Journal of pharmaceutical and biomedical analysis最新文献

{"title":"Comprehensive characterization of the chemical composition of Ela tablet and its metabolites, with molecular docking screening of potential bioactive components for erectile dysfunction treatment","authors":"Ruqi Xi ,&nbsp;Rahima Abdulla ,&nbsp;Haji Akber Aisa ,&nbsp;Yongqiang Liu","doi":"10.1016/j.jpba.2025.116778","DOIUrl":"10.1016/j.jpba.2025.116778","url":null,"abstract":"<div><div>Erectile dysfunction (ED) is a disorder involving both physiological and psychological implications, and phosphodiesterase 5 (PDE5) inhibitors serve as the primary treatment. The Ela tablet, a traditional herbal formulation, has demonstrated promising PDE5 inhibitory effects, yet its bioactive constituents and metabolic fate remain unclear. A four-step strategy combining offline two-dimensional supercritical fluid chromatography (SFC) and ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) was established to characterize the chemical composition of the Ela tablet. This was followed by <em>in vivo</em> metabolic profiling in rats to determine prototype compounds and metabolites. Molecular docking analysis was then conducted to evaluate the interaction of blood-absorbable prototype compounds with PDE5. A total of 362 compounds were identified, including flavonoids, alkaloids, terpenoids, organic acids and other classes of compounds. <em>In vivo</em> metabolic profiling revealed 70 compounds of prototype and metabolites, with hydrolysis and glucuronidation as the predominant metabolic pathway. Icaritin glycoside analogs underwent extensive metabolism producing Icaritin glucuronide conjugates as the major circulating forms, while <em>N</em>-alkylamides primarily remained in their prototype state. Molecular docking analysis revealed <em>N</em>-isobutyl-(2<em>E</em>,4<em>E</em>)-tetradecadienamide, Anacyclin, Dodeca-2<em>E</em>,4<em>E</em>-dienoic acid isobutylamide, Pellitorine, <em>N</em>-isobutyl-2–6–8-decatrienamide, Icaritin, and Desmethylicaritin as potential PDE5 inhibitors. These findings provide insights into the metabolic transformation and pharmacological mechanisms of the Ela tablet, supporting its potential clinical application in ED treatment. Future research should focus on pharmacokinetics, mechanistic validation, and clinical evaluation to further establish its therapeutic efficacy.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"260 ","pages":"Article 116778"},"PeriodicalIF":3.1,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143562077","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 ultra-sensitive, multiplexed, and cost-effective POCT system for the detection of co-infecting respiratory viruses, including SARS-CoV-2, Flu A, Flu B, and RSV, within 30 min","authors":"Zhongfu Chen ,&nbsp;Lizhen Yan ,&nbsp;Jumei Liu ,&nbsp;Weilun Zuo ,&nbsp;Qunshan Xu ,&nbsp;Shan Qiao ,&nbsp;Shengda Liu ,&nbsp;Yuxiang Zheng ,&nbsp;Hao Lin ,&nbsp;Lianwei Yang ,&nbsp;Bin Wang ,&nbsp;Liuwei Song ,&nbsp;Tingdong Li ,&nbsp;Dongxu Zhang ,&nbsp;Shuizhen He ,&nbsp;Huiming Ye ,&nbsp;Jun Zhang ,&nbsp;Shengxiang Ge ,&nbsp;Shiyin Zhang ,&nbsp;Ningshao Xia","doi":"10.1016/j.jpba.2025.116765","DOIUrl":"10.1016/j.jpba.2025.116765","url":null,"abstract":"<div><div>The co-circulation of respiratory viruses, including SARS-CoV-2, Influenza A (Flu A), Influenza B (Flu B), and respiratory syncytial virus (RSV), poses a significant public health threat. Timely recognition of these viruses allows healthcare professionals to implement effective infection control measures, allocate medical resources properly, and prevent complications from incorrect treatments. Multiplex nucleic acid testing Point-of-care test (mNAT-POCT) circumvents issues of traditional tests, such as high demands on laboratory environments, personnel, and equipment, and limited target analyses, allowing its use in point-of-care settings. However, challenges include primer-primer interactions during fast amplification, high automation requirements, configuring multiple fluorescence channels to avoid spectral overlap, and balancing rapid thermal cycling with sensitive fluorescence signal collection. To address these issues, we developed the multiplexed reverse transcription-quantitative PCR (RT-qPCR) POCT system iNAT SARS-CoV-2/Flu A/Flu B/RSV Assay. This assay enables quick, automatic, and accurate detection of multiple pathogens, improving diagnostic and treatment efficiency for syndromic infectious diseases. The limit of detection (LoD) is 45 copies/mL for SARS-CoV-2, 133 copies/mL for Flu A, 57 copies/mL for Flu B, and 212.5 copies/mL for RSV, with a turnaround time (TAT) of 30 min. Clinical sample analysis showed a 99.36 % agreement with National Medical Products Administration (NMPA) approved reference tests. In conclusion, the iNAT SARS-CoV-2/Flu A/Flu B/RSV Assay performs excellently in detecting and differentiating SARS-CoV-2, Flu A, Flu B, and RSV in respiratory infections, which is crucial for accurate diagnoses.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"260 ","pages":"Article 116765"},"PeriodicalIF":3.1,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143549089","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 sensitive reverse-phase ultra performance liquid chromatography-tandem mass spectrometry method for the determination of tobramycin in Pseudomonas aeruginosa cell lysate","authors":"Woravimol Krittaphol ,&nbsp;Lois W. Martin ,&nbsp;Iain L. Lamont ,&nbsp;Greg F. Walker","doi":"10.1016/j.jpba.2025.116743","DOIUrl":"10.1016/j.jpba.2025.116743","url":null,"abstract":"<div><div>Measurement of antibiotic accumulation in bacteria is essential for full understanding of the mechanisms of antimicrobial resistance but requires a highly sensitive analytical assay. A suitable ultra performance reverse phase liquid chromatographic tandem mass spectrometry (UPLC-MS/MS) method was developed in the electrospray negative ionisation mode for quantifying tobramycin in <em>Pseudomonas aeruginosa</em> cell extracts. <em>P. aeruginosa</em> cell lysate extracts were prepared and spiked with tobramycin and kanamycin (internal standard). Following a protein precipitation extraction procedure, the sample was applied to a reverse phase C18 column, equilibrated in 10 mM ammonium hydroxide at pH 11. Tobramycin and kanamycin were eluted using an acetonitrile gradient and detected in the electrospray negative ionisation mode. The retention times for kanamycin and tobramycin were 1.9 and 2.5 min, respectively and total run time of 10 min. The assay demonstrated linearity in the range of 0.02 – 1 µg mL⁻¹ (R = 0.9999), with limits of detection and quantitation at 1.42 ng mL⁻¹ and 10 ng mL⁻¹, respectively. The precision, expressed as the coefficient of variation, ranged from 1.4 % to 6.5 %, and the accuracy, expressed as bias, ranged from 0.4 % to 17.1 % (ICH, 1996). Using a simple protein precipitation method, the recoveries (%) of tobramycin from cell lysate supernatant for quality controls were 99.7–105.2 % and this result shows that the assay is quantitative. This validated analytical protocol will facilitate future studies aimed at determining the cellular uptake kinetics of tobramycin by <em>P. aeruginosa</em> under various controlled conditions and it could be used for other applications.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"260 ","pages":"Article 116743"},"PeriodicalIF":3.1,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143562078","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":"Comparative analysis of chemical constituents in the stems and roots of different Ephedra species and their in vitro anti-acute lung injury effects","authors":"Bing Guo ,&nbsp;Hengyang Li ,&nbsp;Qi An ,&nbsp;Lina Yang ,&nbsp;Yongli Liu ,&nbsp;Hao Yuan ,&nbsp;Haiyang Wang ,&nbsp;Yuguang Zheng ,&nbsp;Long Guo ,&nbsp;Dan Zhang","doi":"10.1016/j.jpba.2025.116777","DOIUrl":"10.1016/j.jpba.2025.116777","url":null,"abstract":"<div><div><em>Ephedra</em>, the sole genus in the Ephedraceae family, comprises 75 species distributed across Asia, North America, and South America. Ephedrae herba, a well-established traditional Chinese medicine, has been used in clinical practice for over two millennia. <em>Ephedra</em> is widely used to treat asthma, colds, and coughs. Given the diversity of <em>Ephedra</em> species, studying their chemical composition and biological activity is essential for their comprehensive utilization. The chemical profiles of different <em>Ephedra</em> stem (MHS) and root (MHR) species were analyzed using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MSMS). Network pharmacology was applied to investigate the mechanisms by which <em>Ephedra</em> influences acute lung injury (ALI). An <em>in vitro</em> ALI model, induced by LPS, was created to assess the activities of various MHS and MHR species. The anti-inflammatory mechanisms were validated through RT-qPCR and molecular docking studies. The results showed that a total of 27 and 35 constituents were identified in MHS and MHR, respectively, with 12 and 13 compounds screened by chemometric analysis as distinguish chemicals for different species of MHS and MHR Network pharmacology identified 7 core components in MHS and MHR, respectively, that intervene in ALI. Moreover, quercetin-4′-<em>O</em>-glucoside, (+)-catechin, and <em>l</em>-norpseudoephedrine from MHS, along with ephedradine A, epigallocatechin, and (-)-epicatechin from MHR, were identified as key components distinguishing the different parts of <em>Ephedra</em>. A comparison of <em>in vitro</em> anti-inflammatory activities of various MHS and MHR species in an ALI model revealed varying degrees of anti-inflammatory activity. Correlation analyses indicated that apigenin-6,8-<em>C</em>-dihexoside, vitexin-2-<em>O</em>-rhamnoside, quercetin-4′-<em>O</em>-glucoside, and pseudoephedrine from MHS, and ephedradine A, feruloylhistamine, kaempferol-3-<em>O</em>-glucoside-7-<em>O</em>-rhamnoside, and diphenydramine from MHR may be the core compounds contributing to the differences in the activity of various MHS and MHR species. RT-qPCR and molecular docking results indicate that <em>Ephedra</em> may exert anti-inflammatory effects on the MAPK/NF-κB targets through 14 core components. This study serves as a foundational reference for identifying <em>Ephedra</em> species, assessing their quality, and evaluating their pharmacodynamics.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"259 ","pages":"Article 116777"},"PeriodicalIF":3.1,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143521019","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 stability-indicating high performance liquid chromatography method based on a core-shell octadecyl column to determine Avermectin and its related substances in bulk Avermectin batches","authors":"Shane N. Berger,&nbsp;Abu M. Rustum","doi":"10.1016/j.jpba.2025.116773","DOIUrl":"10.1016/j.jpba.2025.116773","url":null,"abstract":"<div><div>The Avermectins are a family of macrocyclic lactones isolated from the soil bacterium <em>Streptomyces avermitilis</em> that possess broad spectrum activity as anthelmintics and are employed throughout the agricultural and animal health industries. Avermectin is the parent molecule from which several important parasiticides have been derived, but currently the Ph. Eur. and USP do not describe an HPLC monograph for analysis of Avermectin. Here, we detail the development and validation of a reversed-phase HPLC method for determination of Avermectin and its related substances in bulk batches of Avermectin. The separation of all peaks of interest was accomplished by a gradient elution on an ACE UltraCore (core shell) 2.5 Super C18 column (150 mm × 4.6 mm, 2.5 µm particle size) maintained at 45 °C. The mobile phase for the gradient is 5 mM NH₄OAc in water at pH 9.5 (mobile phase-A) and acetonitrile/methanol/dichloromethane (52/40.5/7.5, v/v/v) (mobile phase-B). Avermectin and its related substances were detected and quantified at 245 nm with a quantitation limit of 0.05 % of the analytical concentration. Method validation following the International Conference on Harmonization guidelines was carried out with respect to specificity, linearity, accuracy, intermediate precision, and robustness and all acceptance criteria were met. The new HPLC method will provide reliable analysis of Avermectin and its related substances, while the method development data and forced degradation data herein will provide critical information applicable to the Avermectin family as a whole.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"259 ","pages":"Article 116773"},"PeriodicalIF":3.1,"publicationDate":"2025-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143521018","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":"Fast quality assessment and origin identification of Gentianae Macrophyllae Radix using fourier transform infrared photoacoustic spectroscopy coupled with chemometrics","authors":"Xiang Liu ,&nbsp;Jing Huang ,&nbsp;Wenlong Li ,&nbsp;Rongqin Chen ,&nbsp;Liuye Cao ,&nbsp;Tiantian Pan ,&nbsp;Fei Liu","doi":"10.1016/j.jpba.2025.116774","DOIUrl":"10.1016/j.jpba.2025.116774","url":null,"abstract":"<div><div>This study explores the application of Fourier Transform Infrared Photoacoustic Spectroscopy (FTIR-PAS) combined with chemometric analysis for rapid quality assessment and origin species verification of <em>Gentianae Macrophyllae Radix</em> (Qinjiao). Qinjiao, a traditional Chinese medicinal herb, faces significant challenges in quality control due to frequent adulteration and substitution. In this study, 195 Qinjiao samples were analyzed using FTIR-PAS, and chemometric techniques were employed for both classification and regression analysis. The results demonstrate that FTIR-PAS, integrated with chemometric methods, effectively differentiates Qinjiao samples by species. The combination of Detrending (DT) preprocessing and a K-Nearest Neighbors (KNN) classification model achieved an accuracy of 97.4 % in species identification. For quantitative analysis, Savitzky-Golay (SG) smoothing was the optimal preprocessing method for gentiopicroside, while DT was best for loganic acid, and no preprocessing was necessary for roburic acid. Additionally, the Competitive Adaptive Reweighted Sampling (CARS) algorithm, combined with models such as XGBoost and Random Forest (RF), significantly improved the prediction accuracy of key active components, with the highest <span><math><msubsup><mrow><mi>R</mi></mrow><mrow><mi>P</mi></mrow><mrow><mn>2</mn></mrow></msubsup></math></span> reaching 0.84. This research underscores the potential of FTIR-PAS as a rapid, non-destructive approach for the quality assessment and authentication of traditional Chinese medicinal materials.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"259 ","pages":"Article 116774"},"PeriodicalIF":3.1,"publicationDate":"2025-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143527314","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":"Serum metabolic signatures and MetalnFF diagnostic score for mild and moderate metabolic dysfunction-associated steatotic liver disease","authors":"Yang Chen ,&nbsp;Yiran Wang ,&nbsp;Tianjiao Shen ,&nbsp;Nan Wang ,&nbsp;Xiao Bai ,&nbsp;Qiyang Li ,&nbsp;Siyue Fang ,&nbsp;Zhe He ,&nbsp;Changhao Sun ,&nbsp;Rennan Feng","doi":"10.1016/j.jpba.2025.116772","DOIUrl":"10.1016/j.jpba.2025.116772","url":null,"abstract":"<div><div>To explore serum metabolic changes in metabolic dysfunction-associated steatotic liver disease (MASLD) with mild or moderate steatosis and develop a diagnostic index based on liver fat content to differentiate these stages. A total of 149 participants were enrolled from the Nutrition Health Atlas Project in 2019 (Stage 1, n = 92) and 2022 (Stage 2, n = 57). Serum levels of amino acids, free fatty acids (FFAs) and other organic acids were quantified using liquid or gas chromatography-mass spectrometry. The relationships between serum metabolites and magnetic resonance imaging proton density hepatic fat fraction were analyzed and a predictive model fitting fat fraction was constructed in Stage 1 and validated in Stage 2. Patients with moderate MASLD had significantly higher pyruvic acid, 2-ketoglutaric acid, malic acid, 2-hydroxyisocaproic acid and FFA(C14:0) than mild MASLD. Pathway analysis indicated that liver fat accumulation is associated with alterations in amino acid, FFA metabolism and tricarboxylic acid cycle (TCA). The MetalnFF score was developed to discriminate among three groups, achieving an area under the curve (AUC) of 0.956 (95 %CI:0.905, 1.00) for MASLD and 0.857 (95 %CI:0.745, 0.968) for moderate MASLD in Stage 1, and was further validated in Stage 2 with an AUC of 0.986 (95 %CI: 0.951, 1.00) and 0.759 (95 %CI:0.607, 0.921), respectively. In the early stages of MASLD, disrupted amino acid, FFAs metabolism and TCA cycle have occurred. As the disease progresses, metabolic disturbances in pyruvate metabolism become more severe. These findings enhance a deeper understanding of pathogenesis and propose MetalnFF score as a potential diagnostic tool.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"260 ","pages":"Article 116772"},"PeriodicalIF":3.1,"publicationDate":"2025-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143549088","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 16S rDNA sequencing analysis and targeted metabolomics to explore the mechanism of Xiexin Tang in treating atherosclerosis mice induced by high-fat diet","authors":"Junling Li ,&nbsp;Qianru Gao ,&nbsp;Hongtao Liu ,&nbsp;Songlin Liu ,&nbsp;Yanchun Wang ,&nbsp;Xiongjie Sun ,&nbsp;Junping Zheng ,&nbsp;Huabing Yang ,&nbsp;Baifei Hu","doi":"10.1016/j.jpba.2025.116760","DOIUrl":"10.1016/j.jpba.2025.116760","url":null,"abstract":"<div><div>Xiexin Tang (XXT) is a classic Chinese medicine formula that can be used to treat Atherosclerosis (AS). This study aimed to investigate the mechanism by which XXT regulated AS lipid levels. Firstly, the mixture components of XXT were analyzed by High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Then, the AS model based on Apolipoprotein E knockout (ApoE^-/-) mice was established. Cytokines related to lipid metabolism and bile acid metabolism were detected by Quantitative Real-time PCR (qRT-PCR). 16S rDNA gene sequencing was performed to analyze differential bacterial populations, and the mechanism of XXT regulation of bile acids affecting lipid metabolism was further explored by targeted metabolomics. Further, antibiotic-treated mice were used to investigate the role of gut microbiota in the anti-AS effect of XXT. The results showed that XXT attenuated the lipid levels and reversed the abnormal elevation of cytokines, such as hepatic lipid metabolism and inflammatory reaction in AS mice. XXT also repaired the gut barrier damage and reversed gut microbiota disorders in AS mice. Furthermore, the metabolic levels of bile acids were reshaped by XXT. Whereas, in the absence of gut microbiota, XXT failed to attenuate lipid levels and inhibit the expression of cytokines related to inflammation and bile acid metabolism in AS mice and failed to play a role in ultimately treating AS. In conclusion, XXT could effectively inhibit the inflammatory reaction and lipid accumulation in AS mice, and this effect was closely related to its remodeling of gut microbiota to regulate bile acid metabolism.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"259 ","pages":"Article 116760"},"PeriodicalIF":3.1,"publicationDate":"2025-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143510527","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":"Illuminating green fluorescent protein: Characterizing tri-peptide fluorescent chromophore, probing reactivity of cysteines, and unveiling site-directed modifications through mass spectrometry","authors":"Jianmin Zhang,&nbsp;Bing Wang","doi":"10.1016/j.jpba.2025.116771","DOIUrl":"10.1016/j.jpba.2025.116771","url":null,"abstract":"<div><div>Bioconjugation technologies enable covalent attachment of diagnostic or therapeutic effectuators onto biological targets, allowing for the precise delivery of desired drugs to the intended targets with enhanced potency, selectivity, specificity, and prolonged duration of action. As the number of bioconjugation techniques has grown enormously, identification and in-depth characterization of in-process products play a critical role in the development of covalent drug conjugates. This is especially significant in light of the increased complexity of novel biotherapeutics derived from biological matrices. This paper describes liquid chromatography-mass spectrometry (LC-MS/MS)-based studies that have contributed to the development of site-specific genetic incorporation of non-natural amino acids (nnAAs) into proteins. A holistic approach was implemented to characterize a wild type green fluorescent protein (wtGFP) and an enhanced green fluorescent protein (eGFP). By using the wtGFP as a pilot and model system, the reactivity of cysteine residues was investigated under different sample processing conditions, followed by a stability evaluation using intact mass measurement. The subsequent complementary proteolytic peptide mappings were performed to achieve full sequence coverage of the proteins, identification of predominant modifications, and granular details of the fluorescent chromophore. The developed method was successfully applied to isolate the eGFP incorporated with nnAA from cells. This enables the verification of the specific site of nnAA incorporation, and the characterization of complex variants using <em>de novo</em> sequencing techniques. MS studies demonstrated that <em>p</em>-azido-phenylalanine <em>(p</em>AzF) was specifically incorporated into the desired site of eGFP with high efficiency and fidelity.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"259 ","pages":"Article 116771"},"PeriodicalIF":3.1,"publicationDate":"2025-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143527318","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":"The active metabolite hydroxyitraconazole has substantially higher in vivo free fraction and free concentrations compared to itraconazole","authors":"Motoshi Iwao ,&nbsp;Ryota Tanaka ,&nbsp;Yosuke Suzuki ,&nbsp;Ryosuke Tatsuta ,&nbsp;Takehiro Hashimoto ,&nbsp;Kazufumi Hiramatsu ,&nbsp;Hiroki Itoh","doi":"10.1016/j.jpba.2025.116775","DOIUrl":"10.1016/j.jpba.2025.116775","url":null,"abstract":"<div><div>Itraconazole (ITCZ) is a triazole antifungal agent that is metabolized to many products. Hydroxyitraconazole (OH-ITCZ) is the major metabolite with antifungal activity comparable to that of ITCZ. Protein-free drug concentration has been reported to be a better biomarker for pharmacodynamics compared with total drug concentration. We developed an assay for quantification of free ITCZ and free OH-ITCZ concentrations using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) with equilibrium dialysis. The assay fulfilled the requirements of the US Food and Drug Administration guideline for assay validation, with lower limit of quantification of 0.025 and 0.25 ng/mL for free ITCZ and OH-ITCZ, respectively, than previous studies. Using this validated assay, we measured plasma free ITCZ and OH-ITCZ concentrations in 18 samples of 11 adult patients who received oral ITCZ between July 2016 and November 2022 at Oita University Hospital, and compared the <em>in vivo</em> percent free fraction and free concentration between the two compounds. Average plasma free concentrations and percent free fraction in 18 samples were, respectively, 0.188 ± 0.123 ng/mL and 0.024 ± 0.016 % for ITCZ, and 1.449 ± 1.017 ng/mL and 0.251 ± 0.109 % for OH-ITCZ, indicating that OH-ITCZ was 8.52-fold higher in percent free fraction and 10.42-fold higher in free concentration compared to ITCZ. Given that OH-ITCZ and ITCZ have similar <em>in vitro</em> antifungal activity, OH-ITCZ may contribute more to <em>in vivo</em> antifungal efficacy than ITCZ, suggesting that monitoring OH-ITCZ would be more beneficial.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"259 ","pages":"Article 116775"},"PeriodicalIF":3.1,"publicationDate":"2025-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143509853","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}