":null,"abstract":"<div><div>Peppermint essential oil (PEO) is a valuable natural product widely used in food, cosmetics, and therapeutics. However, its quality and authenticity are frequently compromised by adulteration and compositional variability. This study explored the application of Nuclear Magnetic Resonance (NMR) technique for the characterization and differentiation of PEO, as well as for the identification of adulterants in commercial PEO samples. Comprehensive analyses of 1D and 2D NMR spectra allowed for the identification of characteristic ¹H NMR signals associated with the key components of PEO, and significant compositional variations between PEOs from different geographical origins were revealed. To facilitate differentiation, a straightforward indicator ratio method was developed to distinguish between PEOs from the United States and India, the two primary production regions. A total of 50 commercial PEO samples were evaluated using NMR in combination with chemometric tools, uncovering a high adulteration rate (42 %). Adulterants, including synthetic chemicals, de-mentholized cornmint oil, and lower-cost oils, were identified. This work demonstrates the potential of NMR as a useful tool for quality assessment and authenticity testing of essential oils. The methodology presented may also be extended to other essential oils to ensure product integrity.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116941"},"PeriodicalIF":3.1,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143916509","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":"Quantitative analysis of trace-level bleomycin in complex matrices: Application to in vitro electrochemotherapy","authors":"Helena Plešnik ,&nbsp;Angelika Vižintin ,&nbsp;Damijan Miklavčič ,&nbsp;Jack Steed ,&nbsp;Jianru Stahl-Zeng ,&nbsp;Tina Kosjek","doi":"10.1016/j.jpba.2025.116927","DOIUrl":"10.1016/j.jpba.2025.116927","url":null,"abstract":"<div><div>Bleomycin, a cytotoxic antibiotic, poses substantial challenges for mass spectrometry-based analysis due to its extreme polarity, chelating properties, heterogeneity of fractions, and propensity to form multiple charged species during electrospray ionization. As one of the few effective drugs used in electrochemotherapy, the ability to quantify trace levels of bleomycin is critical for evaluating treatment efficacy, often requiring sensitivity beyond the capabilities of existing analytical methods. Such precise quantification would facilitate the evaluation of electrochemotherapy efficacy, such as comparing the in vitro effects of nanosecond electric pulses with conventional microsecond pulses. To address these challenges, we integrated cell viability assays with a robust chemical analytical approach. This approach employed solid-phase extraction for sample preparation, combined with HILIC-LC-MS/MS, achieving exceptional sensitivity with LLOQ of 0.075 µg/L and overcoming analyte and matrix complexity. Although a significant reduction in cell survival was confirmed when combining nanosecond pulses (25 × 400 ns) with bleomycin, chemical analysis revealed discrepancies, underscoring the complex interaction between electric pulse parameters and drug action. These findings highlight the need for further refinement of treatment protocols and the development of advanced analytical techniques.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116927"},"PeriodicalIF":3.1,"publicationDate":"2025-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143928377","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 and quantification of falsified Viagra using cloud-based portable NIR technology and machine learning","authors":"Hervé Rais ,&nbsp;Pierre Esseiva ,&nbsp;Olivier Delémont ,&nbsp;Cédric Schelling ,&nbsp;Stefan Stanojevic ,&nbsp;Serge Rudaz ,&nbsp;Florentin Coppey","doi":"10.1016/j.jpba.2025.116940","DOIUrl":"10.1016/j.jpba.2025.116940","url":null,"abstract":"<div><div>The prevalence of falsified medications remains a global health challenge, intensified by globalization, internet accessibility, and the high profitability associated with low risks for this type of trafficking. This study demonstrates the innovative integration of portable Near-Infrared (NIR) spectroscopy with a cloud-based advanced data processing and management architecture, offering a rapid, non-destructive, and reliable solution for on-site detection and quantification of falsified Viagra tablets. Leveraging the advantages of portable NIR technology—such as its speed, ease of use, and ability to deliver nearly instantaneous results—this approach not only differentiates authentic from falsified tablets but also accurately determines their absolute sildenafil content. Utilizing data from authentic and seized falsified samples, Principal Component Analysis (PCA), Euclidean distance measurements and Support Vector Machine (SVM) highlight the capability of portable NIR devices to effectively distinguish between these groups. Such models can be seamlessly integrated into an online system paired with a mobile application, enhancing accessibility and efficiency in field settings. Furthermore, machine learning models were developed to quantify sildenafil content in falsified tablets, achieving excellent accuracy compared to a reference chromatographic method. These findings underscore the potential of portable NIR spectroscopy, combined with advanced data treatment, as a transformative tool for field deployment, empowering regulatory bodies and healthcare providers to ensure medication quality and safety with greater speed and precision.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116940"},"PeriodicalIF":3.1,"publicationDate":"2025-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143891606","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":"Evaluating the quality consistency of Nuangong Qiwei Pills based on integrated HPLC, DSC and electrochemistry fingerprints","authors":"Sha Yang,&nbsp;Mengfan Bao,&nbsp;Lingkui Chen,&nbsp;Mengfan Zhang,&nbsp;Ming Cai,&nbsp;Lili Lan,&nbsp;Guoxiang Sun","doi":"10.1016/j.jpba.2025.116939","DOIUrl":"10.1016/j.jpba.2025.116939","url":null,"abstract":"<div><div>Nuangong Qiwei Pill (NQP) is a classic Mongolian formula consisting of seven herbs, which is widely used in the treatment of gynecological diseases. To provide an integrated and comprehensive quality assessment method for NQP, multidimensional fingerprint was creatively applied to NQP. Firstly, the contents of three compounds-eugenol, agarotetrol and dehydrodiisoeugenol in 18 batches of NQPs were quantitatively analyzed. Then, the multidimensional fingerprints were established, which included the establishment of three-wavelength fusion HPLC fingerprints and their reliability, the obtaining of electrochemical fingerprints by B-Z oscillation and the analysis of nine parameters, and the obtaining of differential scanning calorimetry (DSC) quantum fingerprints by fixed-point merger method. Subsequently, systematically quantitative fingerprint method (SQFM) was employed to evaluate similarities, Finally, the qualitative and quantitative results of the fingerprint were synthesized using the coefficient of variation weighting method (CVWM), and the samples were classified into 1–5 levels, which satisfied the evaluation criteria (<em><strong>S</strong></em>_m ≥ 0.70, 70 % ≤ <em><strong>P</strong></em>_m ≤ 130 %). Overall, this study provides valuable information for ensuring the quality of NQPs and will promote the application of fingerprint technology in the Mongolian medicine filed.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116939"},"PeriodicalIF":3.1,"publicationDate":"2025-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143898556","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 label-free HPLC-CAD method to determine total sialic acid in therapeutic proteins","authors":"Yifei Wang ,&nbsp;Yuechuan (Alex) Xu ,&nbsp;Björn-Hendrik Peters ,&nbsp;Michael Peddicord ,&nbsp;Difei Qiu ,&nbsp;Julie Cheng ,&nbsp;Ming Zeng ,&nbsp;Zhi Chen","doi":"10.1016/j.jpba.2025.116937","DOIUrl":"10.1016/j.jpba.2025.116937","url":null,"abstract":"<div><div>Sialylation plays a crucial role in influencing the pharmacokinetic, chemical, physical, and immunogenic properties of therapeutic proteins and is commonly tracked through total sialic acid analysis. In this study, we developed a novel label-free HPLC method to monitor total sialic acid levels in protein drug products. The method utilizes a replaceable, pre-column C18 cartridge that removes protein in-line, preventing protein interference with sialic acid and contamination of the analytical column and enabling direct sample injection. The optimized mixed-mode hydrophilic interaction-ion exchange liquid chromatography (HILIC-IEX) separation condition includes a volatile mobile phase for charged aerosol detection (CAD) of sialic acids. The method was validated to demonstrate satisfactory specificity, linearity (R &gt; 0.999), precision (RSD between 0.4 % and 2.1 %), sensitivity (QL &lt; 25 ng), and accuracy (recovery between 93 % and 102 %). It was successfully applied to determine total sialic acids in a model protein drug product, demonstrating promising potential as an accurate, robust, efficient, and cost-effective approach for sialylation evaluation.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116937"},"PeriodicalIF":3.1,"publicationDate":"2025-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143898557","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 on the mechanism of action of Penehyclidine hydrochloride on LPS-induced acute lung injury by regulating autophagy through the mTOR/Keap1/Nrf2 signaling pathway","authors":"Junting Weng ,&nbsp;Zhicheng Chen ,&nbsp;Shuoyun Weng ,&nbsp;Rongjie Guo ,&nbsp;Bingbing Shi ,&nbsp;Danjuan Liu ,&nbsp;Shanjiao Huang","doi":"10.1016/j.jpba.2025.116938","DOIUrl":"10.1016/j.jpba.2025.116938","url":null,"abstract":"<div><div>Acute lung injury (ALI) is a clinical syndrome characterized by pulmonary inflammation and edema, leading to impaired oxygenation and respiratory failure. Penehyclidine hydrochloride (PHC) has anticholinergic, anti-inflammatory, and antioxidant properties. In this paper, we investigated the protective role of PHC in ALI and explored its mechanism of action. Both <em>in vivo</em> and <em>in vitro</em> experiments were performed using LPS induction to establish an ALI model. Following PHC intervention, the assessment of lung injury was conducted using pathological section examination, mouse lung injury scoring, and ELISA to measure oxidative stress markers including myeloperoxidase (MPO), malondialdehyde (MDA), Super Oxide Dismutase (SOD), and Glutathione Peroxidase (GSH-Px), as well as inflammatory cytokine levels of TNF-α, IL-1β, and IL-18. Immunoblotting and immunofluorescence assays were employed to detect autophagy markers and the mTOR/Keap1/Nrf2 signaling pathway. To confirm the role of autophagy in the protective effects of PHC against ALI, we administered PHC in combination with Rapamycin (RAPA) or 3-Methyladenine (3-MA) to the model groups and evaluated the aforementioned parameters. Our findings revealed that in the LPS-induced ALI model, there was significant pulmonary histopathological damage and increased levels of MPO, MDA, TNF-α, IL-1β, and IL-18, along with decreased levels of SOD and GSH-Px in lung tissue or serum. These alterations were all reversed following PHC treatment. Additionally, compared to the ALI group, PHC administration reversed the expression of mTOR/Keap1/Nrf2 and autophagy proteins LC3, Beclin-1 and p62 induced by LPS. Treatment with the mTOR inhibitor (autophagy inducer RAPA) blocked the protective effects of PHC on lung injury, the mTOR/Keap1/Nrf2 signaling pathway, and autophagy, while co-treatment with the autophagy inhibitor 3-MA showed a significant protective effect on ALI. The results suggest that PHC has a notable protective effect on ALI, which may be achieved by modulating the mTOR/Keap1/Nrf2 signaling pathway to inhibit autophagy.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116938"},"PeriodicalIF":3.1,"publicationDate":"2025-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143883075","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 3D-printed integrated maneuverable device for sensitive colorimetry of Pseudomonas aeruginosa","authors":"Jizhou Li,&nbsp;Shiruoyu Zhou,&nbsp;Ruining Bai,&nbsp;Zhifeng Fu","doi":"10.1016/j.jpba.2025.116934","DOIUrl":"10.1016/j.jpba.2025.116934","url":null,"abstract":"<div><div>Timely and sensitive monitoring of pathogens in clinical specimen is highly demanded for efficient control and precise treatment of infectious diseases. Herein, a 3D-printed maneuverable device integrating incubation, washing, and detection functions was manufactured for colorimetry of <em>Pseudomonas aeruginosa</em> (<em>P. aeruginosa</em>) using transparent resin. The device combined with magnetic beads (MBs) can achieve specific separation and efficient enrichment of <em>P. aeruginosa</em>. Furthermore, it can be directly fixed onto a 96-well plate holder for colorimetry. Specifically, a <em>P. aeruginosa</em> bacteriophage termed as JZ1 acquired from river water was applied as a recognition reagent to functionalize the separation vectors MBs. Then, a nanoconfinement MOFs material termed as PCN-222(Pt) with remarkable peroxidase-like activity was conjugated with polymyxin B to act as a signal tracer. With the formation of target bacterial complexes, the bound PCN-222(Pt) catalyzed the color reaction of 3,3′,5,5′-tetramethylbenzidine to enable quantitative colorimetry of <em>P. aeruginosa</em> by the maneuverable device. With this device, <em>P. aeruginosa</em> can be quantified within 40 min, with a dynamic range of 1.9 × 10² ∼ 1.9 × 10⁶ cfu mL⁻¹. The results for colorimetry of <em>P. aeruginosa</em> in diverse sample matrixes demonstrated its satisfactory practicability. This work provides a facile, timely, and cost-effective technique for point-of-care testing of pathogens.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116934"},"PeriodicalIF":3.1,"publicationDate":"2025-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143891607","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":"Impurity profiling and stability analysis of enzalutamide: Identification, genotoxicity assessment, and development of UHPLC methods for critical impurities","authors":"Burcu Oktar Uzun ,&nbsp;Pelin Kaygu ,&nbsp;Elif Keskin ,&nbsp;Gamze Bostancıoğlu ,&nbsp;Okşan Soyer Can ,&nbsp;Melike Ceren Miser ,&nbsp;Çağan Ağtaş ,&nbsp;Esen Bellur Atici ,&nbsp;Sibel A. Özkan","doi":"10.1016/j.jpba.2025.116926","DOIUrl":"10.1016/j.jpba.2025.116926","url":null,"abstract":"<div><div>This study aims to comprehensively evaluate and control the impurity profile of enzalutamide, an androgen receptor signaling inhibitor used to treat metastatic castration-resistant prostate cancer. Using liquid chromatography-mass spectrometry (LC-MS), twenty impurities were identified based on their mass-to-charge ratios (<em>m/z</em>) during synthetic method development and stress testing studies. Classification of these compounds according to ICH M7 guidelines revealed two potentially genotoxic impurities: Enzal-2 (4-isothiocyanato-2-(trifluoromethyl)benzonitrile), classified as a Class 3 impurity due to its isothiocyanate group, and Enzal-2A (4-amino-2-(trifluoromethyl)benzonitrile), classified as a Class 2 impurity due to a positive Ames test result. Based on the threshold of toxicological concern (TTC) of 1.5 µg/day and the maximum daily dose of enzalutamide of 160 mg, a control limit of 9.4 ppm was established for Enzal-2 and Enzal-2A to mitigate safety risks. Both Enzal-2 and Enzal-2A were identified as process-related impurities, with Enzal-2A also recognized as a hydrolysis degradation product. Specific ultra-high-performance liquid chromatography (UHPLC) methods were developed and validated for the precise, accurate, and robust quantification of enzalutamide, Enzal-2, Enzal-2A, and related impurities. These methods were applied to enzalutamide samples subjected to various stress conditions, including elevated temperature, daylight, UV radiation, and exposure to oxidative, neutral, alkaline, and acidic environments, as well as under accelerated and long-term stability testing. The findings underscore the importance of comprehensive impurity profiling and validated analytical methods to ensure the safe and effective manufacture, quality control, and use of enzalutamide.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116926"},"PeriodicalIF":3.1,"publicationDate":"2025-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143886868","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}