Breviscapine injection (BI), a standardized botanical drug for cardiovascular and cerebrovascular diseases, lacks adequate characterization of its pharmacokinetics and tissue distribution. A UPLC-MS/MS method was developed and validated for simultaneous quantification of scutellarin and its metabolite iso-scutellarin in rat plasma and tissues after intravenous BI administration. Sample preparation used methanol protein precipitation, with detection via positive electrospray ionization MRM. Validation followed the Chinese Pharmacopoeia and bioanalytical PK guidance. The assay showed LLQs of 10.0 ng/mL (scutellarin) and 5.0 ng/mL (iso-scutellarin), good linearity (r[2] ≥ 0.990), precision (RSD ≤ 11.1%), accuracy (91.7%-106.8%), and acceptable recovery and matrix effects. PK analysis revealed moderate-to-rapid systemic elimination of scutellarin. Both analytes distributed to tissues within 0.25 h postdose, with exposure ranking: bladder > small intestine > kidney > small intestine > kidney > stomach > liver > plasma > heart > skeletal muscle > pancreas > skin > fat > lung > ovary > testis > adrenal > heart > skeletal muscle > pancreas > thymus > spleen > brain. The method is suitable for BI studies. The limited brain penetration suggests formulation optimization to prolong plasma exposure may be worth exploring, and the relatively high hepatic distribution points to a potential need for monitoring liver and kidney function.

This study aims to elucidate the chemical composition of the dichloromethane extract from the aerial parts of Ferula ferulaeoides (DEAFF) and to explore its biological activities. The composition of DEAFF was analyzed by UHPLC-Q-Orbitrap-MS/MS. Then, the carrageenan-induced tail thrombosis model, xylene-induced ear swelling model, and acetic acid-induced writhing model were established to evaluate the relevant activities of DEAFF. Network pharmacology research and molecular docking were conducted to elucidate the pathways and key targets underlying the multiple activities of DEAFF. A total of 30 chemical constituents were tentatively identified in DEAFF based on UHPLC-Q-Orbitrap-MS/MS analysis. Pharmacodynamic results showed that DEAFF significantly reduced the proportion of thrombus in the tail of mice, effectively alleviated ear swelling, improved the degree of ear tissue lesions, and significantly reduced the number of writhing movements in mice, indicating that it has multiple pharmacological effects such as antithrombosis, anti-inflammation, and analgesia. Network pharmacology studies have shown that multiple pathways such as the PI3K-Akt signaling pathway and the MAPK signaling pathway play a crucial role in the pharmacological effects of DEAFF. In this study, the chemical components of DEAFF were preliminarily identified, and its effects on thrombosis prevention, anti-inflammation, and analgesia were systematically investigated.

Morindae Officinalis Radix (MOR), the dried root of Morinda officinalis How, contains abundant saccharides and is used clinically after different processing procedures. However, the anti-inflammatory material basis of MOR and the chemical consequences of Lycium barbarum processing remain insufficiently defined. In this study, high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD) was developed for fingerprint analysis of 15 batches of raw MOR and the corresponding L. barbarum-processed MOR (LB-MOR). Seventeen common peaks were resolved, and 11 saccharides were assigned by comparison with reference standards. Anti-inflammatory activity was evaluated in LPS-stimulated RAW 264.7 macrophages by quantifying nitric oxide (NO), TNF-α, IL-1β, and IL-6. Grey relational analysis and partial least squares regression linked the HPLC fingerprints with bioactivity and identified glucose, sucrose, kestose, and nystose as the principal activity-associated saccharides. Subsequent validation showed that these saccharides inhibited inflammatory mediator release, with kestose and nystose showing the strongest effects. Western blotting further indicated that kestose and nystose restored IκBα expression and decreased COX-2 expression and the p-p65/p65 ratio, consistent with suppression of sustained NF-κB signaling. This separation-based fingerprint and spectrum-effect workflow identify anti-inflammatory oligosaccharides in MOR and provides candidate quality markers for raw and processed MOR products.

Sucralose, an artificial sweetener widely used in sugar-free foods and beverages, raises human exposure and potential accumulation in tissues and organs. Given the profound long-term impact of maternal diet on offspring, its safety during pregnancy is of particular concern. Existing detection methods primarily target food matrices and lack reliable, efficient approaches for biological sample analysis. To address this gap, we developed an ultrahigh-performance liquid chromatography-multiple reaction monitoring tandem mass spectrometry (UHPLC-MS/MS) method based on existing food detection methods to quantify sucralose in mouse serum and placenta. Method validation showed excellent linearity in the range of 20-1250 nmol/L, with limit of detection (LOD) of 4.88 nmol/L and limit of quantification (LOQ) of 9.77 nmol/L. The precision, accuracy, matrix effects, and freeze-thaw stability were all within acceptable limits. Using the method described above, we detected sucralose in both serum and placenta of mice following subchronic exposure, confirming its bioaccumulation, systemic absorption, and transplacental transfer, suggesting the need for further reproductive toxicity studies. In summary, this study developed and preliminarily applied a method for determining sucralose in mouse serum and placenta, providing a methodological reference for the toxicological evaluation and safety assessment of sucralose, and aids in improving food safety risk systems and the formulation of evidence-based public health policies.

Pomegranate peel (PP) is a traditional Chinese medicinal material. PP has multiple biological activities such as anti-inflammatory and anticancer activities. Although biological activity investigations of PP are growing, few studies have focused on its chemical constituents, and there is a lack of research on its in vivo metabolic characteristics. In this study, UHPLC-Orbitrap Exploris-MS was used to analyze constituents of PP and, for the first time, its metabolic profiles. Network pharmacology and molecular docking were used to elucidate anti-colorectal cancer mechanisms of PP. As a result, we identified 131 compounds in PP. Among them, multiple isomeric forms of different types of ellagitannins and gallotannins were simultaneously identified for the first time. Furthermore, we identified 14 absorbed components and their eight metabolites in rat serum after oral administration of PP. In network pharmacology and molecular docking analysis, using 14 absorbed compounds, we obtained six core targets and five main absorbed compounds that were considered possible anti-colorectal cancer targets and bioactive compounds of PP. Our findings provide systematic knowledge on the material basis, metabolic profiles, and anti-colorectal cancer mechanisms of PP. This study also provides further understanding of ellagitannins and gallotannins in medicinal plants/foods, as well as their in vivo metabolic characteristics.

This acidic ethanol extraction method was adopted to extract total alkaloids from lotus leaves. Single-factor experiments combined with response surface methodology (RSM) were employed to optimite extraction conditions. The optimal extraction parameters were determined as follows: 55 °C, 77% ethanol, 0.66% hydrochloric acid, under which the extraction yield of total alkaloids reached 4.14%. D101 macroporous resin was selected for the purification of crude alkaloid extracts. The resin exhibited an adsorption capacity of 8.60 mg/g and a desorption rate of 43.39%. The optimized purification conditions were as follows: 4 mg/mL sample solution, 50% ethanol eluent, 1.5 BV/h flow rate, 30 mL loading/100 mL elution volume. After purification, total alkaloid purity reached 89.31 ± 1.33%. Adsorption obeyed pseudo-second-order kinetics and Temkin isotherm, being spontaneous and endothermic. This provides improved methods for isolating bioactive alkaloids from lotus leaf.

Callicarpa nudiflora Hook. et Arn. (C. nudiflora), a traditional Chinese medicine, is traditionally used to treat bacterial infections, hepatitis, and hemorrhage. However, its chemical constituents and in vivo metabolic profile have not yet been systematically elucidated, and comparisons of different extracts are still lacking. This study aimed to systematically characterize and compare the in vitro chemical constituents and in vivo metabolites of aqueous (CNW) and ethanol (CNE) extracts of C. nudiflora. A total of 86 chemical constituents were identified in C. nudiflora using UHPLC-Q-TOF-MS/MS combined with molecular networking (MN), including 33 flavonoids, 18 phenylethanoid glycosides, 17 iridoids, 9 phenylpropanoids, 8 terpenoids, and 1 other compound. Based on the identified in vitro constituents and metabolic rules, 12 prototypes and 53 metabolites were further confirmed and annotated in vivo. Furthermore, PCA and OPLS-DA revealed 21 differential in vitro constituents and 16 in vivo compounds between CNW and CNE. These findings indicated that extraction solvent markedly influences the chemical composition and metabolic behavior of C. nudiflora. Overall, this study provides a comprehensive and integrated characterization of phytochemicals and in vivo metabolic profiles of C. nudiflora, offering a robust analytical framework for the comparative evaluation and quality control of different extracts.

RU 58841 is a nonsteroidal antiandrogen, developed for the treatment of androgen-dependent conditions. This study aimed to characterize the human metabolism of RU 58841 for the first time and to assess its systemic exposure following topical administration. In an initial in vitro metabolism study, six potential metabolites were identified using liquid chromatography-high resolution (tandem) mass spectrometry (LC-HRMS/MS). Subsequently, an in vivo administration study was conducted with a RU 58841-containing hair treatment product, applied once to the scalp of six healthy male volunteers. Dried blood spots (DBS) and urine samples were collected and analyzed by LC-low resolution MS/MS. Five in vivo-derived metabolites of RU 58841 were detected in human urine, including products formed via oxidation, N-dealkylation, and glucuronidation of the parent compound and its phase I metabolites. In DBS, the parent compound and the oxidized metabolite were detected, indicating considerable systemic exposure to topically applied RU 58841. The steroid profile (SP) as part of the Athlete Biological Passport (ABP) was not affected by the administration. These findings provide information about the metabolism and the systemic exposure of RU 58841, and further indicate that a single topical dose of 30 mg does not interfere with SP parameter determinations in routine doping controls.

Shenmai Formula (SMF) is a classic traditional Chinese medicine for cardiovascular diseases. Its multicomponent complexity and obscure in vivo metabolic fate hinder understanding of its therapeutic material basis. Because many constituents undergo extensive gastrointestinal and hepatic biotransformation, in vitro profiling alone cannot reveal the actual circulating entities; systematic in vitro-in vivo mapping is therefore essential to define SMF's true bioactive material basis. An in-house constituent database was built from literature (PubMed/CNKI). SMF extract and rat plasma (blank and drug-containing) were analyzed by UHPLC-Orbitrap MS, and constituents were identified by integrating database matching with published literature. Fifty-eight in vitro constituents (mainly saponins) were characterized. Integrating in vitro and in vivo data, 19 circulating prototypes and 14 biotransformed metabolites were mapped in rat plasma. Their biotransformation was driven mainly by deglycosylation, oxidation, methylation, desaturation, and sulfation. The 19 prototypes and 14 metabolites define SMF's in vivo profile and represent the primary potential bioactive candidates for its efficacy. Mapping these active forms and pathways provides a targeted material basis for future pharmacological, pharmacokinetic, and mechanistic studies.

CDC-like kinases (CLK) and dual-specificity tyrosine-regulated kinases (DYRK) are protein kinases involved in various cellular functions, mRNA splicing, and DNA damage repair. CLK/DYRK kinases have been implicated in many disorders such as diabetes, neurodegenerative diseases, and cancer. Cirtuvivint (SM08502) is an orally bioavailable, first-in-class pan-CLK and pan-DYRK inhibitor that modulates pre-mRNA splicing and has shown the ability to inhibit cancer cell growth in vitro and reduce tumor burden in vivo. This has led to the administration of cirtuvivint in phase I clinical trials. To quantitate cirtuvivint, we have developed and validated an LC-MS/MS method in human plasma. The assay is simple and robust and consists of a protein precipitation, dilute-and-shoot extraction method using 20 μL of plasma, chromatographic separation with a Phenomenex Kinetex C18, and a gradient mobile phase system consisting of 0.1% formic acid in water and acetonitrile. The chromatographic method is followed by mass spectrometric detection with a SCIEX 4500 tandem mass spectrometer. The method has a 5-min run time and is linear from 5 to 1000 ng/mL. The assay met the criteria outlined by the US Food and Drug Administration guidance for bioanalytical method validation and will support ongoing and future clinical studies defining cirtuvivint pharmacokinetics.

This study presents a holistic "White Analytical Chemistry" approach for the stability-indicating evaluation of dexamethasone in complex povidone-iodine (PVP-I) ophthalmic suspensions. Moving beyond traditional validation, the method's "whiteness" was quantitatively assessed via the RGB model, balancing analytical excellence, ecological impact, and operational practicality. The redness (analytical) of the strategy is highlighted by a robust RP-HPLC method integrated with LC-MS/MS for the structural elucidation of degradation products. This was further augmented by in silico toxicological mapping and molecular docking against the human glucocorticoid receptor, providing a comprehensive "risk map" of degradants. The greenness (ecological) was optimized through a design of experiments (DoE) approach and the use of core-shell column technology at a reduced flow rate (0.4 mL/min), achieving an AGREE score of 0.72 and a favorable GAPI profile. The blueness (practicality) was ensured by streamlining the routine analysis for standard UPLC-PDA/UV systems, ensuring cost-effective tech-transfer for high-throughput quality control. Ultimately, this integrative framework provides a superior safety diagnostic tool for complex ophthalmic formulations, aligning high-tier pharmaceutical accuracy with environmental and economic consciousness.

An AQbD-based approach was employed to develop and validate a reliable, stability-indicating RP-HPLC method for quantifying sotagliflozin in API and tablet dosage forms. Sotagliflozin, a dual SGLT-1/2 inhibitor used in diabetes management and cardiovascular risk reduction, requires a sensitive and robust analytical method for quality control and stability assessment. Critical method parameters were screened using a Taguchi design and optimized through Box-Behnken Design (BBD). Chromatographic separation was achieved on an Agilent Zorbax SB-C18 column using an eco-friendly mobile phase comprising ethanol and 0.1% TFA (pH 2.25) in a 38:62% v/v ratio at a flow rate of 1 mL/min, yielding excellent system performance. The method was validated according to ICH guidelines, demonstrating good linearity (25-150 μg/mL), precision, accuracy, robustness, and sensitivity with low LOD and LOQ values. Forced degradation studies confirmed its stability-indicating capability, with notable degradation under acidic (11.03%), alkaline (14.13%), oxidative (12.39%), and thermal (14.49%) conditions, while minimal degradation occurred under hydrolytic and photolytic stress. LC-MS/MS enabled identification and structural characterization of four major degradation products. Greenness assessment using AGREE and MoGAPI tools yielded scores of 0.89 and 91, respectively, confirming the method's eco-friendly nature for routine pharmaceutical analysis.

Amphotericin B (AmB), a first-line antifungal agent, is highly protein-bound, and its efficacy and toxicity are primarily determined by the unbound fraction. However, current therapeutic drug monitoring mainly relies on total plasma concentrations, and clinical data on unbound AmB remain limited. In this study, a rapid and sensitive LC-MS/MS method was developed and validated in accordance with ICH M10 guidelines for the simultaneous quantification of total and unbound AmB concentrations. The method was successfully applied to 52 clinical samples from 26 patients. Correlation analysis combined with locally estimated scatterplot smoothing demonstrated that the protein binding rate (PBR) of AmB was associated with multiple biochemical parameters as well as total drug concentration. Subgroup analyses further revealed significant differences in PBR stratified by total concentration, albumin levels, and eGFR. The method requires only a small plasma volume and shows good applicability in clinical samples, providing a practical tool for investigating unbound pharmacokinetics and supporting individualized therapeutic drug monitoring, particularly in patients with altered protein-binding capacity.

The rise of triple-negative breast cancer (TNBC) warrants the urgent need to identify novel therapies. Herein, untargeted metabolomics-guided isolation revealed the metabolome fingerprints of Polygonum bistorta (PB) and Polygonum viviparum (PV) in correlation with their TNBC inhibitory activity. Phytochemical analysis annotated 209 metabolites, with flavonoids (45 compounds) as the major class, followed by phenolic acids (25), tannins (24), and hydroxycinnamic acids (18). Multivariate data analysis identified chlorogenic acid, 6-O-galloylarbutin, and procyanidin B1 as key markers of PV, whereas naringenin-O-glucoside, catechin, and crataegunin D characterized PB. Among different polar extracts, ethyl acetate extract of PB (PB-EA) and the n-butanol extract of PV (PV-NB) exhibited the strongest cytotoxicity against 4T1 cells, with IC50 values of 23.28 ± 0.90 and 39.11 ± 2.36 μg/mL, respectively. Subsequently, the same extracts significantly suppressed tumor growth in the 4T1-Luc orthotopic xenograft mouse model compared with their respective model groups (p < 0.001, p < 0.01). Bioassay-guided isolation yielded 8 compounds, including crataegunin D, which isolated from Polygonaceae plants for the first time, displaying IC50 values of 24.91 ± 2.43 and 20.65 ± 1.77 μM against 4T1 and MDA-MB-231 cells, respectively. Molecular docking revealed binding free energies (ΔG) ranging from -52 to -48 kcal/mol targeting EGFR and CDK receptors.

Coccidiosis is a protozoan parasitic disease that necessitates sustainable therapeutic alternatives due to increasing resistance to conventional coccidiostats. This study evaluated the anticoccidial efficacy of Coriandrum sativum methanolic extract (CME) against Eimeria papillata infection in mice. Phytochemical profiling of CME was conducted using liquid chromatography-mass spectrometry (LC-MS), and identified compounds were subjected to in silico molecular docking. Twenty-five male Swiss albino mice were divided into the following five groups as follows: Group 1: noninfected, nontreated (control), Group 2: CME administered control, Group 3: infected with E. papillata, Group 4: infected+CME treated group (300 mg/kg), and Group 5: infected+amprolium treated group (120 mg/kg). Mice were orally infected with 1 × 10[3] sporulated oocysts and treated for 5 days. Docking analysis revealed strong binding affinities of several chromatographic CME constituents toward elongation factor Tu (tufA), with docking scores exceeded amprolium's binding affinity. In vivo, CME significantly restored goblet cell counts and upregulated MUC-2 expression. It reduced pathogenic Salmonella typhi and Staphylococcus aureus colonization, downregulated inducible nitric oxide synthase and CD4 expression, enhanced catalase activity, as well as suppressed pro-inflammatory cytokines interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α). These findings demonstrate that CME exerts anticoccidial, antioxidant, anti-inflammatory, and culture-based bacterial effects, highlighting its potential as a natural therapeutic agent against murine coccidiosis.