Biomedical Chromatography最新文献

A novel reverse-phase high-performance liquid chromatography (RP-HPLC) method for validating the amount of HES in dissolving microneedle patches (DMNs) loaded with HES-based nanostructured lipid carriers (HES-NLC)s has been developed. The method was developed and validated according to the guidelines published by the International Council for Harmonization (ICH), using a mobile phase of methanol:water (90:10 v/v) at a flow rate of 0.5 mL/min, with UV detection at 284 nm. This method demonstrated accuracy from 94% to 108%, with a precision of 2% across all evaluated concentrations, along with high selectivity, sensitivity, and stability for transdermal application studies. The method also showed good linearity in rat plasma and skin homogenate, with no interference from biological matrices, which aligns with FDA guidelines. Permeation studies revealed enhanced HES delivery via DMNs with nearly 20-fold higher drug deposition compared to passive diffusion. Pretreatment with DMNs significantly boosted the transdermal permeation of HES (112.86 ± 23.58 μg/cm² after 24 h, p < 0.01), by increased flux (1.31 ± 0.11 μg/cm²/h) and permeability coefficient (0.36 × 10⁻6 cm/s) values. This validated method offers a promising tool for advancing transdermal evaluation in innovative pharmaceutical formulations.

The present investigation was carried out to study the “Residue Dynamics of Flusilazole on Chilli (Capsicum annuum L.) fruits in Kashmir.” Flusilazole 40 EC at the recommended dosage (0.02%) and double the recommended dosage (0.04%) were applied on chilli. The sampling was done on 0, 1, 3, 5, 7, 10, 15, 20, 25, and 30 days after application. Flusilazole was analyzed by GC-MS/MS. The linearity for flusilazole was achieved, with the determination coefficients (R²) of 0.998. The recovery percentage of flusilazole was found to be within the acceptable range of 70%–120%. The dissipation of flusilazole on chilli fruit at the recommended dose (0.02%) left an initial deposit of 1.713 ± 0.004 ppm, which dissipated by 99.35% in 15 days and was below LOQ (0.02 mg kg⁻¹) beyond this period. The half-life period (t_½) and Pre-Harvest Interval (PHI) (T_tol) of 1.55 days and 15.19 days were recorded. Flusilazole at double the recommended dose (0.04%) left an initial deposit of 2.634 ± 0.004 ppm, which dissipated by 99.46% in 25 days and was below LOQ (0.02 mg kg⁻¹) beyond this period. The half-life period (t_½) and PHI (T_tol) of 3.01 days and 19.83 days were recorded.

Osteoporosis (OP) is characterized by reduced bone mass and deteriorated bone structure, leading to an increased risk of fractures. Xiao Chai Hu Tang (XCHT), a traditional Chinese medicine, has shown promise in alleviating OP, but its underlying mechanisms remain unclear. This study aimed to identify the active compounds in XCHT, screen core targets related to postmenopausal osteoporosis (PMOP), and explore its therapeutic mechanisms. Active compounds in XCHT were identified using LC-Q-MS/MS, and PMOP-related targets were screened via protein–protein interaction (PPI) network analysis. Gene Ontology (GO) and KEGG pathway analyses were conducted to explore core signaling pathways, while in vivo validation was performed using ovariectomized rats. Network pharmacology revealed 278 XCHT targets, with 145 related to PMOP. PPI analysis identified 20 core targets. GO and KEGG analyses suggested that XCHT may regulate gene expression, apoptosis, and inflammation via the MAPK signaling pathway. LC–MS identified 49 active compounds. Animal studies confirmed that XCHT mediates MAPK signaling to treat OP. Overall, the findings suggest that XCHT improves PMOP by regulating apoptosis and inflammation through the MAPK pathway, promoting osteoblast formation and reducing osteoblast apoptosis, providing valuable insights into its potential therapeutic role in PMOP.

Polygonatum sibiricum (PS) has great medicinal value as a frequently used Chinese herbal medicine that can be used to treat fatigue, diabetes, and non-alcoholic fatty liver disease (NAFLD). However, the mechanism related to NAFLD is poorly understood. The present work investigated the mechanism by which PS affects high-fat diet (HFD)–mediated NAFLD using an untargeted metabolomics approach. To establish an NAFLD rat model, an HFD was administered to the rats for 12 weeks, followed by treatment with different doses of PS for 12 weeks. The model and therapeutic efficacy were evaluated using blood biochemical parameters, liver lipid measurements, and histopathological examinations, and rat serum samples were analyzed using UPLC-Q-TOF/MS metabolomics methods. The results showed that PS could reverse HFD-induced metabolic disorders, and 25 biomarkers related to the mechanism of PS's effects on NAFLD were identified, involving metabolic pathways such as tyrosine metabolism, glycerophospholipid metabolism, and arachidonic acid metabolism. Our results shed more light on the multitarget therapeutic mechanism of PS in influencing NAFLD and information on how to prevent NAFLD.

Lung surfactant is a lipid–protein complex essential for maintaining alveolar stability by reducing surface tension. In surfactant-deficient states such as pneumonia or acute respiratory distress syndrome, alveolar collapse and impaired gas exchange may occur. Exogenous supplementation of surfactant phospholipids has been proposed to improve lung function. In this work, we performed LC–MS/MS analysis of phosphatidylcholines from a commercially available Essentiale phospholipid preparation in bronchoalveolar lavage (BAL) fluid. The dynamics of BAL phosphatidylcholine levels in mice following intravenous and aerosol administration of Essentiale were investigated for the first time, revealing the time course and extent of pulmonary accumulation for each route. A 20-min aerosol inhalation of Essentiale (≈3 mg/kg phospholipids) achieved BAL phosphatidylcholine concentrations comparable to those obtained with a 500 mg/kg intravenous dose. Repeated daily intravenous dosing caused a gradual increase in BAL phosphatidylcholine over 3–4 days, whereas prolonged aerosol exposure beyond 20 min did not further elevate BAL levels, likely due to uptake by alveolar cells. These findings highlight aerosol inhalation as a more efficient delivery method for surfactant phospholipids and suggest an optimal dosing regimen (≈ 3 mg/kg, 20 min) for adjunctive therapy in conditions associated with surfactant deficiency.

Tegoprazan (TEG) is a commonly used drug in the treatment of gastroesophageal reflux disease. This work aimed to develop and validate an ultra-high-performance liquid chromatography–tandem mass Spectrometry (UPLC–MS/MS) method to determine the levels of TEG in rat plasma and to apply it for a rat pharmacokinetic study. Electrospray ionization source (ESI) positive and multiple reaction monitoring (MRM) mode were selected. The internal standard revaprazan (REV) and TEG were analyzed separately on a Waters ACQUITY UPLC BEH column. Gradient elution with a flow rate of 0.5 mL/min was used. Accuracy and precision were from −8.5% to 12.2%. Linearity was from 2 to 1000 ng/mL. IS-normalized recovery ranged from 109.3% to 113.6%. IS-normalized matrix effect was from 99.0% to 102.8%. The coefficient of variation of matrix effect was less than 10%. Dilution integrity showed that a tenfold dilution also met the guidelines. In the rat pharmacokinetic study, AUC_{(0 − ∞)} and C_max of TEG were 9129.6 ± 1823.3 μg/L*h and 2513.2 ± 707.0 ng/mL, respectively. T_max and T_1/2 were 1.8 ± 1.1 h and 1.6 ± 0.8 h, respectively. We finally established a rapid and robust UPLC–MS/MS quantitative analysis of TEG in rat plasma. Rat pharmacokinetics indicated that TEG was absorbed quickly and fast reached the maximum concentration.

The discovery of the bioactive constituents from herbal medicines remains a huge challenge in front of us. Here, a rapid approach was established by immobilizing full-length calcium sensing receptor (CaR) on the solid supports to screen the bioactive constituents from Epimedii Folium. The results indicated that the immobilized CaR can recognize specifically agonist Ca²⁺ as well as allosteric modulators cinacalcet and NPS2143, and the four bioactive compounds including epimedin A, epimedin B, epimedin C, and icariin were simultaneously screened from over 100 constituents in one injection of the Epimedii Folium extract. The chromatographic competitive binding experiment in combination with molecular docking gave the evidence that epimedin B and epimedin C were bound to the CaR's transmembrane domain whereas epimedin A and icariin interacted with the CaR's extracellular domain. These findings contributed to understanding the action mechanism of the four bioactive compounds with the CaR at the molecular level. Our work broke through the conventional way to isolate the interested molecules first and then testify their pharmacological and pharmacodynamic effects, establishing a rapid approach to discover simultaneously multiple active constituents from an herbal medicine.

The total flavonoid extract of Lithocarpus litseifolius (Hance) Chun (TFLLC), a medicinal and edible plant of the Fagaceae family, has been developed as a preclinical drug candidate in China due to its hypoglycemic effect. The metabolism of TFLLC is closely associated with both its pharmacological activity and potential toxicity. This study investigated the in vitro and in vivo metabolism of TFLLC, which mainly comprises phlorizin and trilobatin. Ultrahigh-performance liquid chromatography coupled with UV spectrometry and high-resolution mass spectrometry (UHPLC–UV–HRMS) was used to detect and characterize TFLLC metabolites in rats following oral administration at 50 mg/kg. TFLLC underwent extensive metabolic transformation, and 13 metabolites were identified. The major drug-related substances included phloretin—derived from glycosidic cleavage of phlorizin and trilobatin—and its conjugated derivatives, such as glucuronidated metabolite M450, sulfated metabolite M354, and dual-conjugated metabolite M530. These metabolites were primarily excreted in bile and urine. Intestinal incubation studies confirmed that phloretin formation could occur in the gut. These findings provide a pharmacokinetic basis to support the pharmacodynamic interpretation and future drug development of TFLLC.

An ultra-high-performance liquid chromatography with fluorescence detection (UPLC-FL) method was developed and applied for the quantitative analysis of D-alanine (D-Ala) and L-alanine (L-Ala), D-leucine (D-Leu) and L-leucine (L-Leu), and D-valine (D-Val) and L-valine (L-Val) in human saliva. Enantiomers were derivatized using the fluorescent reagent R-(−)-4-(3-isothiocyanatepyrrolidine-l-methyl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R-(−)-DBD-PyNCS] at 55°C in the presence of 3% triethylamine for 20 min, yielding aminothiocarbonyl diastereomeric derivatives. The derivatized products were detected using UPLC-FL at an excitation wavelength (Ex) of 460 nm and a fluorescence wavelength (Em) of 550 nm. The limit of detection was 0.010–0.200 μM, and the resolution (Rs) > 1.5 within 20 min. The R² of D-type amino acids in the range of 0.5–50 μM and L-type amino acids in the range of 1.0–500 μM was greater than or equal to 0.9988. Intraday and interday assay precisions were below 6.94%, with average recoveries ranging from 95.20% to 109.6%. This method was succeeded in quantifying amino acid enantiomers in the saliva of healthy volunteers and patients with diabetes. Significant differences in D-Ala, L-Ala, L-Leu, D-Val, and L-Val levels were observed between the two groups (p < 0.01). The proposed method holds potential for diagnosing diabetes and may assist as an early warning indicator of diabetes.

Garcinia oblongifolia, used by the Li ethnic group native to southern China, is valued for its anti-inflammatory, antimicrobial, and wound-healing properties, yet its bioactive metabolites remain poorly characterized. This study aimed to elucidate tissue-specific metabolic mechanisms underlying the pharmacological effects of G. oblongifolia through untargeted metabolite profiling combined with bioactivity correlation analysis. Untargeted UHPLC-QE-MS metabolomics mapped phytochemical variations across leaves, twigs, bark, and fruits, while antioxidant (ABTS/DPPH), antitumor (CCK-8, apoptosis staining), anti-inflammatory (LPS-triggered cytokine assay), and antibacterial assays evaluated functional activities. Metabolomic analysis identified 402 tissue-specific metabolites, with flavonoids and xanthones as major bioactive candidates. Functional assays revealed tissue-dependent efficacy: leaves exhibited superior ABTS radical scavenging, bark showed the strongest DPPH inhibition, and fruits demonstrated potent antitumor/anti-inflammatory effects. All tissues displayed antibacterial activity against Gram-positive pathogens. Correlation analysis highlighted kaempferol-3-O-rutinoside, marucoflavone, and isovitexin as key contributors to antioxidant activity, while daidzin, kaempferol, and cirsimarin were primarily associated with antitumor, anti-inflammatory, and antibacterial activities. Integrated metabolomic-functional profiling demonstrates that tissue-specific bioactivity profiles of G. oblongifolia arise from differential accumulation of key flavonoid/xanthone metabolites. This metabolite-activity matrix provides a scientific basis for optimizing medicinal utilization, quality standardization, and drug discovery from this ethnopharmacological resource. This study characterized tissue-specific metabolites and bioactivities of Garcinia oblongifolia, an ethnomedicine. Untargeted metabolomics (UHPLC-QE-MS) identified 402 metabolites, predominantly flavonoids/xanthones. Bioassays showed leaves excelled in ABTS scavenging, bark in DPPH inhibition, fruits in antitumor/anti-inflammatory effects, and all tissues had antibacterial activity. Key correlated metabolites included kaempferol-3-O-rutinoside (antioxidant), daidzin (antitumor), and kaempferol (anti-inflammatory/antibacterial). This metabolite-activity matrix guides optimized medicinal use and quality control.