Biomedical Chromatography最新文献

Bile acids function both as facilitators of biliary lipid transport and as signaling mediators that maintain metabolic homeostasis. Perturbations in bile acid metabolism accompany diverse forms of liver injury, yet conventional clinical assays lack the sensitivity and specificity required for comprehensive bile acid profiling. To address this, we aimed to develop and validate a rapid liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of 20 bile acids using minimal serum volume and shortened analysis time. The developed method requires only 20 μL of serum and completes analysis within 13 min. Following protein precipitation, analytes were separated on a C18 column with gradient elution. The method demonstrated excellent sensitivity with limits of detection of 1–2 ng/mL and a uniform limit of quantification of 5 ng/mL for all analytes. Validation results showed satisfactory performance: accuracy within ± 14.8%, intra-assay and interassay precision below 16.5%, extraction recovery of 72.6%–108.4%, and acceptable matrix effects. Application to murine hepatotoxicity models induced by α-amanitin (α-AMA), ethanol, and acetaminophen (APAP) revealed distinct, compound-specific bile acid alterations. This work establishes a rapid, miniaturized, and validated LC-MS/MS approach, demonstrating its utility in revealing toxin-specific bile acid signatures, which provides a valuable tool for preclinical hepatotoxicity research and potential translational applications.

“Shengma–Gegen Decoction” (SMGGD), a traditional Chinese herbal formula, is composed of four medicinal herbs, namely, Cimicifugae Rhizoma, Puerariae Lobatae Radix, Paeoniae Radix Alba, and Glycyrrhizae Radix et Rhizoma, and has been proven effective in alleviating headache symptoms. However, the material basis of its pharmacological effects and the metabolic pathways through which it regulates diseases remain unclear. In this study, ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was used to detect the aqueous decoction of SMGGD, as well as plasma and brain tissue samples of animals after administration. Combined with desorption electrospray ionization mass spectrometry imaging (DESI-MSI), the distribution of brain-penetrating components was analyzed, and the differential endogenous metabolites in brain tissues were enriched. The results showed that a total of 104 chemical components, 39 blood-entering components, and two brain-penetrating components were identified from SMGGD. These two brain-penetrating components were distributed in the prefrontal cortex and thalamus, consistent with the localization of differential metabolites. The differential metabolites were enriched in sphingolipid and glycerophospholipid metabolic pathways. This study clarifies the active components of SMGGD for treating migraine and the metabolic pathways it regulates, providing a theoretical basis for its quality standard research, secondary development, and clinical application.

Vitex negundo L. (Verbenaceae) is a well-known medicinal plant used in traditional medicine for the treatment of inflammatory, microbial, and oxidative stress–related disorders. The present study aimed to evaluate antibacterial and antioxidant activities of Vitex negundo leaf extracts, along with their chemical characterization using Fourier transform infrared (FT-IR) spectroscopy and gas chromatography–mass spectrometry (GC–MS). Antibacterial activity of acetone, methanol, and aqueous extracts was assessed by agar well diffusion method against four bacterial strains. The acetone extract exhibited notable antibacterial activity, particularly against Bacillus cereus and Bacillus subtilis. Antioxidant potential was evaluated using the DPPH radical scavenging assay, where acetone and methanol extracts demonstrated the highest activity in a dose-dependent manner. FT-IR analysis confirmed the presence of various functional groups associated with bioactive compounds. GC–MS profiling identified 24 compounds in the acetone extract and 20 compounds in the methanol extract, with major constituents such as isoambreinolide, catechol, n-hexadecanoic acid, kojic acid, karanjin, and phytol, many of which are known for their antimicrobial, antioxidant and anti-inflammatory properties. Overall, the findings indicate that Vitex negundo leaves are rich in bioactive phytochemicals and possess significant antibacterial and antioxidant activities, supporting their traditional medicinal use and highlighting their potential for pharmaceutical applications.

Calycosin is an active compound of Danggui Buxue Decoction (DBD) with various pharmacological effects including anti-inflammatory and immunomodulatory activities. However, it remains unclear whether long-term administration and the multi component of DBD influence the disposition of calycosin. This study aimed to investigate the absorption, distribution, and metabolism of calycosin following long-term oral administration of DBD. SD rats were divided into six groups. Plasma was collected at 0.083–24 h after the last administration and separated for the determination of calycosin. For the tissue distribution study, rats were sacrificed 1 h after the last dose and tissues were collected. The concentrations of analytes were determined after sample preparation. Plasma collected within 1 h was used for metabolite identification of calycosin. Pharmacokinetic results indicated that the SH group exhibited the highest plasma exposure of calycosin. Calycosin accumulation was detected in the liver of six groups with levels ranking as MH > SH > ML > SL > MD > SD group. Metabolism studies revealed that the MD group had the highest number of calycosin metabolites. Both long-term administration and the complex composition influence the absorption, distribution, and metabolism of calycosin in rats after oral administration of DBD. Therefore, this study provides a theoretical basis for the rational clinical use of DBD and calycosin.

Chimonanthus salicifolius leaves (CSL), a traditional She medicine, are used both as food and medicine. Conventionally, tender leaves are used as tea beverages, while mature leaves serve as medicinal materials. However, their difference in chemistry and activity remains unknown. In this study, HPLC fingerprints of 20 batches of both tender and mature CSL were established, revealing a shared metabolite backbone, yet hierarchical cluster analysis clearly split the two stages into discrete chemotypes. DPPH and ABTS assays demonstrated that mature leaves possessed significantly higher antioxidant capacity than tender leaves. Integration of Pearson correlation analysis and partial least squares regression analysis uncovered the spectrum–bioactivity relationship, guiding three flavonoid glycosides, isoquercitrin (IQ), kaempferol-7-O-rutinoside, and nicotiflorin, as antioxidant quality markers (Q-markers). Moreover, the quantitation of Q-markers illustrated that IQ emerged as the pivotal differential compound: Its abundance in mature leaves was 1.7-fold higher than in tender leaves, whereas the content of the three glycosides was nearly twice as high in tender leaves. The established strategy for identifying antioxidant Q-markers, based on HPLC fingerprinting and spectrum–effect relationships, effectively reveals quality differences between mature and tender leaves and provides a robust scientific basis for the tailored exploitation of CSL in both medicinal and nutraceutical applications.

Ru-Pi-Ning granules were a classic traditional Chinese medicine (TCM) prescription with good therapeutic effects on mammary gland hyperplasia (MGH). However, the main components and potential mechanisms of action of Ru-Pi-Ning were still unclear. This study aimed to preliminarily elucidate the potential mechanism of Ru-Pi-Ning in the treatment of MGH based on ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and network pharmacology. Compounds in Ru-Pi-Ning were determined by UPLC-Q-TOF-MS. A Venn diagram was created with drug targets and disease targets, and a PPI network diagram was constructed with shared targets using the STRING database. GO and KEGG pathway analysis were performed using the Metascape platform; molecular docking was carried out using AutoDockTools. A total of 127 active components and 75 overlapping genes were screened out. The main biological processes included cellular response to hormone stimulus and positive regulation of cell migration. Ru-Pi-Ning in treating MGH could regulate several signaling pathways, including the relaxin-signaling pathway and HIF-1 signaling pathway. The core targets were TP53, EGFR, and AKT1. Molecular docking indicated that the active compounds had good binding ability with the screened targets. Ru-Pi-Ning might exert its therapeutic effect on MGH through multiple components, multiple targets, and multiple pathways.

Genotoxic impurities (GTIs) in pharmaceutical products represent a critical safety concern because they can induce DNA damage and may lead to mutagenic and carcinogenic effects even at trace concentrations. Consequently, regulatory authorities such as EMA, FDA, and ICH require a risk-based and impurity-specific control strategy, as defined in ICH M7, for active pharmaceutical ingredients (APIs) and finished dosage forms. Due to their structural and physicochemical diversity, GTIs cannot be adequately monitored using a single analytical technique, and method selection must be tailored to the characteristics of each impurity. In this study, impurity-specific analytical methods were developed and validated to demonstrate the practical application of this regulatory approach in different pharmaceutical matrices. GC–MS method was optimized for the determination of volatile alkylating agents, namely methyl, ethyl, and isopropyl methanesulfonate in imatinib mesylate API. LC–MS/MS method was established for the quantification of 5-(4′-(azidomethyl)-[1,1′-biphenyl]-2-yl)-1H-tetrazole, a polar and thermolabile azido impurity in valsartan formulations. Both methods were validated in accordance with ICH Q2(R1) guidelines, demonstrating excellent specificity, linearity (R² > 0.999), accuracy, and precision, with detection limits sufficient to meet regulatory requirements. Application to commercial products confirmed the robustness of methods and their suitability for routine quality control supporting effective GTI risk management.

Chlorogenic acid (CA) exhibits significant anti-inflammatory potential for acute disease treatment, but suffers from low oral bioavailability and nonlinear pharmacokinetic behavior due to hepatic rapid metabolism, low solubility and instability. This study developed a sustained-release capsule (CA-CAPC, 1:2) containing both immediate-release CA powder and sustained-release CA-phospholipid complex (CA-PC, 1:1) to enhance bioavailability. Comprehensive characterization (IR, DSC, hygroscopicity, angle of repose, and in vitro dissolution) confirmed the stable formation of the complex through hydrogen bonding and hydrophobic interactions, with improved flowability and low hygroscopicity. The established UPLC-MS/MS method showed good linearity (r² = 0.9967) in the range of 12.5–3200 ng/mL, and its accuracy, precision, recovery rate, matrix effect, and stability all met the requirements for biological sample analysis. Quantify the concentration of CA in plasma using this validated UPLC-MS/MS method. Pharmacokinetic studies in beagle dogs showed CA-CAPC significantly increased AUC_(0-t), AUC_(0-∞), and C_max, advanced T_max, and prolonged MRT_(0-t) and T_1/2 versus CA or CA-PC, indicating enhanced absorption and extended therapeutic coverage. Cross-species consistency in rats further validated the formulation. The CA-CAPC strategy leverages rapid onset from CA powder and sustained release from CA-PC, overcoming CA's pharmacokinetic limitations and providing a promising preclinical foundation for acute disease therapy.

A sensitive and robust HPLC-MS/MS method was developed and validated for the simultaneous determination of YWS22026 and its major metabolite M3 in rat plasma using protein precipitation. Chromatographic separation was achieved on a Shim-pack Scepter C18 column (4.6 × 50 mm, 3 μm) with a gradient elution program (mobile phase A: 0.2% formic acid in water; mobile phase B: methanol) at a flow rate of 0.9 mL/min. Detection was performed via positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. The method demonstrated excellent specificity, accuracy (relative error, %RE < 9.1%), precision (relative standard deviation, %RSD < 9.9%), and linearity (5–2000 ng/mL for both analytes). The lower limit of quantification (LLOQ) was 5 ng/mL. Stability studies confirmed that plasma samples remained stable under various storage conditions (at room temperature for 10 h, −20°C for 22 days, three freeze–thaw cycles, and 4°C autosampler storage for 73 h). This validated method was successfully applied to a 26-week concomitant toxicokinetic study in Sprague–Dawley rats, which revealed significant sex-related differences in exposure for both YWS22026 and M3. These findings provide valuable insights for informing the safety assessment of YWS22026 in subsequent clinical trials.