Biomedical Chromatography最新文献

Population growth and improved industrialization have led to a sharp rise in the demand for plant medicine. In recent years, there has been a general concern about developing new medicinal resources, cutting down on pharmaceutical waste, and discovering new, effective components of traditional Chinese medicine. A novel medication called Wuteng tablets is made from Schisandra chinensis stems and shows promise as a treatment for Alzheimer's disease. This work is the first development of an overall identification technique based on ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF-MS). Using the MS-DIAL integrated informatics platform and UNIFI software, the chemical components of Wuteng tablets were identified, and the amount of lignin in the tablets was ascertained. This study will identify the chemical components of such medications, aid in the development and utilization of medicinal plant resources, and serve as a foundation for the analysis of the components of their biopharmaceutical origin.

This groundbreaking study introduces a pioneering development of multi-method approach for the first-ever detection and quantification of 13 genotoxic impurities (GTIs) in Apixaban (Apx) drug substance using ultra-performance liquid chromatography (UPLC) with ultraviolet (UV) detector. In this novel endeavor, two distinct UPLC-UV methods, Method A (for impurities A to G) and Method B (for impurities H to M), were meticulously developed and validated as per International Council for Harmonization (ICH) guidelines to address the challenge of identification and control of 13 GTIs in Apx drug substance. The validation process included rigorous assessment of linearity, accuracy, specificity, precision, limit of quantification (LOQ), and limit of detection (LOD) for each impurity in each method which marks a significant advancement in pharmaceutical analysis. The developed methods address the regulatory requirements set forth by ICH M7(R2) guidelines by providing a reliable approach for quantifying GTIs in Apx drug substance at trace levels to minimize the potential carcinogenic risk to the patients.

The classical traditional Chinese medicine formula Huangqi-Guizhi-Wuwutang (HGW) has been shown to enhance sperm production. However, the bioactive components and comprehensive mechanisms underlying the therapeutic effects remain unclear. The present study investigates the potential active ingredients and underlying mechanisms of HGW against spermatogenesis dysfunction. The chemical components of HGW were analyzed by mass spectrometry. And then the “components-targets-pathway-disease” network was constructed using network pharmacology research methods, which aimed to identify the key active components and potential targets of HGW in treating oligospermia. Experimental validation was finally conducted in animal model. The male-specific pathogen-free Kunming mice were divided into five groups: Sham group, Model group, and HGW groups (8, 16, and 32 g/kg of HGW by gavage for 35 days). Chemical profile and network pharmacology results revealed that potential bioactive compounds were dihydrocinnacasside, isomucronulatol, and 6-gingerol, and the mechanism of which was enriched in regulating spermatogenic stem cells (SSCs), endocrine function, and apoptosis. The administration of HGW significantly improved oligospermia in mice. HGW significantly upregulated the expression of marker proteins in SSCs and the potential targets within the testis simultaneously. Our data indicates that HGW enhances the proliferation of SSCs, and HGW can be a promising therapeutic candidate for oligospermia.

Astragali Radix (AR) is one of the famous traditional Chinese medicines (TCMs) for boosting immunity, whereas the quality markers (Q-markers) of AR have not been clearly researched. The immunomodulatory activities of the bioactive extractions and components were evaluated by NO inhibition rate; phagocytic index; IL-10, TNF-α, IL-1β, and IL-6 cytokines in RAW264.7 cells; and the relative proliferation rate of spleen cells. The total saponins (TS) and the grade 2 (Xiaoxuan, XX) of AR showed the strongest immunomodulatory activities. At the concentration of 40 μg/mL, the TS increased spleen cells proliferation by 48.0% and upregulated the level of IL-1β and IL-6. Cytokines in the XX-treated group were at least 1.6 times higher than the control group. A total of 190 common peaks were detected in AR by ultrahigh-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF-MS). The multivariate statistical analyses revealed that 41 compounds were positively correlated with immune responses, and bioactive compounds were verified by using RAW264.7 cell assay. Subsequently, the contents of six compounds in different commercial grades were determined, and the results showed the same trend in contents and activities. Finally, calycosin-7-O-β-D-glucoside, astragaloside IV, astragaloside II, astragaloside I, isomucronulatol-7-O-glucoside, and 9,10-dimethoxypterocarpan-3-O-glucoside were screened out as immunomodulatory Q-markers of AR.

This study focuses on characterizing the forced degradation products of antidiabetic drugs glimepiride (GMD) and glyburide (GBD), with previously unexplored genotoxicity. Drugs underwent stress induced by acid, base, and hydrogen peroxide. For GMD, impurities were profiled and isolated using Hypersil Gold C8 (250 × 10 mm, 5 μ) through semi-preparative HPLC with a fraction collector. For GBD, impurity profiling was performed using semi-preparative HPLC (Hypersil GOLD C18, 250 × 10 mm, 5 μ), and reverse-phase flash chromatography (FP ECOFLEX C18 4 g column) for isolation. Although five GMD and three GBD impurities were detected, only three GMD and two GBD impurities were separated and assessed for purity using analytical RP-HPLC with the purity percentages ranging from 96.6% to 99.9%. LC-Orbitrap MS was used to identify these three GMD impurities (m/z: 408.122, 338.340, 381.160) and two GBD impurities (m/z: 369.065, 325.283). ProTox-II in silico predictions classified all impurities as class 4 and 5, with no positive genotoxicity indications. In vitro comet assays, using HEK cells, indicated that for GMD, impurity 2 and impurity 5 were less genotoxic, whereas impurity 4 exhibited genotoxicity. For GBD, both impurities 1 and 3 were found to be genotoxic, with impurity 3 showing a higher level of genotoxicity than impurity 1.

This work aimed to establish an HILIC-MS/MS method to simultaneously determine the levels of 13 endogenous amino acids and trimethylamine oxide in the biological samples from the mice. Electrospray ion source was used for the analysis of mass spectrometry. The 20 min separation was applied in a Dikma Inspire Hilic column (2.1 × 100.0 mm, 3 μM). Positive ion mode under an MRM model gave a satisfying response value. The limits of quantitation were evaluated by accuracy from −12.59% to 7.89% and precision from 1.77% to 14.00% as well as acceptable interday and intraday precision, matrix effect, recovery, and stability. Later, the assay was successfully used to measure the concentrations of the determinands in the biological samples. Individual and tissue distribution differences for these metabolites were observable. The amino acids had a consistent highest content in the spleens, while the lowest levels were found in the livers. Alanine was the most abundant amino acid in the serum, and taurine kept the highest content in all of the tissues. Trimethylamine oxide remained low level, especially in the liver samples.

The DNA-dependent protein kinase (DNA-PK) is an abundant nuclear protein that mediates DNA double-strand break repair by nonhomologous end joining (NHEJ). As such, DNA-PK is critical for V(D)J recombination in lymphocytes and for survival in cells exposed to ionizing radiation and clastogens. Peposertib (M3814) is a small molecule DNA-PK inhibitor currently in preclinical and clinical development for cancer treatment. We have developed a high-performance liquid chromatography-mass spectrometry method for quantitating peposertib and its metabolite in 0.1 mL human plasma. After MTBE liquid–liquid extraction, chromatographic separation was achieved with a Phenomenex Synergi polar reverse phase (4 μm, 2 × 50 mm) column and a gradient of 0.1% formic acid in acetonitrile and water over an 8 min run time. Mass spectrometric detection was performed on an ABI SCIEX 4000 with electrospray, positive-mode ionization. The assay was linear from 10 to 3000 ng/mL for peposertib and 1–300 ng/mL for the metabolite and proved to be both accurate (97.3%–103.7%) and precise (<8.9%CV) fulfilling criteria from the Food and Drug Administration (FDA) guidance on bioanalytical method validation. This liquid chromatography-tandem mass spectroscopy (LC–MS/MS) assay will support several ongoing clinical studies by defining peposertib pharmacokinetics.

Bone healing is crucial in managing osteomyelitis after fracture fixation. Understanding the mechanism of extensive callus formation in pediatric osteomyelitis is highly important. This study aims to analyze bone and periosteum samples from pediatric patients to elucidate the essential processes involved in callus formation during osteomyelitis. The study included eight patients from our hospital: four with positive microbial culture who underwent osteomyelitis debridement and four who had osteotomy surgery as contral. We used tandem mass tag quantitative proteomics to investigate proteomic changes in bone and periosteum tissues obtained from these patients. Differential expression proteins were analyzed for their pathways through Gene Ontology (GO) annotation, GO enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and protein–protein interaction networks. A total of 4737 proteins were successfully identified. About 2224 differentially expressed proteins were detected in the bone tissues group and periosteum tissues group. Among the differentially expressed proteins, 10 protein genes in the bone group were associated with inflammation and osteogenesis, while in the periosteum group were nine. Cytochrome b-245, beta polypeptide (CYBB), nicotinamide phosphoribosyltransferase (NAMPT), tissue inhibitor of metalloproteinases 1 (TIMP-1), Raf-1 proto-oncogene, serine/threonine kinase (RAF-1), RELA proto-oncogene, NF-KB subunit (RELA), and sphingomyelin synthase 2 (SGMS2) may play an important role in callus formation in patients with osteomyelitis. This study provides novel clues for understanding callus formation in pediatric patients with osteomyelitis.

Mass spectrometry (MS) plays a crucial role in metabolomics, especially in the discovery of disease biomarkers. This review outlines strategies for identifying metabolites, emphasizing precise and detailed use of MS techniques. It explores various methods for quantification, discusses challenges encountered, and examines recent breakthroughs in biomarker discovery. In the field of diagnostics, MS has revolutionized approaches by enabling a deeper understanding of tissue-specific metabolic changes associated with disease. The reliability of results is ensured through robust experimental design and stringent system suitability criteria. In the past, data quality, standardization, and reproducibility were often overlooked despite their significant impact on MS-based metabolomics. Progress in this field heavily depends on continuous training and education. The review also highlights the emergence of innovative MS technologies and methodologies. MS has the potential to transform our understanding of metabolic landscapes, which is crucial for disease biomarker discovery. This article serves as an invaluable resource for researchers in metabolomics, presenting fresh perspectives and advancements that propels the field forward.

Nimbolide is a major furanoid compound isolated from Azadirachta indica. The aim of this study was to characterize the metabolites of nimbolide in rats and to propose the metabolic pathways. The metabolites were generated by incubating nimbolide (10 μM) with rat liver microsomes, nicotinamide adenine dinucleotide phosphate (NADPH), and nucleophiles (glutathione [GSH] or N-acetyl-lysine [NAL]) at 37°C for 60 min. For the in vivo study, nimbolide was intravenously administered to rats at a single dose of 10 mg/kg, and the bile and urine were collected. The metabolites were identified by ultra-high-performance liquid chromatography-quadrupole/orbitrap mass spectrometry (UPLC-Q/Orbitrap-MS) using electrospray ionization in positive ion mode. Totally, nine metabolites were detected, and their identities were characterized by accurate MS and MS/MS data. In GSH-supplemented liver microsomes, GSH conjugation was the primary elimination pathway. The furan ring was bioactivated into cis-butene-1,4-dial that can be trapped by GSH. In NAL-supplemented liver microsomes, two NAL conjugates (M4 and M5) derived from cis-butene-1,4-dial were observed. In rat bile and urine, N-acetyl-cysteine, cysteine-glycine, and GSH conjugate were also found. The current study provides an overview of the metabolism and the bioactivation profiles of nimbolide in rats, which aids in understanding its safety and activity.