Journal of pharmaceutical and biomedical analysis最新文献

{"title":"Integrated screening of quality markers from Paris polyphylla var. yunnanensis using UHPLC-Q/TOF-HRMS, spectrum-effect relationship analysis, network pharmacology, and quantitative analysis","authors":"Pengjuan Li,&nbsp;Hongbo Xu,&nbsp;Shuming Li,&nbsp;Jingyu Weng,&nbsp;Yuangui Yang","doi":"10.1016/j.jpba.2025.117140","DOIUrl":"10.1016/j.jpba.2025.117140","url":null,"abstract":"<div><div>This study aims to comprehensively screen quality markers using an integrated multi-strategy approach combining plant metabolomics, spectrum-effect relationship analysis, network pharmacology, and quantitative analysis, thereby providing a basis for quality control of <em>Paris polyphylla</em> var. <em>yunnanensis</em> and its closely related species. Firstly, 14 differential metabolites were screened from the roots, stems, and leaves of <em>Paris polyphylla</em> var. <em>yunnanensis</em> using UHPLC-Q/TOF-HRMS combined with principal component analysis and partial least squares-discriminant analysis. Secondly, fingerprint profiles of the roots, stems, and leaves of <em>Paris polyphylla</em> var. <em>yunnanensis</em> were established by UHPLC-Q/TOF-HRMS, identifying 22, 26, and 15 common peaks, respectively. By integrating grey relational analysis, bivariate correlation analysis, and partial least squares regression, five potential quality markers (<em>β</em>-Ecdysone, polyphyllin VII, II, I, and V) were preliminarily screened from <em>Paris polyphylla</em> var. <em>yunnanensis</em>. Subsequently, through network pharmacology and molecular docking, four quality markers (polyphyllin VII, II, I, and V) were ultimately identified. Finally, HPLC-DAD quantitative analysis revealed differential capabilities in accumulating and storing these quality markers among <em>Paris polyphylla</em> var. <em>yunnanensis</em> and its closely related species. In summary, this study holds significant implications for elucidating the pharmacodynamic material basis of <em>Paris polyphylla</em> var. <em>yunnanensis</em>, screening core efficacy-related quality markers through spectrum-effect relationships, and ensuring quality control and rational utilization of Paridis Rhizoma.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"268 ","pages":"Article 117140"},"PeriodicalIF":3.1,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145015706","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":"Unraveling the bridging region of Lenacapavir atropisomers by two-dimensional liquid chromatography","authors":"Yi Shen,&nbsp;Sarju Adhikari,&nbsp;Gavin Carr,&nbsp;Chris Foti,&nbsp;Alban R. Pereira","doi":"10.1016/j.jpba.2025.117129","DOIUrl":"10.1016/j.jpba.2025.117129","url":null,"abstract":"<div><div>Atropisomers are stereoisomers that arise from restricted bond rotation and undergo dynamic conformational change depending on time and temperature. The interconversion of atropisomers could result in a non-baseline “plateau” or “bridging area” between the isomer peaks in liquid chromatography (LC), which diminishes the chromatographic resolution and reduces the assurance of the analyte’s purity. Conventional one-dimensional LC (1D-LC) separation relies on one or two major interactions among the analyte, mobile and stationary phase in an analytical column, which may not be sufficient for deciphering the bridging area. In contrast, two-dimensional LC (2D-LC) leverages orthogonal separation mechanisms in two dimensions, significantly enhancing peak capacity to resolve co-eluting impurities and ensure peak purity. Herein, we present a comprehensive 2D-LC study to investigate the on-column interconversion of Lenacapavir (LEN), which exists as a mixture of two atropisomers. Firstly, different columns, temperature, mobile phase pH, and cation additives were investigated using 1D-LC to assess their potential effects toward on-column interconversion of LEN atropisomers. The bridging area of LEN atropisomers was then unraveled using the heart-cutting mode in 2D-LC, presenting varying ratios for the two atropisomers across the retention time window of LEN. Furthermore, orthogonal conditions for the second dimension were developed to disentangle possible impurities in the bridging area using a regioisomer as surrogate. Lastly, and most importantly, by implementing the developed second-dimension methods, a co-eluting regioisomer of LEN can be separated using 2D-LC. With the aid of high-resolution mass spectrometry (HRMS), we were able to distinguish the co-eluting regioisomer down to 0.05 % (w/w).</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117129"},"PeriodicalIF":3.1,"publicationDate":"2025-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144913258","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":"Liquid chromatography mass spectrometry of segmented hair cortisol – A Bayesian retrospective window","authors":"Pavel Šmak ,&nbsp;Robin Kotouček ,&nbsp;Katarína Kostolanská ,&nbsp;Eliška Bartečková ,&nbsp;Jan Juřica ,&nbsp;Libor Ustohal ,&nbsp;Jana Hořínková ,&nbsp;Pavla Linhartová ,&nbsp;Ondřej Peš ,&nbsp;Martin Čuta ,&nbsp;Eva Táborská ,&nbsp;Jana Gregorová","doi":"10.1016/j.jpba.2025.117127","DOIUrl":"10.1016/j.jpba.2025.117127","url":null,"abstract":"<div><div>This study aimed to develop a robust methodology for quantifying cortisol decay in segmented human hair and to establish accurate retrospective estimates of initial cortisol levels. Two independent probabilistic approaches were employed: i) a Bayesian multilevel analysis across 37 individuals with 3–6 hair segments each and ii) a Bayesian repeated sampling approach in a single individual with 10 segments collected over eight months. All hair segments were analyzed for cortisol concentration using liquid chromatography-mass spectrometry with on-line solid phase extraction. Both approaches demonstrated an exponential decay pattern of cortisol in hair, with estimated decay constants (k) of 0.16 (95 % Credible interval: 0.10–0.22) and 0.11 (95 % Credible interval: 0.06–0.15), respectively. A correction factor was introduced to significantly enhance the accuracy of initial cortisol level estimation, enabling more reliable comparisons with reference intervals obtained from proximal hair segments. This innovative method has the potential to significantly improve long-term cortisol monitoring and advance clinical research especially in psychiatry and endocrinology.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117127"},"PeriodicalIF":3.1,"publicationDate":"2025-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144917606","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Multi-omics analysis revealing the taste characteristics and formation mechanism of Ocimum","authors":"Lingliang Guan ,&nbsp;Jingtian Yang ,&nbsp;Chao Yuan ,&nbsp;Xiaoli Xie ,&nbsp;Mei Huang ,&nbsp;Xiaolu Chen ,&nbsp;Xuan Hu ,&nbsp;Shidong Li ,&nbsp;Guoli Jing ,&nbsp;Zhenxia Chen ,&nbsp;Kai Wang ,&nbsp;Fulai Yu ,&nbsp;Lei Liu","doi":"10.1016/j.jpba.2025.117130","DOIUrl":"10.1016/j.jpba.2025.117130","url":null,"abstract":"<div><div><em>Ocimum</em>, a plant of significant economic value, finds extensive applications in the food, spice, and pharmaceutical industries. This study integrated sensory evaluation, metabolomics, and transcriptomics to systematically analyze taste differentiation in four <em>Ocimum</em> accessions (G126, G081, G096, G124). Standardized quantitative descriptive analysis (QDA) revealed distinct taste profiles: G126 (<em>O. basilicum</em>) exhibited high sweetness and low bitterness, G081 (<em>O. kilimandscharicum</em>) showed prominent minty notes, G096 (<em>O. gratissimum</em>) demonstrated intense piquancy, and G124 (<em>O. gratissimum</em>) displayed unique numbness. UPLC-MS/MS-based metabolomics identified 2275 metabolites, with key taste-metabolite correlations established: N-acetyl-tryptophan positively correlated with sweetness, N-isobutyl decanamide with numbness, flavonoids (particularly flavanones) with bitterness, and terpenoids (monoterpenoids) with minty perception. Transcriptomics uncovered 18 bitterness-associated DEGs (<em>PAL</em>, <em>4CL</em>, <em>C4H</em>, <em>CHS</em>) in flavonoid pathways and 8 minty-linked DEGs (DXS, ispG, ispH, TPS) in terpenoid biosynthesis. Crucially, we constructed an integrated regulatory network linking sensory attributes, key metabolites, and genetic determinants. Our specific conclusions are: Non-sugar metabolite N-acetyl-tryptophan is the primary sweetness contributor in <em>Ocimum</em>; Genotype-specific expression of terpenoid/flavonoid pathway genes drives minty/bitter taste divergence; N-isobutyl decanamide represents a novel chemosensory marker for numbness. These findings provide molecular targets for flavor optimization in pharmaceutical and food applications of <em>Ocimum</em>.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117130"},"PeriodicalIF":3.1,"publicationDate":"2025-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144904087","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":"Integrated proteomic and lipidomic analysis revealed potential plasma biomarkers for cervical cancer","authors":"Yong Chen ,&nbsp;Junhua Xie ,&nbsp;Bingfan Xie ,&nbsp;Hang Zhong ,&nbsp;Zhiyan Zhang ,&nbsp;Qixiao Lin ,&nbsp;Enru Li ,&nbsp;Zhixiang Yan ,&nbsp;Li Cong ,&nbsp;Chunrong Qin ,&nbsp;Feng Wang ,&nbsp;Panpan Wang","doi":"10.1016/j.jpba.2025.117131","DOIUrl":"10.1016/j.jpba.2025.117131","url":null,"abstract":"<div><div>Cervical cancer (CC) remains the most prevalent malignant tumor in the female reproductive system. However, the absence of specific biomarkers and typical clinical manifestations in the early stages significantly impedes early diagnosis, prevention, and treatment efforts. Sensitive, non-invasive biomarkers are urgently necessary for the early detection of CC. This study leveraged proteomic and lipidomic analyses on plasma samples from 115 high-grade squamous intraepithelial lesion (HSIL) patients, 133 CC patients, and 88 healthy controls (CT) and develops robust models for effectively predicting CC. Proteomic profiles revealed 48 differentially abundant proteins in HSIL and CC patients, respectively, most of which were duplicated in two patient groups. HSIL displayed specific lipid accumulation in both discovery and validation cohorts. The elevated lipids were enriched in triglycerides (TG) and phosphatidylcholines (PC), while the lower lipids were enriched in fatty acids (FA). Random Forest classifier results suggest that 4 CC-specific plasma proteins [fibrinogen, complement C3 (C3), hemoglobin subunit alpha, alpha-1-antitrypsin (SERPINA1)], 2 of which were core lipid-interacted proteins (C3, SERPINA1), show predictive ability for CC in both discovery and validation cohorts (AUC 0.97 and 0.64). Our results suggest that lipid and protein perturbations exhibit differential sensitivity towards HSIL and CC and may be used as non-invasive diagnostic markers.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117131"},"PeriodicalIF":3.1,"publicationDate":"2025-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144906796","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":"Comprehensive identification and characterization of in vitro and in vivo metabolites of the novel GLP-1 receptor agonist danuglipron using UHPLC-QToF-MS/MS","authors":"Anupam Jaiswal ,&nbsp;Rushikesh Biradar ,&nbsp;Vaibhav Deshmukh,&nbsp;Rashmi Deshpande,&nbsp;Sukhendu Nandi","doi":"10.1016/j.jpba.2025.117128","DOIUrl":"10.1016/j.jpba.2025.117128","url":null,"abstract":"<div><div>Small-molecule glucagon-like peptide-1 receptor (GLP-1R) agonists are emerging as promising therapeutic agents for type 2 diabetes mellitus (T2DM) and obesity. Danuglipron, a novel investigational GLP-1R agonist, has demonstrated notable efficacy in clinical trials. This study aimed to evaluate the <em>in vitro</em> metabolic stability of danuglipron and to identify its metabolites both <em>in vitro</em> and <em>in vivo</em>. Metabolite profiling was conducted using ultra-high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-QToF-MS/MS). <em>In vitro</em> studies were performed in human liver microsomes (HLM), rat liver microsomes (RLM), and human S9 (HS9) fractions, while <em>in vivo</em> metabolites were identified from rat plasma, urine, and faeces. Seven novel phase I and phase II metabolites were characterized through high-resolution MS data, employing both data-dependent and data-independent acquisition, alongside <em>in silico</em> prediction tools. Key biotransformations included hydroxylation, <em>O</em>-dealkylation, oxetane ring hydrolysis followed by acetylation and methylation. The <em>in vitro</em> half-life (t₁/₂) of danuglipron was 208 ± 31 min in HLM and 81 ± 16 min in RLM, with corresponding intrinsic clearance (CL_int) values of 7.49 µL/min/mg and 35.57 µL/min/mg, respectively, indicating moderate hepatic metabolism and low clearance in both species. Molecular docking suggested that certain metabolites (M-1, M-2, M-7) may have stronger GLP-1R binding affinity than the parent compound, implying potential bioactivity. This comprehensive analysis offers valuable insights into danuglipron metabolic fate and supports further investigation of its metabolites in the treatment of T2DM and obesity.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117128"},"PeriodicalIF":3.1,"publicationDate":"2025-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144904086","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 anti-pulmonary fibrosis action of amygdalin based on transcriptomics and metabolomics","authors":"Jiaxing Gao ,&nbsp;Hong Chang ,&nbsp;Ling Ding ,&nbsp;Hongbing Zhou ,&nbsp;Jiaqi Liu ,&nbsp;Jia Wang ,&nbsp;Yingchun Bai ,&nbsp;Shufang Niu ,&nbsp;Shuyuan Jiang ,&nbsp;Liya Fan ,&nbsp;Kun Xin ,&nbsp;Wanfu Bai ,&nbsp;Songli Shi","doi":"10.1016/j.jpba.2025.117126","DOIUrl":"10.1016/j.jpba.2025.117126","url":null,"abstract":"<div><div>Pulmonary fibrosis (PF) is closely associated with many chronic lung diseases. Amygdalin has antitussive and asthmatic properties and may play a role in intervening in PF. This study was to investigate the effects of amygdalin on bleomycin-induced PF in rats by integrating pharmacodynamics, transcriptomics, and untargeted metabolomics. The experimental findings indicated that the 20 mg/kg amygdalin group exhibited a significant reduction in inflammatory and fibrotic markers, thereby demonstrating the most pronounced anti-PF effect. Following amygdalin treatment, levels of 117 mRNA and 21 metabolites, including linoleic acid, citrulline and sphingosine 1-phosphate, were significantly restored in PF rats. These mRNAs are enriched in pathways such as neuroactive ligand-receptor interactions, phospholipase D signaling, and linoleic acid metabolism, and these metabolites are closely related to linoleic acid metabolism, arginine biosynthesis, and sphingolipid metabolism. Combined transcriptomic and untargeted metabolomic analyses revealed that amygdalin 's mechanism of action is closely linked to five pathways, including neuroactive ligand-receptor interactions, the phospholipase D signaling pathway and linoleic acid metabolism. These pathways may be the key pathways for the anti-PF of amygdalin. Furthermore, 3 key metabolites and 11 key genes in the key pathway may be closely related to the anti-PF mechanism of amygdalin and are key biomarkers. Furthermore, the study revealed that amygdalin has the capacity to modulate the TGF-β1/Smad signaling pathway and the fibrotic protein marker α-SMA, which may contribute to its anti-PF properties.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"267 ","pages":"Article 117126"},"PeriodicalIF":3.1,"publicationDate":"2025-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144896606","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":"Advances and challenges in sample preparation and analytical methods for water-soluble vitamins: Application in food, clinical, pharmaceutical samples","authors":"Tikeshwari ,&nbsp;Kamlesh Shrivas ,&nbsp;Khushali Tandey ,&nbsp;Anuradha Sharma ,&nbsp;Nagendra Kumar Chandrawanshi ,&nbsp;Kallol K. Ghosh","doi":"10.1016/j.jpba.2025.117124","DOIUrl":"10.1016/j.jpba.2025.117124","url":null,"abstract":"<div><div>This review provides a comprehensive analysis of advances and challenges in the sample preparation and analytical methods for determining water-soluble vitamins (WSVs) in foods, clinical, and pharmaceutical samples. WSVs, including vitamin C and B-complex vitamins, are essential for various physiological functions such as energy metabolism, immune response, and neurological health. The accurate analysis of these vitamins is crucial for ensuring nutritional adequacy and preventing deficiencies. The paper explores the different separation and extraction techniques, such as acid and enzymatic hydrolysis, solid-phase extraction (SPE), ultrasound-assisted extraction (UAE), and Soxhlet extraction, highlighting their strengths and limitations. Analytical techniques like spectrophotometry, fluorometry, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and electrochemical methods are reviewed for their applicability in quantifying the WSVs. The review also addresses the challenges in WSV analysis in complex sample matrices, including vitamin stability, matrix effects, and method standardization, which impact the reliability of results. Furthermore, the revolutionary role of nanomaterials, particularly nanoparticles and quantum dots, in enhancing sensitivity for vitamin detection through colorimetric and fluorometric methods is discussed. Finally, different sample preparation and analytical techniques are employed for quantitative measurement of WSVs in foods (vegetables, fruits, and grains) clinical (urine, plasma, tears), pharmaceuticals (syrups, drugs) and metabolic studies. The review concludes by emphasizing the significance of these methodologies in improving the accuracy of WSV analysis, contributing valuable knowledge to the fields of nutrition, clinical and analytical chemistry and supporting public health strategies for preventing vitamin deficiencies.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"266 ","pages":"Article 117124"},"PeriodicalIF":3.1,"publicationDate":"2025-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144886807","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":"Pharmacokinetic assessment of twenty compounds from Bufei Huoxue capsule in rats using HPLC-MS/MS","authors":"Wenwen Li ,&nbsp;Jinyue Ma ,&nbsp;Zijing Zhang ,&nbsp;Kaili Zhang ,&nbsp;Wenhan Lin ,&nbsp;Lu Chen ,&nbsp;Zhenguo Lv ,&nbsp;Yameng Zhu ,&nbsp;Huizi Ouyang ,&nbsp;Jihong Feng ,&nbsp;Jun He","doi":"10.1016/j.jpba.2025.117123","DOIUrl":"10.1016/j.jpba.2025.117123","url":null,"abstract":"<div><div>Bufei Huoxue capsule (BHC), consisting of Astragali Radix, Paeoniae Radix Rubra, and Psoraleae Fructus, demonstrates remarkable efficacy in treating various pulmonary conditions. In this research, an original approach using high-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-MS/MS) was devised to quantitatively determine twenty components (calycosin-7-<em>O</em>-<em>β</em>-D-glucoside, methylnissolin, calycosin, genistein, formononetin, daidzein, angelicin, psoralen, astragaloside IV, 6′-<em>O</em>-galloyl paeoniflorin, benzoylpaeoniflorin, albiflorin, paeoniflorin, oxypaeoniflorin, psoralenoside, isopsoralenoside, catechin, kaempferol, <em>p</em>-coumaric acid, and protocatechuic acid) of BHC in rat plasma. For the twenty selected quantitative prototypes, all calibration curves exhibited excellent linearity (r ≥ 0.9926) within their respective linear ranges. All analytes showed intra-day and inter-day precision (RSD) below 13.41 %, with the accuracy varying between –13.56 % and 12.97 %. The extraction recoveries were measured in the interval of 62.17–96.05 %, and the matrix effects exhibited a range from 74.42 % to 115.96 %. The stability tests indicated that the twenty compounds maintained stability across four distinct storage conditions, and their relative standard deviations did not exceed 14.34 %. Following validation, this methodology was applied to determine the pharmacokinetic behavior of twenty compounds in BHC after oral administration to rats, with relevant pharmacokinetic parameters derived. The obtained data provided a valuable reference for future protocol enhancements and clinical applications.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"266 ","pages":"Article 117123"},"PeriodicalIF":3.1,"publicationDate":"2025-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144878594","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":"Chromatographic separation of dihydroxylated vitamin D3 and accurate quantification of 1α,25(OH)2D3 in human serum","authors":"Koji Takahashi ,&nbsp;Masaki Takiwaki ,&nbsp;Rino Tsutsumi ,&nbsp;Ryota Sakamoto ,&nbsp;Kazuo Nagasawa ,&nbsp;Yutaka Kuroda ,&nbsp;Seketsu Fukuzawa","doi":"10.1016/j.jpba.2025.117125","DOIUrl":"10.1016/j.jpba.2025.117125","url":null,"abstract":"<div><div>The most bioactive form of dihydroxylated vitamin D (VD) is 1<em>α</em>,25-dihydroxyvitamin D₃ (1<em>α</em>,25(OH)₂D₃), which is implicated in various physiological processes and disease development. Therefore, accurate quantification of 1<em>α</em>,25(OH)₂D₃ is essential for clinical diagnosis, VD-related disease management, and infection control. However, 1<em>α</em>,25(OH)₂D₃ quantification in biological samples is challenging because of its low concentration and interference from other dihydroxylated VD metabolites. In this study, we developed a novel assay for 1<em>α</em>,25(OH)₂D₃ quantification, based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) in combination with protein precipitation, solid-phase extraction (SPE), and derivatization of VD metabolites with 14-(4-dimethylaminophenyl)-9-phenyl-9,10-dihydro-9,10-[1,2]epitriazoloanthracene-13,15-dione (DAP-PA). In the derivatization process, the 6 <em>R</em> and 6<em>S</em> isomers of 1<em>α</em>,25(OH)₂D₃ obtained in a 1:1 ratio were quantitatively separated and detected using a pentafluorophenyl-octadecylsilyl mix mode column. This approach enabled the complete separation of 1<em>α</em>,25(OH)₂D₃ from other dihydroxylated VD₃s, including 1<em>β</em>,25-dihydroxyvitamin D₃ (1<em>β</em>,25(OH)₂D₃), 3-<em>epi</em>-1<em>α</em>,25-dihydroxyvitamin D₃ (3-<em>epi</em>-1<em>α</em>,25(OH)₂D₃), 2<em>α</em>,25-dihydroxyvitamin D₃ (2<em>α</em>,25(OH)₂D₃), 2<em>β</em>,25-dihydroxyvitamin D₃ (2<em>β</em>,25(OH)₂D₃), 4<em>α</em>,25-dihydroxyvitamin D₃ (4<em>α</em>,25(OH)₂D₃), and 4<em>β</em>,25-dihydroxyvitamin D₃ (4<em>β</em>,25(OH)₂D₃). To demonstrate the applicability of this optimized method, we analyzed human serum and compared the results with a widely used immunoaffinity extraction-based LC–MS/MS assay for 1<em>α</em>,25(OH)₂D₃, showing equivalent quantitative values. The optimized LC–MS/MS method achieved a run time of 7.7 min, with a lower limit of quantification of 2.5 pg/mL using a sample serum volume of 100 µL. This method is highly sensitive, precise, and capable of high-throughput analysis, providing a robust approach for accurate quantification of 1<em>α</em>,25(OH)₂D₃ while effectively separating interfering dihydroxylated VD₃ isomers.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"266 ","pages":"Article 117125"},"PeriodicalIF":3.1,"publicationDate":"2025-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144864279","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}