Biomedical Chromatography最新文献

The traditional formulation Hanchuan zupa granules (HCZPs) have been widely used for controlling coronavirus disease 2019 (COVID-19). However, its active components remain unknown. Here, HCZP components targeting the spike receptor-binding domain (S-RBD) of SARS-CoV-2 were investigated using a surface plasmon resonance (SPR) biosensor-based active ingredient recognition system (SPR-AIRS). Recombinant S-RBD proteins were immobilized on the SPR chip by amine coupling for the prescreening of nine HCZP medicinal herbs. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) identified gallic acid (GA) and methyl gallate (MG) from Rosa rugosa as S-RBD ligands, with K_D values of 2.69 and 0.95 μM, respectively, as shown by SPR. Molecular dynamics indicated that GA formed hydrogen bonds with G496, N501, and Y505 of S-RBD, and MG with G496 and Y505, inhibiting S-RBD binding to angiotensin-converting enzyme 2 (ACE2). SPR-based competition analysis verified that both compounds blocked S-RBD and ACE2 binding, and SPR demonstrated that GA and MG bound to ACE2 (K_D = 5.10 and 4.05 μM, respectively), suggesting that they blocked the receptor and neutralized SARS-CoV-2. Infection with SARS-CoV-2 pseudovirus showed that GA and MG suppressed viral entry into 293T-ACE2 cells. These S-RBD inhibitors have potential for drug design, while the findings provide a reference on HCZP composition and its use for treating COVID-19.

A sensitive, rapid, and simple HPLC–MS/MS method was first developed and fully validated to determine the icaritin (ICT) and its novel 3-methylcarbamate prodrug (3N) simultaneously in rat plasma. Analytes were extracted from rat plasma using a liquid–liquid extraction (LLE) method. Chromatographic separation was performed on ACE Excel 2 C18-Amide column. Quantitation of analytes was conducted on an LCMS-8060 triple-quadrupole tandem mass spectrometer. The quantitation mode was the multiple reaction monitoring via positive electrospray ionization. The calibration curve was linear over the concentration range of 1 to 200 ng/ml for ICT with a correlation coefficient of r = 0.9950 and 1 to 400 ng/ml for 3N with a correlation coefficient of r = 0.9956. The intra-precision RSDs were ≤12% for ICT and 3N. The inter-day precision RSDs were ≤10% for ICT and 3N. The accuracy RE was between −2.6% and 7.8% for ICT and 3N. The average ICT, 3N and IS recoveries were 87.9%, 83.6%, and 84.3%. The plasma matrix of ICT and 3N complied with the guidelines. ICT and 3N were stable in rat plasma under various tested conditions. This work has been successfully applied to studying the pharmacokinetics of ICT and 3N.

Shengmai Jianghuang San (SMJHS) is a traditional Chinese herbal compound reported to inhibit Nasopharyngeal Carcinoma (NPC) progression and enhance radiosensitivity. However, the specific active ingredients and regulatory mechanisms of SMJHS against NPC, particularly under hypoxic conditions, remain unclear. In this study, Sprague–Dawley (SD) rats were gavaged with Shengmai Jianghuang San (SMJHS), and their blood was collected from the abdominal aorta. UHPLC-Q-Exactive orbitrap MS/MS was used to identify the metabolite profiles of SMJHS drug-containing serum. A molecular network of the active compositions in SMJHS targeting NPC was constructed through network pharmacology and molecular docking. The HIF-1α/VEGF pathway was in key positions. The effects of SMJHS on the proliferation, migration, and radiosensitivity of hypoxic NPC cells were assessed by in vitro experiments. NPC cell lines stably overexpressing HIF-1α were established using a lentivirus to investigate the regulation of HIF-1α/VEGF signaling in hypoxic NPC cells by SMJHS. Through a combination of network pharmacological analysis, cellular biofunctional validation, and molecular biochemical experiments, our study found that SMJHS had an anti-proliferative effect on NPC cells cultured under hypoxic conditions, inhibiting their migration and increasing their radiosensitivity. Additionally, SMJHS suppressed the expression of HIF-1α and VEGFA, exhibiting potential as an effective option for improving NPC treatment.

Euphorbiae pekinensis Radix (EPR) is a traditional Chinese herb commonly used to treat edema, pleural effusion, and ascites. However, counterfeit and adulterated products often appear in the market because of the homonym phenomenon, similar appearance, and artificial forgery of Chinese herbs. This study comprehensively evaluated the quality of EPR using multiple methods. The DNA barcode technique was used to identify EPR, while the UPLC-Q-TOF-MS technique was utilized to analyze the chemical composition of EPR. A total of 15 tannin and phenolic acid components were identified. Furthermore, UPLC fingerprints of EPR and its common counterfeit products were established, and unsupervised and supervised pattern recognition models were developed using these fingerprints. The backpropagation artificial neural network and counter-propagation artificial neural network models accurately identified counterfeit and adulterated products, with a counterfeit ratio of more than 25%. Finally, the contents of the chemical markers 3,3′-di-O-methyl ellagic acid-4′-O-β-D-glucopyranoside, ellagic acid, 3,3′-di-O-methyl ellagic acid-4′-O-β-d-xylopyranoside, and 3,3′-di-O-methyl ellagic acid were determined to range from 0.05% to 0.11%, 1.95% to 8.52%, 0.27% to 0.86%, and 0.10% to 0.42%, respectively. This proposed strategy offers a general procedure for identifying Chinese herbs and distinguishing between counterfeit and adulterated products.

Peptide aggregation is one of the key challenges associated with the development of therapeutic peptides. Peptide and protein aggregates are considered as one of the most important critical quality attributes (CQA). Therapeutic liraglutide (LGT) is proteinaceous in nature, and aggregation can be triggered by various environmental stress condition. Therefore, it is essential to separate and identify aggregation states of such drugs. In this study, we have established size exclusion chromatography-liquid chromatography-ultraviolet/high resolution mass spectrometry (SEC-LC-UV/HRMS) method to separate and identify the stress induced LGT aggregates. LGT samples were subjected to photolytic, thermal, freeze thaw and shaking stress conditions. Additionally, LGT solution was incubated with surfactant and excipient that are commonly used in peptide formulation, to evaluate their impact on aggregation level and physicochemical stability over time. The developed SEC method was also validated for specificity, accuracy, precision and linearity. The results of this study will be useful for investigators to monitor LGT aggregates during product development.

Medicinal fungi Phellinus igniarius exhibited hypoglycemic effects; however, the protective mechanisms of P. igniarius on type 2 diabetes are not yet fully understood. Herein, the anti-diabetic effect of P. igniarius was investigated via gas chromatography–mass spectrometry (GC/MS)-based metabolome analysis. The rats were divided into normal group; model group; positive group; and groups treated with low, medium, and high dose of P. igniarius. After the treatments, a significant decrease in blood glucose concentration was observed. The levels of total cholesterol and triglyceride were dramatically decreased, whereas the level of insulin was increased. Multivariate statistical analysis revealed 31 differential endogenous metabolites between model group and normal group. A total of 14, 28, and 31 biomarkers were identified for low, medium, and high dose of P. igniarius treated groups, respectively. Twenty-one of the biomarkers were validated by using standard substances. The linear correlation coefficients ranged from 0.9990 to 1.0000. The methodology exhibited good repeatability, recoveries, and stability. The major intervened metabolic pathways covered glyoxylate and dicarboxylic acid metabolism; alanine, aspartate, and glutamate metabolism; and glycine, serine, and threonine metabolism. Our metabolome analysis has provided insights into the underlying mechanism of P. igniarius on type 2 diabetes.

In this research, the study utilized the root, leaf, and petiole parts of in vitro grown Salvia hispanica plants as explants. Following UV-C treatment applied to developing callus, methanol extracts were obtained and analyzed using liquid chromatography–mass spectrometry (LC/MS) to investigate their anticancer properties. First, the seeds of S. hispanica were soaked in commercial bleach for 6 min to ensure surface sterilization. The most effective antimicrobial activity on Gram-negative bacteria, with a zone diameter (11 ± 0.82 mm), was noticed in callus extracts obtained from the petiole explant in the second protocol against Klebsiella pneumoniae EMCS bacteria. Anticancer activities on SH-SY5Y human neuroblastoma cells were investigated by using 1000, 500, 250, 125, 62.5, 31.25, 15.62, and 78.12 μg/mL doses of the extracts, and the most effective cytotoxic activity was determined at the 1000 μg/mL dose of the extracts obtained from both protocols. The extracts were determined to inhibit hCAI, hCAII, AChE, and BChE enzymes. The content of 53 different phytochemical components of the extracts was analyzed by liquid chromatography with tandem mass spectrometry (LC–MS/MS). Rosmarinic acid, quinic acid, and caffeic acid were found in the highest concentration. The comprehensive LC–MS/MS analysis of S. hispanica extracts revealed a diverse array of phytochemical compounds, highlighting its potential for therapeutic applications.

Hashimoto's thyroiditis (HT) is an autoimmune disease caused by the immune system attacking healthy tissues. However, the exact pathogenesis of HT remains unclear. Metabolomic analysis was performed to obtain information about the possible pathogenic mechanisms and diagnostic biomarkers of HT. The amino acid profile was analyzed using an LC–MS/MS method using serum samples obtained from 30 patients diagnosed with ultrasonographic imaging and laboratory markers (thyroid stimulating hormone) free thyroxine and thyroid peroxidase) and 30 healthy individuals. There were statistically significant changes in 27 amino acids out of 32 amino acids analyzed (p < 0.05). Based on the receiver operating characteristic curve analysis, the six amino acid (1-methylhistidine, cystine, norvaline, histidine, glutamic acid and leucine) biomarkers showed high sensitivity, specificity (area under the curve > 0.98), positive likelihood ratio and low negative likelihood ratio. Also, according to pathway analysis, degradation of phenylalanine, tyrosine and tryptophan biosynthesis was the highest metabolic pathway according to the impact value (p < 0.001 and impact value = 1.0). We provide serum amino acid profiles of patients with Hashimoto's thyroiditis and identify five potential biomarkers for early diagnosis by clinicians.

Terminalia chebula exhibits a high level of antioxidant capacity and is highly valued in medicine and cosmetics. However, its main efficacy and active ingredients related to antioxidant, whitening, and anti-aging are still unclear. In this study, the active site responsible for its cosmetic efficacy was specified by the biological activity-guided method and further characterized by using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS/MS). T. chebula was ultrasonically extracted by five solvents, and 30% ethanol extract was screened out for subsequent purification by 1,1-D-iphenyl-2-picrylhydrazyl radical (DPPH), 2,2′-Azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS), hydroxyl, and superoxide anion free radical scavenging assays. Five elution fractions were obtained by column chromatography on D101 macroporous adsorbent resin eluted by an increased proportion of ethanol. The 30% ethanol elution fraction was specified as the enrichment site of active ingredients showing good antioxidant capacity and potent inhibitory activity against tyrosinase and elastase. A total of 30 compounds were identified by UHPLC-QTOF-MS/MS in the 30% ethanol elution fraction, including 11 gallotannins, 14 ellagitannins, and 5 other compounds, and these compounds may be the key ingredients in cosmetics beneficial for the skin. Such a biological activity-guided method has provided a simple and rapid venue for specifying the components of medicinal herbs responsible for cosmetic efficacy.

Monitoring antibiotic plasma levels is critical in populations with altered pharmacokinetics, such as critically ill patients in neonatal or adult intensive care units. This study aimed to develop and validate a rapid, reproducible and sensitive liquid chromatography–tandem mass spectrometry assay (LC–MS/MS) for measuring total and unbound concentrations of amoxicillin, ampicillin, ceftazidime, ceftriaxone, ertapenem, fosfomycin and penicillin G in human plasma. The method required 20 and 250 μl sample volumes for measuring total and unbound concentrations, respectively. Sample preparation involved protein precipitation and the addition of an internal standard. Ultrafiltration separated unbound drugs. Method validation covered selectivity, carryover, linearity, accuracy, precision, dilution effects, matrix effects and stability. The LC–MS/MS was performed within a run time of 7.5 min. Calibration curves were linear for ceftazidime and ertapenem (ranges 0.1–50 and 0.05–100 mg/l, respectively) and quadratic for other analytes (0.1–50 mg/l, except for ampicillin: 0.1–20 mg/l; R² > 0.990). Accuracy was within ±15% of the nominal concentration, and precision did not exceed ±15% (relative standard deviation). Samples showed no significant degradation at the tested temperatures and time points. Clinical applicability was demonstrated in a critically ill neonate. This method with minimal sample volume and short analysis time enables the measurement of total and unbound concentrations of selected antibiotics, and is suitable for routine clinical care and studies.