Ultrahigh-performance Liquid Chromatography-tandem Mass Spectrometry Research Articles

Quantitative analysis of erythromycin, its major metabolite and clarithromycin in chicken tissues and eggs via QuEChERS extraction coupled with ultrahigh-performance liquid chromatography-tandem mass spectrometry

A simple, rapid and novel method involving ultrahigh-performance liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry (UHPLC-ESI–MS/MS) was developed to simultaneously detect erythromycin, its major metabolite and clarithromycin in chicken tissues (muscle, liver and kidney) and eggs (whole egg, albumen and yolk). Samples were extracted using acetonitrile–water (80:20, v/v), and a a Cleanert MAS-Q cartridge was used to perform quick, easy, cheap, effective, rugged, and safe (QuEChERS) purification. The average recoveries were 87.78–104.22 %, and the corresponding intraday and interday relative standard deviations were less than 7.10 %. The decision limits and detection capabilities of the chicken tissues and eggs were 2.15–105.21 μg/kg and 2.26–110.42 μg/kg, respectively. For chicken tissues and eggs, the limits of detection and limits of quantification were 0.5 μg/kg and 2.0 μg/kg, respectively. The proposed method was successfully employed to analyse real samples, demonstrating its applicability.

Determination of succinic acid and lactic acid in pigs' serum, intestinal contents, and meat by ultrahigh-performance liquid chromatography-tandem mass spectrometry.

Succinic acid and lactic acid have been associated with diarrhea in weaned piglets. The level of succinic acid and lactic acid in serum, meat, and intestinal contents is important to elucidate the mechanism of diarrhea in weaned piglets. A facile method was developed for the quantification of succinic acid and lactic acid in pigs' serum, intestinal contents, and meat using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS). The serum samples underwent protein precipitation with methanol. The meat and intestinal contents were freeze-dried and homogenized using a tissue grinding apparatus. Methanol-water mixture (80:20, v/v) was used for homogenizing the meat, while water was used for homogenizing the intestinal contents. An additional step of protein precipitation with acetonitrile was required for the intestinal contents. The resulting solution was diluted with water before being analyzed by UHPLC/MS/MS. Separation of succinic acid and lactic acid could be achieved within 3min using a Kinetic XB-C18 column. The coefficients of variation for peak areas of succinic acid and lactic acid were less than 5.0%. The established method demonstrated good linearity as indicated by correlation coefficients exceeding 0.996. Additionally, satisfactory recoveries ranging from 88.58% to 108.8% were obtained. The detection limits (RS/N = 3) for succinic acid and lactic acid were determined to be 0.75 ng/mL and 0.02 μg/mL, respectively. This method exhibited high sensitivity, simplicity in operation, and small sample weight, making it suitable for quantitative determination of succinic acid and lactic acid in pigs' serum, intestinal contents, and meat. The method developed will provide valuable technical support in studying the metabolic mechanisms of succinic acid and lactic acid in pigs.

High-sensitivity liquid chromatography-tandem mass spectrometry quantitative for alkyl imidazolium ionic liquids in human serum: Advancing biomonitoring of human exposure concerns

Alkyl imidazolium ionic liquids (Cn[MIM]), initially heralded as eco-friendly green solvents for diverse industrial applications, have increasingly been recognized fortheir biodegradability challenges and multiple biotoxicity. Despite potential health risks, research into the effects of Cn[MIM] on human health remains scarce, particularly regarding their detection in biological serum samples. This study validated a matrix-matched calibration quantitative method that utilizes solid-phase extraction (SPE) coupled with ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The method was used to analyze the presence of 10 ionic liquids (ILs) with varying alkyl carbon chain lengths (C2–C12) across 300 human serum samples. Efficient separation was achieved using optimized SPE conditions and a BEH C18 column with an appropriate mobile phase. Results demonstrated a strong linear relationship (0.05–100 ng/mL; R2 = 0.995–0.999), with detection and quantification limits with detection and quantification limits ranging from 0.001 to 0.107 ng/mL and 0.003–0.355 ng/mL, respectively. Intraday and inter-day precisions were 0.85–6.99 % and 1.50–7.46 %, with recoveries between 82 and 113 %. The validated method detected C6MIM in 19 % of samples and C8MIM in 8.3 % of samples, with concentrations ranging from 0.02 to 111.70 μg/L and 0.09–16.99 μg/L, respectively, suggesting a potential risk of human exposure. This underscores the importance of robust detection methods in monitoring environmental and human health impacts of alkyl imidazolium compounds.

A rapid solid-phase extraction purification combined with UPLC[sbnd]MS/MS used for simultaneous determination of eight tetracyclines and three metabolite in chicken and pork

A method for rapid detection and identification of 8 tetracyclines (TCs) and 3 metabolite residues in chicken and pork was established and verified by combining rapid solid-phase extraction (SPE) technology with ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLCMS/MS). After a sample was extracted, it was purified with a PRiME HLB cartridge, and the cartridge did not need to be activated, balanced, or eluted. The filtrate was dried under nitrogen, redissolved, and subsequently analyzed via UPLCMS/MS. The limits of detection for chicken and pork were 0.05–0.48 μg/kg and 0.05–0.52 μg/kg, respectively. The limits of quantification were 0.16–1.61 μg/kg and 0.16–1.75 μg/kg, respectively. The recoveries were 85.15%–107.50% and 84.21%–106.37%, respectively. The established method was suitable for qualitative and quantitative analyses of TCs and their metabolite residues in chicken and pork and has been used for the detection of TC in chicken and pork samples from local markets.

Covalent organic frameworks as dispersive solid-phase extraction adsorbents for β-lactam detection in aquatic environments

Currently, antibiotic residues in the environment pose a serious public health risk; therefore, new sensitive approaches for detecting the presence of antibiotics are urgently needed. Ionic-type covalent organic frameworks (TAPB-(TFIT)+Cl−-COFs) were designed and prepared in this work, and these imine-linked COFs were characterized by several techniques, proving of porous structure and rich of nitrogen. The materials were applied to the extraction of β-lactams from aqueous samples, and the important parameters of the adsorption–desorption conditions were optimized. A dispersive solid-phase extraction method coupled with ultrahigh-performance liquid chromatography-tandem mass spectrometry (TAPB-(TFIT)+Cl−-COFs-d-SPE-UPLC-MS/MS) was proposed and utilized to determine the levels of 11 antibiotics in the β-lactams in the extracted samples. Good linearity (1.0–200.0 μg·L−1, r > 0.9999) was achieved together with limits of detection and quantification of 0.01–0.92 μg·L−1 and 0.05–2.78 μg·L−1, respectively; recoveries ranged from 75.57 to 114.20 %, conferring relative standard deviations < 8.99 % and negligible matrix effects (84.88–109.40 %). The synthesized TAPB-(TFIT)+Cl−-COFs were demonstrated to have good reusability and high stability. Furthermore, the proposed method was clearly shown to be sensitive and separable for the reliable simultaneous detection of 11 β-lactam residues in aquatic environments.

Metabolomic analysis reveals the changing trend and differential markers of volatile and nonvolatile components of Artemisiae argyi with different aging years.

Artemisia argyi Folium (AAF) is a traditional medicinal herb and edible plant. Analyzing the differential metabolites that affect the efficacy of AAF with different aging years is necessary. The aim of the study was to investigate the changing trend and differential markers of volatile and nonvolatile metabolites of AAF from different aging years, which are necessary for application in clinical medicine. Metabolites were analyzed using a widely targeted metabolomic approach based on ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and gas chromatography tandem mass spectrometry (GC-MS). A total of 153 volatile metabolites and 159 nonvolatile metabolites were identified. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) could clearly distinguish AAF aged for 1 year (AF-1), 3 years (AF-3), and 5 years (AF-5). Seven flavonoids and nine terpenoids were identified as biomarkers for tracking the aging years. The metabolomic method provided an effective strategy for tracking and identifying biomarkers of AAF from different aging years. This study laid the foundation for analysis of the biological activity of Artemisia argyi with different aging years.

Simultaneous Determination of Selected Steroids with Neuroactive Effects in Human Serum by Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Neuroactive steroids are a group of steroid molecules that are involved in the regulation of functions of the nervous system. The nervous system is not only the site of their action, but their biosynthesis can also occur there. Neuroactive steroid levels depend not only on the physiological state of an individual (person's sex, age, diurnal variation, etc.), but they are also affected by various pathological processes in the nervous system (some neurological and psychiatric diseases or injuries), and new knowledge can be gained by monitoring these processes. The aim of our research was to develop and validate a comprehensive method for the simultaneous determination of selected steroids with neuroactive effects in human serum. The developed method enables high throughput and a sensitive quantitative analysis of nine neuroactive steroid substances (pregnenolone, progesterone, 5α-dihydroprogesterone, allopregnanolone, testosterone, 5α-dihydrotestosterone, androstenedione, dehydroepiandrosterone, and epiandrosterone) in 150 μL of human serum by ultrahigh-performance liquid chromatography with tandem mass spectrometry. The correlation coefficients above 0.999 indicated that the developed analytical procedure was linear in the range of 0.90 nmol/L to 28.46 μmol/L in human serum. The accuracy and precision of the method for all analytes ranged from 83 to 118% and from 0.9 to 14.1%, respectively. This described method could contribute to a deeper understanding of the pathophysiology of various diseases. Similarly, it can also be helpful in the search for new biomarkers and diagnostic options or therapeutic approaches.

Chemoselectivity Strategy Based on B-Label Integrated with Tailored COF for Targeted Metabolomic Analysis of Short-Chain Fatty Acids by UHPLC-MS/MS.

Chemoselective extraction strategy is an emerging and powerful means for targeted metabolomics analysis, which allows for the selective identification of biomarkers. Short-chain fatty acids (SCFAs) as functional metabolites for many diseases pose challenges in qualitative and quantitative analyses due to their high polarity and uneven abundance. In our study, we proposed the B-labeled method for the derivatization of SCFAs using easily available 3-aminobenzeneboronic acid as the derivatization reagent, which enables the introduction of recognition unit (boric acid groups). To analyze the B-labeled targeted metabolites accurately, cis-diol-based covalent organic framework (COF) was designed to specifically capture and release target compounds by pH-response borate affinity principle. The COF synthesized by the one-step Schiff base reaction possessed a large surface area (215.77 m2/g), excellent adsorption capacity (774.9 μmol/g), good selectivity, and strong regeneration ability (20 times). Combined with ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis, our results indicated that the detection sensitivities of SCFAs increased by 1.2-2500 folds compared with unlabeled method, and the retention time and isomer separation were improved. Using this strategy, we determined twenty-six SCFAs in the serum and urine of rats in four groups about osteoporosis and identified important biomarkers related to the tricarboxylic acid cycle and fatty acid metabolism pathways. In summary, UHPLC-MS/MS based on B-labeled derivatization with tailored COF strategy shows its high selectivity, excellent sensitivity, and good chromatographic behavior and has remarkable application prospect in targeted metabolomics study of biospecimens.

Characterization of an Arginine Decarboxylase from Streptococcus pneumoniae by Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Polyamines are polycations derived from amino acids that play an important role in proliferation and growth in almost all living cells. In Streptococcus pneumoniae (the pneumococcus), modulation of polyamine metabolism not only plays an important regulatory role in central metabolism, but also impacts virulence factors such as the capsule and stress responses that affect survival in the host. However, functional annotation of enzymes from the polyamine biosynthesis pathways in the pneumococcus is based predominantly on computational prediction. In this study, we cloned SP_0166, predicted to be a pyridoxal-dependent decarboxylase, from the Orn/Lys/Arg family pathway in S. pneumoniae TIGR4 and expressed and purified the recombinant protein. We performed biochemical characterization of the recombinant SP_0166 and confirmed the substrate specificity. For polyamine analysis, we developed a simultaneous quantitative method using hydrophilic interaction liquid chromatography (HILIC)-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. SP_0166 has apparent Km, kcat, and kcat/Km values of 11.3 mM, 715,053 min-1, and 63,218 min-1 mM-1, respectively, with arginine as a substrate at pH 7.5. We carried out inhibition studies of SP_0166 enzymatic activity with arginine as a substrate using chemical inhibitors DFMO and DFMA. DFMO is an irreversible inhibitor of ornithine decarboxylase activity, while DFMA inhibits arginine decarboxylase activity. Our findings confirm that SP_0166 is inhibited by DFMA and DFMO, impacting agmatine production. The use of arginine as a substrate revealed that the synthesis of putrescine by agmatinase and N-carbamoylputrescine by agmatine deiminase were both affected and inhibited by DFMA. This study provides experimental validation that SP_0166 is an arginine decarboxylase in pneumococci.

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 &gt; 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.