Ultrahigh-performance Liquid Chromatography-tandem Mass Spectrometry Research Articles

Perfluoropentane-based oxygen-loaded nanodroplets reduce microglial activation through metabolic reprogramming.

JOURNAL/nrgr/04.03/01300535-202504000-00032/figure1/v/2024-07-06T104127Z/r/image-tiff Microglia, the primary immune cells within the brain, have gained recognition as a promising therapeutic target for managing neurodegenerative diseases within the central nervous system, including Parkinson's disease. Nanoscale perfluorocarbon droplets have been reported to not only possess a high oxygen-carrying capacity, but also exhibit remarkable anti-inflammatory properties. However, the role of perfluoropentane in microglia-mediated central inflammatory reactions remains poorly understood. In this study, we developed perfluoropentane-based oxygen-loaded nanodroplets (PFP-OLNDs) and found that pretreatment with these droplets suppressed the lipopolysaccharide-induced activation of M1-type microglia in vitro and in vivo, and suppressed microglial activation in a mouse model of Parkinson's disease. Microglial suppression led to a reduction in the inflammatory response, oxidative stress, and cell migration capacity in vitro. Consequently, the neurotoxic effects were mitigated, which alleviated neuronal degeneration. Additionally, ultrahigh-performance liquid chromatography-tandem mass spectrometry showed that the anti-inflammatory effects of PFP-OLNDs mainly resulted from the modulation of microglial metabolic reprogramming. We further showed that PFP-OLNDs regulated microglial metabolic reprogramming through the AKT-mTOR-HIF-1α pathway. Collectively, our findings suggest that the novel PFP-OLNDs constructed in this study alleviate microglia-mediated central inflammatory reactions through metabolic reprogramming.

Profiling of urinary steroids aided by lithium ion adduction-based ultrahigh-performance liquid chromatography-tandem mass spectrometry.

As 3-OH-containing steroids are prone to dehydration by conventional electrospray ionization, reducing detection sensitivity, Li ion adduction-based ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS), developed to prevent dehydration and effectively detect 3-OH steroids, was applied for profiling total and free steroids in urine. Free urinary steroids were isolated directly from urine by solid-phase extraction (SPE) with 80% acetonitrile. The total steroids were prepared by enzymatic treatment of urine with a cocktail of sulfatase and glucronidase, protein precipitation, and separation with the above SPE. In order to detect as many steroid types as possible, UHPLC/MS/MS (Li method) with Li+ solution added after the column was used for analysis in addition to the conventional method of detecting protonated ions (H method). The 13 3-OH steroids and the remaining 16 steroids were quantified by standard curves prepared using product ion transitions derived from [M + Li]+ and MH+ , respectively. Two groups of human urine, male and female urine, were analyzed. 3-OH steroids could be detected with greater sensitivity using the Li method than the conventional method. The absolute amounts of each steroid were normalized based on creatinine levels. The difference between the male and female groups are clearly attributable to sex steroids. Twenty-nine total steroids and 19 free steroids were identified in a limited volume (240 mL) of urine. Of these, 13 3-OH steroids were better detected by Li+ adduction-based UHPLC/MS/MS.

Determination of higenamine in common natural spices by ultrahigh-performance liquid chromatography-tandem mass spectrometry.

Higenamine (HG) is a β2 receptor agonist and was explicitly added to the Prohibited List of the World Anti-Doping Agency in 2017. This compound is prohibited in both in- and out-of-competition athletes and falls under the category of nonthreshold substances. Because of HG presence in numerous plants, as evidenced by a growing body of research data, an exception was made for HG in the TD2017MRPL document, in which adverse analytical findings (AAFs) were not reported if the urinary HG concentration was less than 10ng/mL. In this study, a comprehensive and systematic analysis of the HG content in five batches of samples from each of the 48 natural spices selected for this investigation was conducted using UPLC-MS/MS technology. Method validation was carried out in accordance with the ICH Analytical Procedures and Methods Validation for Drugs and Biologics Guidance, and the experimental results demonstrated that the method provided appropriate sensitivity, precision, stability, linearity, and accuracy. HG was detected for the first time in Houttuynia cordata, Zingiber officinale, Cinnamomum cassia, Stevia rebaudiana, Piper nigrum, Siraitia grosuenorii, Platycodon grandiflorus, and Myristica fragrans. Furthermore, the content of HG was found to vary significantly among the different plant parts of Nelumbo nucifera, such as rhizomes, leaves, seeds, and plumules. This paper provides systematic and comprehensive data to support the safe use of spices in athletes' diets, thereby reducing the risk of food-sourced doping violations.

Rapid Determination of Diverse Ganoderic Acids in Ganoderma Using UPLC–MS/MS

Background:: Ganoderma is known for its pharmaceutical, nutritional, and functional benefits. Its primary bioactive components are ganoderic acids. However, previous quantification methods only analyzed an individual or limited number of ganoderic acids. This study aims to develop a reliable method for simultaneously quantifying the major ganoderic acids to enhance Ganoderma quality control and study its active ingredients. Methods:: We developed a rapid quality assessment method to simultaneously determine the eleven ganoderic acids in Ganoderma using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The sample extraction method, along with mass spectrometric detection and chromatographic separation conditions was optimized. The separation was carried out using the ACQUITY UPLC BEH C18 column with a gradient elution of 0.1% (v/v) formic acid in water and acetonitrile. The mass spectrometry utilized negative mode electrospray ionization (ESI), with quantitative analysis being carried out in the MRM mode. Results:: The calibration curves showed good correlation coefficients (r2 > 0.998). The recovery range was 89.1–114.0%. The intra-day and inter-day relative standard deviation (RSD) were below 6.8% (n = 6) and 8.1% (n = 6), respectively. Furthermore, the detection and quantification limits were 0.66–6.55 μg/kg and 2.20–21.84 μg/kg, respectively. All 11 ganoderic acids in the sample solution remained stable at room temperature for 72 hours. A total of 11 ganoderic acids were quantified in the 13 Ganoderma samples. The levels of ganoderic acids were higher in Ganoderma lucidum than in Ganoderma sinense. Conclusion:: The method developed in this study can quantify ganoderic acids in Ganoderma lucidum, thus establishing a technical foundation for evaluating the Ganoderma quality.

Evaluation of drug interactions of Saposhnikoviae Radix and its major components with astragaloside IV and paeoniflorin using in vitro and in vivo experiments

Saposhnikoviae Radix (SR) may enhance the pharmacodynamics of Huangqi Chifeng Tang (HQCFT) in the treatment of cerebral infarction according to our previous research, but the underlying mechanism is unknown. Herein, an in vivo pharmacokinetic assay in rats and in vitro MDCK-MDR1 cell assays were used to investigate the possible mechanism of SR, its main components, and its interactions with Astragali Radix (AR) and Paeoniae Radix (PR). An ultrahigh-performance liquid chromatography–tandem mass spectrometry (UPLC‒MS/MS)-based analytical method for quantifying astragaloside IV (ASIV) and paeoniflorin (PAE) in microdialysis and transport samples was developed. The pharmacokinetic parameters of SR were determined using noncompartmental analyses CCK-8 assays were used to detect the cytotoxicity of ASIV, PAE, cimifugin (CIM), prim-o-glucosylcimifugin (POG) and their combinations. Moreover, drug transport was studied using MDCK-MDR1 cells. Western blotting was performed to measure the protein expression levels of P-GP and MRP1. Claudin-5, ZO-1, and F-actin expression was determined via immunohistochemical staining of MDCK-MDR1 cells. harmacokinetic studies revealed that, compared with those of Huangqi Chifeng Tang-Saposhnikoviae Radix (HQCFT-SR), the Tmax of ASIV increased by 11.11 %, and the MRT0-t and Tmax of PAE increased by 11.19 % and 20 %, respectively, in the HQCFT group. Transport studies revealed that when ASIV was coincubated with 28 μM CIM or POG, the apparent permeability coefficient (Papp) increased by 71.52 % and 50.33 %, respectively. Coincubation of PAE with 120 μM CIM or POG increased the Papp by 87.62 % and 60.95 %, respectively. Moreover, CIM and POG significantly downregulated P-gp and MRP1 (P < 0.05), inhibited the expression of Claudin-5, ZO-1, and F-actin (P < 0.05), and affected intercellular tight junctions (TJs). In conclusion, our study successfully established a selective, sensitive and reproducible UPLC‒MS/MS analytical method to detect drug‒drug interactions between SR, AR and PR in vivo and in vitro, which is beneficial for enhancing the therapeutic efficacies of AR and PR. Moreover, this study provides a theoretical basis for further research on the use of SR as a drug carrier.

Determination of 55 Veterinary Antibiotics in Chicken Manure Using Ultra-High-Performance Liquid Chromatography – Tandem Mass Spectrometry (UPLC-MS/MS)

A highly efficient method for the detection and quantification of 55 veterinary antibiotics (VAs) in chicken manure belonging to six drug classes using solid-phase extraction (SPE) and ultrahigh-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed. The mixture of EDTA–McIlvaine buffer (pH 3.0) and the organic extractant (methanol–acetonitrile, 1:3, v/v) was used to isolate the target VAs from freeze-dried chicken manure. The extract was purified by a hydrophile–lipophile balance cartridge. All antibiotics showed excellent linear relationships in the range between 1 and 100 µg/kg with the coefficients of determination of the standard curves above 0.990. The recoveries of antibiotics were between 35.66% and 103.54% using matrix-matched calibration for quantification. The limits of detection and quantification were from 0.01 to 1.59 µg/kg and 0.04 to 4.76 µg/kg, respectively. The method was demployed to determine VAs in chicken manure from 12 farms, revealing contamination in all samples. A total of 13 VAs belonging to six classes were detected with concentrations up to 41.47 mg/kg. The presence of high concentrations of antibiotic residues in poultry manure poses a potential risk for environmental contamination. This work has identified significant differences in the types of VAs compared to literature reports. Additionally, it detected pleuromutilin antibiotics in chicken manure for the first time.

Comparison of filter membranes in the analysis of 183 veterinary and other drugs by liquid chromatography-tandem mass spectrometry.

Although filtration is one of the most common steps in sample preparation for chemical analysis, filter membrane materials can leach contaminants and/or retain some analytes in the filtered solutions. In multiclass, multiresidue analysis of veterinary drugs, it is challenging to find one type of filter membrane that does not retain at least some of the analytes before injection in ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). In this study, different filter membranes were tested for use in UHPLC-MS/MS analysis of 183 diverse drugs in bovine muscle, kidney, and liver tissues. Membranes evaluated consisted of polytetrafluoroethylene (PTFE), polyvinylidene difluoride (PVDF), polyethersulfone, nylon, and regenerated cellulose. Drug classes represented among the analytes included β-agonists, β-lactams, anthelmintics, macrolides, tetracyclines, sulfonamides, tranquilizers, (fluoro)quinolones, anti-inflammatories, nitroimidazoles, coccidiostats, phenicols, and others. Although the presence of a matrix helped reduce the binding of analytes on surface active sites, all of the filter types partially retained at least some of the drugs in the final extracts. In testing by flow-injection analysis, all of the membrane filters were also observed to leach interfering components. Ultimately, filtration was avoided altogether in the final sample preparation approach known as the quick, easy, cheap, effective, rugged, safe, efficient, and robust (QuEChERSER) mega-method, and ultracentrifugation was chosen as an alternative.

A Validated Assay to Quantify Osimertinib and Its Metabolites, AZ5104 and AZ7550, from Microsampled Dried Blood Spots and Plasma.

Osimertinib is an oral small-molecule tyrosine kinase receptor inhibitor used to treat non-small cell lung cancer (NSCLC) with a sensitizing epidermal growth factor receptor mutation. Patients may experience drug toxicity and require dose deescalation. The study aimed to quantitate osimertinib and its 2 active metabolites, AZ5104 and AZ7550, in microsampled dried blood spots (DBS) collected from patients with NSCLC using a hemaPEN device and compare them with plasma drug levels. A 6-min ultrahigh-performance liquid chromatography-tandem mass spectrometry method was developed and validated using plasma and DBS. The accuracy, selectivity, matrix effect, recovery, and stability were assessed using bioanalytical validation criteria. The hematocrit effect was investigated in DBS. Drug levels were measured in 15 patients with NSCLC, and the Bland-Altman method was used to compare measurements between plasma and DBS. The validated assay determined accurate and precise quantities, respectively, for osimertinib in both plasma (93.2%-99.3%; 0.2%-2.3%) and DBS (96.7%-99.6%; 0.5%-10.3%) over a concentration of 1-729 ng/mL. The osimertinib metabolites, AZ5104 and AZ7550, were similarly validated in accordance with bioanalytical guidelines. For 30%-60% patient hematocrit, no hematocrit bias was observed with DBS for all analytes. The Bland-Altman method showed high concordance between plasma and DBS analyte levels. Stability experiments revealed that osimertinib and its metabolites were poorly stable in plasma at room temperature, whereas all analytes were stable in DBS for 10 days at room temperature. The measurement of osimertinib, AZ5104, and AZ7550 from hemaPEN microsampled DBS is a convenient and reliable approach for therapeutic drug monitoring that produces measurements consistent with plasma drug levels.

Qi-Ju-Di-Huang-Pill delays the progression of diabetic retinopathy

Ethnopharmacological relevanceQi-Ju-Di-Huang-Pill (QJDH pill) is a Chinese decoction. Although it is commonly used to treat eye conditions, such as diabetic retinopathy (DR), its exact mechanism of action is unknown. Aim of the studyTo investigate the specific mechanism by which QJDH pill slows the progression of diabetic retinopathy (DR) based on animal and cellular experiments. Material and methodsThe major components of QJDH pill were characterized by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLCMS/MS). C57BL/6J mice were randomly divided into five groups as follows: normal group (control group), model group (STZ group), low-dosage QJDH pill group (QJDH-L group), medium-dosage QJDH pill group (QJDH-M group) and high-dosage QJDH pill group (QJDH-H group). Changes in water intake, urination, food intake, and body mass were monitored weekly, while changes in blood glucose were monitored monthly. Fluorescein fundus angiography (FFA), optical coherence tomography angiography (OCTA), and optical coherence tomography (OCT) were utilized to analyze the changes in fundus imaging indications. Hematoxylin & eosin (H&E) and transmission electron microscopy (TEM) were employed to examine histopathologic and ultrastructural changes in retina. The levels of interleukin-6 (IL-6), interleukin-17 (IL-17), tumor necrosis factor-α (TNF-α), and vascular endothelial growth factor (VEGF) in peripheral blood were detected using Enzyme-linked immunosorbent assay (ELISA). The mouse retina apoptotic cells were labeled with green fluorescence via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (Tunel). The protein levels of Bcl-2-Associated X (Bax), B cell lymphoma 2 (Bcl-2), Caspase-3, PI3K, phosphorylated PI3K (p-PI3K), protein kinase B (AKT) and phosphorylated AKT (p-AKT) were quantified by Western blot (WB). The retinal pigment epithelium (RPE) cells were cultured and classified into five groups as follows: normal glucose group (NG group), high glucose group (HG group), high glucose + QJDH pill group (HG + QJDH group), high glucose + inhibitor group (HG + LY294002 group), and high glucose + inhibitor + QJDH pill group (HG + LY294002 + QJDH group). Cell viability and apoptosis were detected via Cell Counting Kit-8 (CCK8) and then analyzed by flow cytometry. ResultsIn vivo experiments revealed that the QJDH pill effectively reduced blood glucose, symptoms of increased water intake, elevated urination, increased food intake and decreased body mass in DR mice. QJDH pill also slowed the development of a series of fundus imaging signs, such as retinal microangiomas, tortuous dilatation of blood vessels, decreased vascular density, and thinning of retinal thickness, downregulated IL-6, IL-17, TNF-α, and VEGF levels in peripheral blood, and inhibited retinal cell apoptosis by activating the PI3K/AKT signaling pathway. Moreover, in vitro experiments showed that high glucose environment inhibited RPE cell viability and activated RPE cell apoptosis pathway. In contrast, lyophilized powder of QJDH pill increased RPE cell viability, protected RPE cells from high glucose-induced damage, and decreased apoptosis of RPE cells by activating the pi3k pathway. ConclusionQJDH pill induces hypoglycemic, anti-inflammatory effects, anti-VEGF and anti-retinal cell apoptosis by activating PI3K/AKT signaling pathway, and thus can protect the retina and slow the DR progression.

Patients with Neurodegenerative Proteinopathies Exhibit Altered Tryptophan Metabolism in the Serum and Cerebrospinal Fluid.

Some pathological conditions affecting the human body can also disrupt metabolic pathways and thus alter the overall metabolic profile. Knowledge of metabolic disturbances in specific diseases could thus enable the differential diagnosis of otherwise similar conditions. This work therefore aimed to comprehensively characterize changes in tryptophan metabolism in selected neurodegenerative diseases. Levels of 18 tryptophan-related neuroactive substances were determined by high throughput and sensitive ultrahigh-performance liquid chromatography-tandem mass spectrometry in time-linked blood serum and cerebrospinal fluid samples from 100 age-matched participants belonging to five cohorts: healthy volunteers (n = 21) and patients with Lewy body disease (Parkinson's disease and dementia with Lewy bodies; n = 31), four-repeat tauopathy (progressive supranuclear palsy and corticobasal syndrome; n = 10), multiple system atrophy (n = 13), and Alzheimer's disease (n = 25). Although these conditions have different pathologies and clinical symptoms, the discovery of new biomarkers is still important. The most statistically significant differences (with p-values of ≤0.05 to ≤0.0001) between the study cohorts were observed for three tryptophan metabolites: l-kynurenine in cerebrospinal fluid and 3-hydroxy-l-kynurenine and 5-hydroxy-l-tryptophan in blood serum. This led to the discovery of distinctive correlation patterns between the profiled cerebrospinal fluid and serum metabolites that could provide a basis for the differential diagnosis of neurodegenerative tauopathies and synucleinopathies. However, further large-scale studies are needed to determine the direct involvement of these metabolites in the studied neuropathologies, their response to medication, and their potential therapeutic relevance.

Plasma lipidomic profiling reveals six candidate biomarkers for the prediction of incident stroke in patients with hypertension.

The burden of stroke in patients with hypertension is very high, and its prediction is critical. We aimed to use plasma lipidomics profiling to identify lipid biomarkers for predicting incident stroke in patients with hypertension. This was a nested case-control study. Baseline plasma samples were collected from 30 hypertensive patients with newly developed stroke, 30 matched patients with hypertension, 30 matched patients at high risk of stroke, and 30 matched healthy controls. Lipidomics analysis was performed by ultrahigh-performance liquid chromatography-tandem mass spectrometry, and differential lipid metabolites were screened using multivariate and univariate statistical methods. Machine learning methods (least absolute shrinkage and selection operator, random forest) were used to identify candidate biomarkers for predicting stroke in patients with hypertension. Co-expression network analysis revealed that the key molecular alterations of the lipid network in stroke implicate glycerophospholipid metabolism and choline metabolism. Six lipid metabolites were identified as candidate biomarkers by multivariate statistical and machine learning methods, namely phosphatidyl choline(40:3p)(rep), cholesteryl ester(20:5), monoglyceride(29:5), triglyceride(18:0p/18:1/18:1), triglyceride(18:1/18:2/21:0) and coenzyme(q9). The combination of these six lipid biomarkers exhibited good diagnostic and predictive ability, as it could indicate a risk of stroke at an early stage in patients with hypertension (area under the curve = 0.870; 95% confidence interval: 0.783-0.957). We determined lipidomic signatures associated with future stroke development and identified new lipid biomarkers for predicting stroke in patients with hypertension. The biomarkers have translational potential and thus may serve as blood-based biomarkers for predicting hypertensive stroke.

Influence of lactic acid fermentation on the phenolic profile, antioxidant activities, and volatile compounds of black chokeberry (Aronia melanocarpa) juice.

Lactic acid fermentation is an effective method for improving the quality of black chokeberry. This study aimed to investigate the influence of lactic acid bacteria on the phenolic profile, antioxidant activities, and volatiles of black chokeberry juice. Initially, 107 cfu/mL of Lactiplantibacillus plantarum, Lactobacillus acidophilus, and Lacticaseibacillus rhamnosus were inoculated into pasteurized black chokeberry juice and fermented for 48h at 37°C. All these strains enhanced the total phenolic and total flavonoid contents, with La. acidophilus showing the highest total phenolic (1683.64mg/L) and total flavonoid (659.27mg/L) contents. Phenolic acids, flavonoids, and anthocyanins were identified using ultrahigh-performance liquid chromatography-tandem mass spectrometry. The prevalent phenolic acid, flavonoid, and anthocyanin in the lactic-acid-fermented black chokeberry juice were cinnamic acid, rutin, and cyanidin-3-O-rutinoside, respectively. Furthermore, following fermentation, the DPPH and ABTS scavenging capacity, as well as the reducing power capacity, increased from 59.98% to 92.70%, 83.06% to 94.95%, and 1.24 to 1.82, respectively. Pearson's correlation analysis revealed that the transformation of phenolic acids, flavonoids, and anthocyanins probably contributed to enhancing antioxidant activities and color conversation in black chokeberry juice. A total of 40 volatiles were detected in the fermented black chokeberry juice by gas chromatography-ion mobility spectrometry. The off-flavor odors, such as 1-penten-3-one and propanal in the black chokeberry juice, were weakened after fermentation. The content of 2-pentanone significantly increased in all fermented juice, imparting an ethereal flavor. Hence, lactic acid fermentation can effectively enhance black chokeberry products' flavor and prebiotic value, offering valuable insights into their production. PRACTICAL APPLICATION: The application of lactic acid bacteria in black chokeberry juice not only enhances its flavor but also improves its health benefits. This study has expanded the range of black chokeberry products and offers a new perspective for the development of the black chokeberry industry.

A novel method to quantify chitosan in aqueous solutions by ultrahigh-performance liquid chromatography–tandem mass spectrometry

In this study, a novel and accurate quantitative analysis method for the direct determination of chitosan (CS) in aqueous solutions using ultrahigh-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) is presented. By detecting the mass spectrum response intensity of a series of CS characteristic ion pairs, the sample concentration (abscissa) was linearly fitted with the total ion current (TIC) response intensity of its characteristic ion pairs (ordinate). A reliable standard curve was derived for quantifying CS in the range of 125–4000 ng/mL. Under the detection conditions, this CS quantification method yielded acceptable specificity (no interference peak), linearity (with correlation coefficient (r2) values >0.999), precision (acceptable limit RSDr < 3 %, RSDR < 6 %), accuracy (RE within the acceptable limits of ±5 %), and stability (acceptable limit RE within ±5 %, RSDr < 3 %). Moreover, the applicability of measurement was verified when a series of substrates did not interact with CS in the solution. Results have verified the applicability of this method for determining CS content in different composites. This study provides a method for determining CS content with significant practical value and economic benefit.

Changes of anthocyanin and amino acid metabolites in saffron petals (Crocus sativus L.) during fermentation based on untargeted metabolomics

In order to investigate the possibility of fermentation for deep processing of saffron petals, the metabolic profiles of anthocyanins and amino acids in saffron petals were studied by untargeted metabolomics method based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS) combined with multivariate statistical analysis. The PCA, OPLS-DA and HCA models were established to analyze the differential metabolites before and after fermentation. Subsequently, the differential metabolites were screened by OPLS-DA with pairwise modeling. The contents of 26 kinds of anthocyanins during fermentation and 23 kinds of anthocyanins after fermentation were significantly different, respectively. After fermentation, the total anthocyanin content increased, and the monomer anthocyanin produced a variety of glucoside anthocyanin. As for amino acid metabolism, there were significant differences in 24 amino acids during fermentation, 27 kinds of amino acids after fermentation, and then the total content of amino acids decreased. In addition, based on the KEEG database, several metabolic pathways during the fermentation process were identified, and the metabolism pathway of anthocyanin is primarily related to anthocyanin biosynthesis, while the metabolic pathway of amino acid is mainly related to metabolic pathways. These findings provide comprehensive insights into the quality improvements of saffron petals wine.

Plasma levels of urea cycle related amino acids in association with risk of ischemic stroke: Findings from a nested case-control study

ObjectivesThe role of urea cycle related amino acids in the development of ischemic stroke (IS) remains unclear. The study aimed to evaluate the association of these amino acids with IS. Materials and MethodsWe conducted a case-control study nested within a cohort study in Changshu, Eastern China. A total of 321 cases and 321 controls matched by age and gender were finally included. Plasma levels of ornithine, arginine, spermidine, and proline were measured using ultra-high performance liquid chromatography–tandem mass-spectrometry (UHPLC–MS/MS). Odds ratios (ORs) and their 95 % confidence intervals (CIs) were calculated by conditional logistic regression analyses. ResultsPlasma ornithine was inversely associated with risk of IS [crude OR: 0.62 (95 % CI: 0.40-0.97)]. After adjustment for body mass index, smoking, hypertension, family history of stroke, estimated glomerular filtration rate, and total cholesterol, the corresponding ORs for the highest compared to the lowest quartiles was essentially unchanged [adjusted OR: 0.62 (95 % CI: 0.39-0.99)]. The risk association remained significant after repeating the analyses by excluding the first two years of follow-up. Plasma arginine, spermidine, and proline were not associated with the risk of IS. ConclusionWe observed that higher plasma levels of ornithine were associated with a lower risk of incident IS. Our novel findings suggest a protective role of ornithine in the pathogenesis of IS.

Development and validation of a sensitive UPLC-MS/MS analytical method for GFH009 in rat plasma and its application to toxicokinetics studies.

GFH009 is a potent, highly selective, small molecule that targets and inhibits the activity of the CDK9/cyclin T1 regulatory complex of P-TEFb. This study aimed to develop and validate a highly selective and sensitive ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for precise quantification of GFH009 in rat plasma. This method was subsequently employed for conducting toxicokinetic studies of GFH009 in rats. Plasma was prepared using a simple protein precipitation method by acetonitrile. Chromatographic separation of the analytes was achieved on a BEH C18 analytical column with a rapid 3.0 min run time and a flow rate of 0.5ml/min. The calibration curves for plasma samples exhibited excellent linearity over a wide concentration range of 1.0-1,000 ng/ml for GFH009. Intra- and inter-day accuracies were within 92.7-105.7%, and precisions were no more than 6.7%. Furthermore, the analyte demonstrated stability under four different storage conditions, with variations of <15.0%. This study pioneers a methodological innovation by introducing a highly reliable, specific and sensitive analytical method for GFH009 in rat plasma. The successful application of this method in toxicokinetic studies further underscores its significance, offering valuable insights for the methodology of clinical pharmacokinetic research.

Antimicrobial Activity and Mechanisms of Walnut Green Husk Extract.

Walnut green husks (WGHs), by-products of walnut production, are believed to possess antimicrobial properties, making them a potential alternative to antibiotics. In this study, the antibacterial activities of three extracts, derived from WGH, against Staphylococcus aureus, Bacillus subtilis, and Escherichia coli were investigated, and the antibacterial mechanisms of an anhydrous ethanol extract of WGH (WGHa) were examined. The results showed that WGHa exhibited inhibitory effects on all tested bacteria. The ultrahigh-performance liquid chromatography-tandem mass spectrometry analysis revealed that the major active compounds present in WGHa were terpenoids, phenols, and flavonoids. Treatment with WGHa resulted in the leakage of intracellular ions and alkaline phosphatase; a reduction in intracellular ATP content, ATPase activity, and nucleic acid content; as well as cellular metabolic viability. The transmission electron microscopy images showed varying degrees of cell deformation and membrane damage following WGHa treatment. The transcriptome sequencing and differentially expressed gene enrichment analyses revealed an up-regulation in pathways associated with RNA degradation, translation, protein export, and oxidative phosphorylation. Conversely, pathways involved in cell movement and localization, as well as cell wall organization and carbohydrate transport, were found to be down-regulated. These findings suggest that WGHa alters cell membrane permeability and causes damage to the cell wall. Additionally, WGHa interferes with cellular energy metabolism, compromises RNA integrity, and induces DNA replication stress, consequently inhibiting the normal growth and proliferation of bacteria. These findings unveiled the antimicrobial mechanisms of WGHa, highlighting its potential application as an antibiotic alternative.

An ultrahigh-performance liquid chromatography-tandem mass spectrometry method for quantification of methotrexate and 7-hydroxy-methotrexate and application for therapeutic drug monitoring in patients with central nervous system lymphoma.

A method using ultrahigh-performance liquid chromatography-tandem mass spectrometry was developed, validated, and applied to simultaneously determine plasma methotrexate (MTX) and 7-hydroxy-methotrexate (7-OH-MTX) in 117 patients with central nervous system (CNS) lymphoma. The ion transitions utilized were m/z 455.2 > 308.2 for MTX and m/z 471.2 > 324.1 for 7-OH-MTX. Samples were prepared through protein precipitation using methanol. Chromatographic separation was achieved within 3.0 min on a CMS9030 column (Ruixi, 2.1 × 50 mm, 3 μm) through a gradient elution of methanol and a 10% ammonium acetate solution at a flow rate of 0.4 mL/min. The method demonstrated linearity in the concentration range of 0.05-10 μM for MTX and 0.25-50 μM for 7-OH-MTX. The intra- and inter-day inaccuracy ranged from -7.38% to 7.83%, and the imprecision was less than 6.00% for both analytes. The recovery and matrix effect normalized by the internal standard (MTX-D3 ) remained consistent. Both analytes remained stable under nine different storage conditions. In patients with CNS lymphoma, MTX levels at 12 h and 7-OH-MTX levels at 12, 36, and 60 h after dosing in individuals with impaired renal function were significantly higher compared with those with normal renal function. 7-OH-MTX could potentially serve as a superior indicator for nephrotoxicity compared with MTX.

HuangQi ChiFeng decoction maintains gut microbiota and bile acid homeostasis through FXR signaling to improve atherosclerosis

Huangqi Chifeng Decoction (HQCFT), a traditional Chinese medicine preparation, has long been used to treat cardiovascular and cerebrovascular diseases. However, the mechanism of the beneficial effect of HQCFT on atherosclerosis remains to be explored. In this work, to investigate the effects of HQCFT on bile acid (BA) metabolism and the gut microbiome in atherosclerosis, ApoE−/− mice were fed a with high-fat diet for 16 weeks to establish the AS model. HQCFT(1.95 g kg−1 and 3.9 g kg−1 per day) was administered intragastrically for 8 weeks to investigate the regulatory effects of HQCFT on gut microbiota and bile acid metabolism and to inhibit the occurrence and development of AS induced by a high-fat diet. Histopathology, liver function and blood lipids were used to assess whether HQCFT can reduce plaque area, regulate lipid levels and alleviate liver steatosis in AS mice. In addition, 16S rDNA sequencing was used to screen the gut microbiota structure, and ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC‒MS/MS) was used to determine the bile acid profile. The mRNA and protein expression levels of bile acid metabolism were detected by RT‒PCR and WB to find the potential correlation. Results: HQCFT can regulate gut microbiota disorders, which was achieved by increasing gut microbiota diversity and altering Proteobacteria, Desulfobacterota, Deferribacteres, Rodentibacter, Parasutterella, and Mucispirillum interference abundance to improve AS-induced gut microbiota. HQCFT can also adjust the content of bile acids (TCA, LCA, DCA, TDCA, TLCA, UDCA, etc.), regulate bile acid metabolism, relieve liver fat accumulation, and inhibit the process of AS. In addition, HQCFT can restore the abnormal metabolism of bile acid caused by AS by regulating the expression of farnesoid X receptor (FXR), liver X receptor α (LXRα), ABCA1, ABCG1 and CYP7A1. Conclusion: HQCFT may play a part in the prevention of atherosclerosis by inhibiting the FXR/LXRα axis, increasing the expression of CYP7A1 in the liver, and regulating the interaction between the gut microbiota and bile acid metabolism.

Plasma metabolomics profiles in Black and White participants of the Adventist Health Study-2 cohort

BackgroundBlack Americans suffer disparities in risk for cardiometabolic and other chronic diseases. Findings from the Adventist Health Study-2 (AHS-2) cohort have shown associations of plant-based dietary patterns and healthy lifestyle factors with prevention of such diseases. Hence, it is likely that racial differences in metabolic profiles correlating with disparities in chronic diseases are explained largely by diet and lifestyle, besides social determinants of health.MethodsUntargeted plasma metabolomics screening was performed on plasma samples from 350 participants of the AHS-2, including 171 Black and 179 White participants, using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and a global platform of 892 metabolites. Differences in metabolites or biochemical subclasses by race were analyzed using linear regression, considering various models adjusted for known confounders, dietary and/or other lifestyle behaviors, social vulnerability, and psychosocial stress. The Storey permutation approach was used to adjust for false discovery at FDR < 0.05.ResultsLinear regression revealed differential abundance of over 40% of individual metabolites or biochemical subclasses when comparing Black with White participants after adjustment for false discovery (FDR < 0.05), with the vast majority showing lower abundance in Blacks. Associations were not appreciably altered with adjustment for dietary patterns and socioeconomic or psychosocial stress. Metabolite subclasses showing consistently lower abundance in Black participants included various lipids, such as lysophospholipids, phosphatidylethanolamines, monoacylglycerols, diacylglycerols, and long-chain monounsaturated fatty acids, among other subclasses or lipid categories. Among all biochemical subclasses, creatine metabolism exclusively showed higher abundance in Black participants, although among metabolites within this subclass, only creatine showed differential abundance after adjustment for glomerular filtration rate. Notable metabolites in higher abundance in Black participants included methyl and propyl paraben sulfates, piperine metabolites, and a considerable proportion of acetylated amino acids, including many previously found associated with glomerular filtration rate.ConclusionsDifferences in metabolic profiles were evident when comparing Black and White participants of the AHS-2 cohort. These differences are likely attributed in part to dietary behaviors not adequately explained by dietary pattern covariates, besides other environmental or genetic factors. Alterations in these metabolites and associated subclasses may have implications for the prevention of chronic diseases in Black Americans.