Ultra-high Performance Liquid Chromatography Research Articles

Exploring the Potential Lipid-Lowering and Weight-Reducing Mechanisms of FH06 Fermented Beverages Based on Non-Targeted Metabolomics and Network Pharmacology

Investigating the intricate pathways through which FH06 fermentation broth exerts lipid-lowering and weight-loss effects is pivotal for advancing our comprehension of metabolic regulation and therapeutic interventions. Ultrahigh-performance liquid chromatography quadrupole electrostatic field orbit trap mass spectrometry (UHPLC-QE-MS) detection and the ChEMBL database were used to determine the effective compounds in the FH06 fermentation broth and predict their targets. The TTD database and DisGeNET database were used to query obesity-related targets. The STRING database was used to construct protein interaction information. The Gene Ontology (GO) database and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used to perform biological function annotation (GO) and KEGG pathway enrichment analyses of the targets. Results: A total of 85 effective compounds were screened from the fermentation broth of FH06; these compounds may act on TP53, PPARG, TNF, and other targets through 10 signaling pathways, such as the chemical carcinogenesis-receptor activation and lipid and atherosclerosis pathways, and exert pharmacological effects, such as hypoglycemic effects and weight loss. They also have anti-inflammatory, antioxidant, antitumor, and immunoregulatory effects. These findings reveal the active ingredients of FH06 fermentation broth and its multi-target and multi-channel characteristics in lipid lowering and weight loss. This study has positive implications for the clinical treatment of obesity using FH06, providing a theoretical and scientific basis for further developing of FH06-assisted lipid-lowering products.

PRM1201 effectively inhibits colorectal cancer metastasis via shaping gut microbiota and short- chain fatty acids

BackgroundPRM1201 is a traditional medicine with beneficial effects against colorectal cancer (CRC) metastasis. However, the underlying mechanism of this action remains to be determined. HypothesisRemodeling microbiota and short-chain fatty acids (SCFAs) metabolism might be a potential mechanism to explain the anti-metastatic action of PRM1201, as this gut-microbiota dependent effect involves downregulation of histone deacetylation and EMT. MethodsTo investigate this possibility, clinical specimens were sequenced and the correlation between the anti-metastatic efficacy of PRM1201 and the restoration of SCFA-producing bacteria was studied. To obtain solid causal evidence, a mouse metastasis model was established to detect the influence of PRM1201 on cancer metastasis. Specifically, 16S amplicon sequencing, ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) analysis, and bacterial manipulation were used to examine the gut microbiota-driven anti-metastatic action of PRM1201. ResultsClinical data showed that PRM1201 increased both the number of SCFA-producing bacteria and generation of SCFAs in the feces of CRC patients. A positive correlation between the anti-metastatic efficacy of PRM1201 and the restoration of SCFAs observed. The animal experiments demonstrated that PRM1201 effectively blocked CRC metastasis in a dose-dependent manner. PRM1201 treatment modulated the composition of gut microbiota, and promoted the proliferation of beneficial SCFAs producers such as Akkermansia, Lachnospiraceae_NK4A136_group and Blautia, while simultaneously reducing the abundance of pathogenic bacteria like Escherichia-Shigella. In addition, PRM1201 led to augmentation of SCFAs content. Further results indicated that the anti-cancer metastatic mechanism of PRM1201 was linked to inhibition of histone deacetylation and suppression of epithelial-to-mesenchymal transition (EMT) in metastatic lesions. Microbiota depletion treatment and fecal microbiota transplantation (FMT) underscored the microbiota-dependent nature of this phenomenon. Moreover, this anti-colorectal cancer metastatic effect and mechanism of total SCFAs and single SCFA were also confirmed. ConclusionIn summary, PRM1201 exerts its anti-metastatic effects by modulating SCFA-producing bacteria and enhancing the production of SCFAs. Furthermore, the prebiotic-like actions of PRM1201, along with the PRM1201-treated bacteria, function as inhibitors of histone deacetylases (DHACs) thereby effectively suppressing EMT events.

The mother-offspring transfer of chlorothalonil through human breast milk: A multi-city cross-sectional study

4-Hydroxychlorothalonil (4-OH CHT), the main metabolite of chlorothalonil and the most widely used fungicide, has been frequently detected in human samples during monitoring. 4-OH CHT may exhibit higher toxicity and persistence in the environment compared to its prototype. In this study, a total of 540 paired serum and breast milk samples from pregnant women in three provinces in China were monitored for contaminant residues. 4-OH CHT was analyzed in the samples using ultra high-performance liquid chromatography - high-resolution mass spectrometry with a detection limit of 20 ng/L. The study investigated the effects of demographic factors, such as BMI, region of residence, and education level, on the levels of 4-OH CHT residues in serum and breast milk. Among the three provinces, the highest median concentration of 4-OH CHT in serum samples was observed in Hebei (1.04 × 103 ng/L), while the highest median concentration of 4-OH CHT in breast milk samples was observed in Hubei and Guangdong (491 ng/L). Multiple linear regression was used to investigate the significant positive correlation between 4-OH CHT in serum and breast milk (p = 0.000) after adjusting for personal characteristics. Based on this, the study further explored the influencing factors of transfer efficiencies (TEs) in conjunction with the individual TEs and the personal characteristics of the participants. Our results demonstrated that the age of the volunteers and their exercise habits had an effect on TEs, but further studies are needed to determine whether exercise leads to an increase in TEs.

Comprehensive Quality Evaluation of Danggui-Jianzhong Decoction by Fingerprint Analysis, Multi-Component Quantitation and UPLC-Q-TOF-MS.

Danggui-Jianzhong decoction (DGJZ) is a famous classical traditional Chinese medicine formula, which ingredients are complex and the quality is difficult to control. Our study aimed to identify the overall chemical profile of DGJZ qualitatively by ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and UPLC. A total of 77 components, including terpenoids, flavonoids, phenolic acids, gingerols and other components, were firstly detected and characterized by UPLC-Q-TOF-MS and 18 peaks marked after analyzing the UPLC fingerprint. Finally, paeoniflorin, liquiritin, ferulic acid, cinnamic acid, glycyrrhizic acid and 6-gingerol were quantified, which was validated in terms of linearity, precision, accuracy, repeatability and recovery. Taken together, the chemical constitutes of DGJZ were systematically identified and a reliable quantitative method coupled with fingerprint analysis was successfully employed for evaluating the holistic quality, which will provide a robust foundation for the quality control of DGJZ.

Stability of clozapine tablets repackaged in dose administration aids using repackaging machines

BackgroundThe use of dose administration aids in automated ward dispensing devices requires the repackaging of medications, which may impact their stability compared with the original manufacturer’s packaging.ObjectivesThis study aimed to...

Chemical characterization and comparative analysis of different parts of Cocculus orbiculatus through UHPLC-Q-TOF-MS.

Cocculus orbiculatus (L.) DC. (C. orbiculatus) is a medicinal herb valued for its dried roots with anti-inflammatory, analgesic, diuretic, and other therapeutic properties. Despite its traditional applications, chemical investigations into C. orbiculatus remain limited, focusing predominantly on alkaloids and flavonoids. Furthermore, the therapeutic use of C. orbiculatus predominantly focuses on the roots, leaving the stems, a significant portion of the plant, underutilized. This study employed ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS) with in-house and online databases for comprehensive identification of components in various plant parts. Subsequently, untargeted metabolomics was employed to analyze differences in components across different harvest periods and plant sections of C. orbiculatus, aiming to screen for distinct components in different parts of the plant. Finally, metabolomic analysis of the roots and stems, which contribute significantly to the plant's weight, was conducted using chemometrics, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares discriminant analysis (OPLS-DA), and heatmaps. A total of 113 components, including alkaloids, flavonoids, and organic acids, were annotated across the root, stem, leaf, flower, and fruit, along with numerous previously unreported compounds. Metabolomic analyses revealed substantial differences in components between the root and stem compared to the leaf, flower, and fruit during the same harvest period. PLS-DA and OPLS-DA annotated 10 differentiating components (VIP > 1.5, P < 0.05, FC > 2 or FC < 0.67), with 5 unique to the root and stem, exhibiting lower mass spectrometric responses. This study provided the first characterization of 113 chemical constituents in different parts of C. orbiculatus, laying the groundwork for pharmacological research and advocating for the enhanced utilization of its stem.

Comparative study on chemical constituents of different medicinal parts of Lonicera japonica Thunb. Based on LC-MS combined with multivariate statistical analysis

Lonicerae japonicae flos (LJF), Lonicerae japonicae caulis (LJC), Lonicerae folium (LF) and Lonicerae fructus (LFR) are derived from Lonicera japonica Thunb., which are formed due to different medicinal parts. The efficacy of the 4 medicinal materials has similarities and differences. However, little attention has been paid to illustrate the differences in efficacy from the perspective of phytochemistry. In this study, ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap mass spectrometry (UPLC-Q-Exactive-Orbitrap-MS) was used to qualitatively analyze the ingredients in 4 herbs. A total of 86 compounds were plausibly or unambiguously identified, there were 54 common components among the 4 medicinal materials, and each kind of medicinal materials had its own unique components. On the basis of qualitative analysis, ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UPLC-QQQ-MS/MS) was used to quantitatively analyze 31 components contained in 4 medicinal materials, and principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and other multivariate statistical analysis were furtherly performed for comparing the component contents. The results showed that the samples from the same parts were clustered into one group, and the samples from different medicinal parts were significantly different. The analysis of variable importance projection (VIP) value of the OPLS-DA model showed that 10 components including chlorogenic acid, secologanic acid, isochlorogenic acid A, loganin, lonicerin, loganic acid, secoxyloganin, sweroside, luteolin and rhoifolin were the main difference components among the 4 medicinal materials. The study not only lays a solid foundation for the intrinsic quality control of 4 medicinal materials and the study of different effects of the 4 medicinal materials at the phytochemical level, but also provides a basis for more rational utilization of various parts of L. japonica and expansion of medicinal resources.

Investigation of doxycycline residues in bones after oral administration to broiler chickens

Abstract Introduction Chicken bones, a by-product of the poultry industry, can directly or indirectly enter the food chain. Bone meal and bone products could be sources of many contaminants. Considering the wide range of uses made of bones in the culinary and food industries, this material needs to be safe and antibiotic residue–free. To determine if such is the case, the concentration of doxycycline in chicken bones was investigated, this antimicrobial being one of the most commonly used in poultry production. Material and Methods Ross 308 broilers were grouped into three experimental and one control group. Doxycycline was administered in drinking water at therapeutic and sub-therapeutic doses, as well as via spray treatment. The concentration of doxycycline in bones was determined post slaughter by ultra-high performance liquid chromatography–tandem mass spectrometry. Results Doxycycline was quantified at 135 μg/kg 22 days after the last day of antibiotic administration at therapeutic doses; 2,285 μg/kg after sub-therapeutic treatment for 27 days and 9.62 μg/kg 22 days after the end of spray application. Conclusion High concentrations and long persistence of doxycycline in bones were found in this study. Doxycycline can contaminate all bone-derived products in the food and fertiliser industries.

Human metabolism of the semi-synthetic cannabinoids hexahydrocannabinol, hexahydrocannabiphorol and their acetates using hepatocytes and urine samples.

Hexahydrocannabinol (HHC), hexahydrocannabiphorol (HHCP) and their acetates, HHC-O and HHCP-O, respectively, are emerging in Europe as alternatives to tetrahydrocannabinol (THC). This study aimed to elucidate the metabolic pathways of the semi-synthetic cannabinoids HHC, HHCP, HHC-O and HHCP-O from incubation with human hepatocytes. The metabolites of HHC were also identified in authentic urine samples. HHC, HHCP, HHC-O and HHCP-O were incubated with primary human hepatocytes for 1, 3 and 5 h. Authentic urine samples from cases screened positive for cannabis in blood using ELISA but confirmed negative were analysed both non-hydrolysed and hydrolysed for HHC metabolites. Potential metabolites were identified using ultra-high performance liquid chromatography (UHPLC) coupled to a quadrupole time-of-flight mass spectrometer (QToF-MS). HHC and HHCP were primarily metabolised through monohydroxylation (monoOH), followed by oxidation to a carboxylic acid metabolite. HHC-O and HHCP-O were rapidly metabolised to HHC and HHCP, respectively. In authentic urine samples, 18 different metabolites were identified, and 99.3% of hydroxylated metabolites were glucuronidated. 11-OH-HHC, 5'OH-HHC and another metabolite with a monoOH on the side chain were the only metabolites present in all 16 urine samples. The metabolism of HHC and HHCP were similar, although the longer alkyl side chain of HHCP (heptyl) led to greater hydroxylation on the side chain than HHC (pentyl). The use of HHC and HHCP can be differentiated from the use of THC and other phytocannabinoids, but the use of the acetate analogues may not be differentiable from their non-acetate analogues.

Amino acid metabolomics and machine learning for assessment of post-hepatectomy liver regeneration

ObjectiveAmino acid (AA) metabolism plays a vital role in liver regeneration. However, its measuring utility for post-hepatectomy liver regeneration under different conditions remains unclear. We aimed to combine machine learning (ML) models with AA metabolomics to assess liver regeneration in health and non-alcoholic steatohepatitis (NASH).MethodsThe liver index (liver weight/body weight) was calculated following 70% hepatectomy in healthy and NASH mice. The serum levels of 39 amino acids were measured using ultra-high performance liquid chromatography–tandem mass spectrometry analysis. We used orthogonal partial least squares discriminant analysis to determine differential AAs and disturbed metabolic pathways during liver regeneration. The SHapley Additive exPlanations algorithm was performed to identify potential AA signatures, and five ML models including least absolute shrinkage and selection operator, random forest, K-nearest neighbor (KNN), support vector regression, and extreme gradient boosting were utilized to assess the liver index.ResultsEleven and twenty-two differential AAs were identified in the healthy and NASH groups, respectively. Among these metabolites, arginine and proline metabolism were commonly disturbed metabolic pathways related to liver regeneration in both groups. Five AA signatures were identified, including hydroxylysine, L-serine, 3-methylhistidine, L-tyrosine, and homocitrulline in healthy group, and L-arginine, 2-aminobutyric acid, sarcosine, beta-alanine, and L-cysteine in NASH group. The KNN model demonstrated the best evaluation performance with mean absolute error, root mean square error, and coefficient of determination values of 0.0037, 0.0047, 0.79 and 0.0028, 0.0034, 0.71 for the healthy and NASH groups, respectively.ConclusionThe KNN model based on five AA signatures performed best, which suggests that it may be a valuable tool for assessing post-hepatectomy liver regeneration in health and NASH.

Amanitin determination in bile samples by UHPLC-MS: LR-MS and HR-MS analytical performance

Consumption of misidentified foraged mushrooms containing bicyclic amanitin octapeptides is a worldwide public health and veterinary problem, being considered one of the deadliest accidental human and canine food ingestion due to acute liver failure (ALF). Reversal of advanced ALF and complete clinical recovery can be achieved following definitive removal of accumulated amatoxin laden bile from the gallbladder. An accurate means of quantifying amanitin content in aspirated bile is, therefore, urgently needed. Building on our prior work validating a method to detect and quantify amanitin in hepatic autopsy tissue, the development of an accurate method of measuring α- and β-amanitin in aspirated gallbladder bile was performed to evaluate the efficiency of this emergency procedure applied as a clinical treatment for intoxicated patients. A solid-phase extraction (SPE) procedure was optimized followed by detection based on ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS). Low resolution mass spectrometry (LRMS) was compared with high resolution (HRMS) by the validation of UHPLC-MS/MS (triple quadrupole MS) and UHPLC-ToF-MS (time-of-flight MS). Both methods were able to detect amatoxins in bile with limits of detection and quantification ranging from 2.71 to 3.46 µg.kg-1, and 8.36–9.03 µg.kg-1 for α-amanitin and, 0.32–1.69 µg.kg-1 and 0.55–5.62 µg.kg-1 for β-amanitin, respectively. Validation was completed with the evaluation of linearity, specificity, robustness, recovery, and precision following the ICH guidelines and CIR 808/2021. The validated methods were finally applied to bile samples obtained 48–96 hours + post-ingestion from 4 amatoxin poisoning patients who underwent gallbladder drainage procedures in Vietnam, Canada, and California. Gallbladder bile from patients with amatoxin mushroom poisoning contained significant amanitin content, even when aspirated several days post-ingestion, thus confirming the important role of enterohepatic circulation in amatoxin hepatotoxicity. This work represents a high and unique analytical throughput in amanitin poisoning allowing to efficiently respond to this fatal health problem.

Chemical profiling of Zhi-Ke-Bao pills and its potential mechanism against cough by ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry and network pharmacology

Zhi-Ke-Bao pills (ZKB), a traditional Chinese medicine preparation composed of 13 herbs, is generally used to treat cough caused by external wind cold, phlegm, etc in clinical applications, and it plays a core role in relieving cough caused by COVID-19 and influenza in China. Till now, the understanding of its chemical constituents was dramatically limited due to its chemical complexity, restricting its clinical application or development. In this work, a developed ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) method, a targeted and non-targeted strategy and network pharmacology were used to comprehensively characterize the chemical compositions in ZKB and predict its mechanism against cough. A total of 164 compounds (148 targeted compounds and 16 non-targeted ones) were identified or tentatively characterized in ZKB, including 65 flavonoids, 25 alkaloids, 19 organic acids, 41 saponins, 9 coumarins, 2 phenylpropanoids, 2 anthraquinones, and 1 other types. Among them, 37 compounds were unambiguously identified by comparison to reference standards. Meanwhile, the fragmentation behaviors of five main chemical structure types were also summarized. 309 targets and two core signaling pathways of ZKB against cough were predicted by network pharmacology, including MAPK and PI3K-Akt signaling pathways. It was the first time to characterize the chemical compounds of ZKB and reveal its potential mechanism against cough, providing the material basis for further quality control or pharmacodynamic evaluation of ZKB.

Distribution of antibiotic resistance genes and antibiotic residues in drinking water production facilities: Links to bacterial community.

There is a rapid spread of antibiotic resistance in the environment. However, the impact of antibiotic resistance in drinking water is relatively underexplored. Thus, this study aimed to quantify antibiotic resistance genes (ARGs) and antibiotic residues in two drinking water production facilities (NW-E and NW-C) in North West Province, South Africa and link these parameters to bacterial communities. Physicochemical and ARG levels were determined using standard procedures. Residues (antibiotics and fluconazole) and ARGs were quantified using ultra-high performance liquid chromatography (UHPLC) chemical analysis and real-time PCR, respectively. Bacterial community compositions were determined by high-throughput 16S rRNA sequencing. Data were analysed using redundancy analysis and pairwise correlation. Although some physicochemical levels were higher in treated than in raw water, drinking water in NW-E and NW-C was safe for human consumption using the South African Water Quality Guideline (SAWQG). ARGs were detected in raw and treated water. In NW-E, the concentrations of ARGs (sul1, intl1, EBC, FOX, ACC and DHA) were higher in treated water than in raw water. Regarding antimicrobial agents, antibiotic and fluconazole concentrations were higher in raw than in treated water. However, in NW-C, trimethoprim concentrations were higher in raw than in treated water. Redundancy analysis showed that bacterial communities were not significantly correlated (Monte Carlo simulations, p-value >0.05) with environmental factors. However, pairwise correlation showed significant differences (p-value <0.05) for Armatimonas, CL500-29 marine group, Clade III, Dickeya and Zymomonas genera with environmental factors. The presence of ARGs and antibiotic residues in the current study indicated that antibiotic resistance is not only a clinical phenomenon but also in environmental settings, particularly in drinking water niches. Consumption of NW-E and NW-C treated water may facilitate the spread of antibiotic resistance among consumers. Thus, regulating and monitoring ARGs and antibiotic residues in drinking water production facilities should be regarded as paramount.

#1554 The association between gut-derived metabolites and biomarkers of renal and cerebro-vascular damage in type 2 diabetes mellitus patients

Abstract Background and Aims Due to its numerous complications, type 2 diabetes mellitus (T2DM) has a major impact on the global economic burden and overall mortality. This condition modifies the metabolic microenvironment in targeted organs, such as the kidney and the brain, by its hyperglycemic nature. Gut microbiota seems to have significant interference with the renal-cerebral axis, especially in a dysbiosis state. The aim of the study was to quantify gut derived metabolites by metabolomic techniques and to evaluate their association with markers of endothelial, podocyte and proximal tubule damage and with cerebro-vascular hemodynamic indices. Method The metabolomic profiling was realized on serum and urine samples originated from 90 T2DM patients, divided into three subgroups, such as the following: 30 normal to mildly-, 30 moderately-, and 30 severely increased albuminuria, respectively, and 20 healthy subjects. The samples were examined by using untargeted and targeted ultra-high performance liquid chromatography coupled with electrospray-ionization quadroupole time-of-flight mass spectrometry (UHPLC-QTOF-ESI+-MS), in order to obtain the metabolic profile. The markers of renal damage such as synaptopodin (SP), podocalyxin (PD), N-acetyl-β-D-glucosaminidase (NAG), kidney injury molecule 1 (KIM-1), monocyte chemotactic protein 1 (MCP-1), and intercellular adhesion molecule 1 (ICAM-1) were assessed by ELISA technique. The hemodynamic cerebro-vascular indices were obtained by high resolution Doppler ultrasound of common carotid arteries (CCAs), internal carotid arteries (ICAs), and middle cerebral arteries (MCAs), bilaterally. The correlations between the metabolites, the markers of renal damage, and neurosonological indices were realized by univariable and multivariable linear regression analyses. Results UHPLC-QTOF-ESI+-MS targeted analysis and subsequent statistical assessments revealed a subset of metabolites with a specific pattern for the normal to mildly increased albuminuria subgroup. The metabolites identified as significant were, in serum: arginine (sArg), hippuric acid (sHA), indoxyl sulfate (sIS), butenoylcarnitine (sBCA), sorbitol (sSorb); and in urine, arginine (uArg), butenoylcarnitine (uBCA), indoxyl sulfate (uIS) and p-cresyl sulfate (uPCS). The univariable linear regression analysis revealed that sArg correlated positively with the breath-holding index (BHI) and negatively with intima media thickness (IMT) in ACI, MCP-1 and ICAM-1; sIS, sSorb, and sBCA correlated inversely with these parametes. The multivariable linear regression analysis displayed the specific correlations between metabolites, markers of endothelial dysfunction, and neurosonological indices, such as follows: sArg correlated with IMT and ICAM-1; sIS followed a predictive pattern along with resistance index (RI) in ACIs and ICAM-1; sBCA correlated with IMT and MCP-1 and sSorb correlated with ICAM-1 and BHI. The urine metabolites were correlated with markers of podocyte and proximal tubule dysfunction: uIS and uBCA were correlated with PD and urinary albumin to-creatinin ratio (uACR), and uPCR correlated with KIM-1 and uACR. Conclusion This study revealed a set of metabolites potentially derived from the intestinal microflora which may act as biomarkers for the early detection of both CMA and DKD in T2DM patients. SArg, sIS and, sBCA are indicators of renal endothelial dysfunction. Moreover, sArg and sIS reflect incipient common and internal carotid artery atherosclerosis, suggesting vascular remodeling at these levels. SSorb correlated with BHI, indicating blood-brain barrier dysfunction and CMA development. In urine, uBCA and uIS are indicators of podocytopathy, whereas uPCS is associated with proximal tubule damage in incipient DKD.

A simple and sensitive ultra-high performance liquid chromatography tandem mass spectrometry method for the quantitative analysis of VX-548 in monkey plasma: Method validation and application to pharmacokinetic study.

VX-548 is an orally active and highly selective NaV 1.8 inhibitor that is undergoing development for the treatment of acute pain. The aim of this study was to develop a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the measurement of VX-548 in monkey plasma. VX-548 was extracted from the plasma using acetonitrile-mediated protein precipitation. The quantitative analysis was performed on a Thermo Vantage TSQ mass spectrometer with ibrutinib as an internal standard. Chromatography was performed on a Waters ACQUITY UPLC BEH C18 column with 0.1% aqueous formic acid and acetonitrile as mobile phase. The precursor-to-product ion transitions were m/z 474.2 > 165.0 and m/z 441.2 > 138.1 for VX-548 and internal standard, respectively. This developed method was successfully validated in the concentration range of 1-1000 ng/mL. The calibration curve showed excellent linearity with a correlation coefficient of >0.999. The precision expressed as relative standard deviation (RSD) was <8.4%, whereas the accuracy denoted as relative error (RE) ranged from -5.0% to 9.1%. The mean recovery was >84%. VX-548 was stable in monkey plasma after storage under certain conditions. The validated method was successfully applied to the pharmacokinetic study of VX-548 in monkey plasma after single oral (2 mg/kg) and intravenous (1 mg/kg) administrations.

Widely targeted metabolomics analysis of Sanghuangporus vaninii mycelia and fruiting bodies at different harvest stages

Sanghuangprous vaninii is a medicinal macrofungus cultivated extensively in China. Both the mycelia and fruiting bodies of S. vaninii have remarkable therapeutic properties, but it remains unclear whether the mycelia may serve as a substitute for the fruiting bodies. Furthermore, S. vaninii is a perennial fungus with therapeutic components that vary significantly depending on the growing year of the fruiting bodies. Hence, it is critical to select an appropriate harvest stage for S. vaninii fruiting bodies for a specific purpose. With the aid of Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), metabolomics based on ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QQQ-MS) was used to preliminarily determine 81 key active metabolites and 157 active pharmaceutical metabolites in S. vaninii responsible for resistance to the six major diseases. To evaluate the substitutability of the mycelia and fruiting bodies of S. vaninii and to select an appropriate harvest stage for the fruiting bodies of S. vaninii, we analyzed the metabolite differences, especially active metabolite differences, among the mycelia and fruiting bodies during three different harvest stages (1-year-old, 2-year-old, and 3-year-old). Moreover, we also determined the most prominent and crucial metabolites in each sample of S. vaninii. These results suggested that the mycelia show promise as a substitute for the fruiting bodies of S. vaninii and that extending the growth year does not necessarily lead to higher accumulation levels of active metabolites in the S. vaninii fruiting bodies. This study provided a theoretical basis for developing and using S. vaninii.

Rapid Classification and Identification of Chemical Compounds and Semi-Quantitative Metabolism of Huangkui Capsules and the Protective Effects of Its Quercetin Derivatives against Tacrolimus-induced HK-cell Reduction

Abstract Objective: The study aimed to establish an effective strategy for systematically characterizing and verifying compounds in Huangkui capsules (HKCs). Materials and Methods: An ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (MS) method was effectively established and utilized for the chemical compound characterization in HKC, with the support of MS-DIAL, MS-FINDER, and Global Natural Product Social Molecular Network. Multiple rat samples were analyzed after the oral administration. Metabolites were identified based on specific cleavage behaviors, and metabolic pathways were predicted. Semi-quantitative analysis of the metabolome profiles was conducted using post-data processing. High concentrations in vivo were investigated for their role in tacrolimus-induced death of HK-2 cells. Results: In total, 129 compounds were identified in HKC, of which 74 were reported for the first time. In this study, we successfully identified and uncovered 19 prototypes and 123 metabolites from the biosamples. The concentrations of glucuronidation and methylation metabolites of quercetin were the highest in the kidney and intestinal tissues. In contrast, significant glucuronidation of quercetin metabolites was observed with high blood concentrations. Notably, quercetin glucuronidation and methylation metabolites protected HK-cell against tacrolimus-induced injury in a dose-dependent manner. Conclusion: This study successfully established a reliable and efficient strategy for comprehensive characterization of traditional Chinese medicine compounds. This strategy harnessed the power of various intelligent postprocessing technologies to provide a more thorough understanding of active components and their metabolic transformations in the body. These results suggest that quercetin metabolites should be evaluated for their protective capacity against kidney disease.

Rapid chemical characterization and pharmacological mechanism of Fining Granules in the treatment of chronic bronchitis based on UHPLC–Q-exactive orbitrap mass spectrometer and network pharmacology

BackgroundSenecio cannabifolius Less. is a perennial herb belonging to the Compositae family that has been used in traditional medicine as an antitussive and expectorant for treating chronic bronchitis and acute respiratory infections. Traditionally, Feining Granules are prepared from water extracts of the raw plant material. However, the chemical composition and pharmacological mechanisms of Feining Granules have not been thoroughly investigated. MethodsA systematic strategy for the rapid detection and identification of the constituents of Feining Granules was developed using ultrahigh-performance liquid chromatography–quadrupole-exactive orbitrap mass spectrometry (MS) with parallel reaction monitoring. ResultsOverall, 162 compounds, including flavonoids, alkaloids, organic acids, and others, were identified unambiguously and tentatively by comparing the retention times and MS fragmentation with reference standards and literature data. Ninety-nine of these were reported for the first time to the best of our knowledge. Network pharmacology suggests that Feining Granules can be used to treat chronic bronchitis as they contain active components associated with the ALB, VEGFA, and SRC target genes influenced by HIF-1, VEGF, and other signaling pathways. ConclusionThese results provide information that can help understand the effective substances of S. cannabifolius Less. and improve quality control.

Alkylresorcinol detection and identification in archaeological pottery using ultra-high-performance liquid chromatography-quadrupole/Orbitrap mass spectrometry.

Alkylresorcinols (AR) are cereal-specific biomarkers and have recently been found in archaeological pots. However, their low concentrations and high susceptibility to degradation make them difficult to detect using conventional gas chromatography mass spectrometry (GC/MS). Here we describe the development of a more sensitive liquid chromatography mass spectrometry (LC/MS) method to detect these compounds. A method based on the use of ultra-high-performance liquid chromatography (UHPLC) coupled to an Orbitrap mass analyser was established and validated for the detection of low-concentration ARs in pottery. During the preliminary experiments, UHPLC-Q/Orbitrap MS (ultra-high-performance liquid chromatography-quadrupole/Orbitrap mass spectrometry) was demonstrated to be more sensitive, and a wide range of AR homologues in cereal extracts were detected, unlike UHPLC-QTOFMS (ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry) and GC/MS. The developed method was utilised to profile AR homologue distribution in modern cereal samples and reanalyse AR-containing pots from the archaeological site of Must Farm. A highly sensitive LC/MS method with a limit of detection (LOD) of 0.02 μg/g and a limit of quantification (LOQ) of 0.06 μg/g was used to profile ARs in five modern cereal grains. The obtained LOD is 250 times lower than that obtained using the conventional GC/MS approach. AR 21:0 was the most abundant homologue in all four Triticum spp.-einkorn, emmer, Khorasan wheat and common wheat. Meanwhile, AR 25:0 was the predominant homologue in barley, potentially enabling differentiation between wheat and barley. The developed LC/MS-based method was successfully used to analyse ARs extracted from Must Farm potsherds and identified the cereal species most likely processed in the pots-emmer wheat. The described method offers an alternative and more sensitive approach for detecting and identifying ARs in ancient pottery. It has been successfully utilised to detect AR homologues in archaeological samples and discriminate which cereal species-wheat and barley-were processed in the pots.

Exploring the gut microbiota mediated biotransformation of Senecio scandens Buch.-Ham.: Insights from metabolite spectrum with UHPLC-Q-Orbitrap HRMS and bioinformatics analysis of gut microbiota metabolites

Senecio scandens Buch.-Ham., a traditional Chinese medicine commonly used clinically, exhibits various pharmacological properties, including anti-inflammatory, anti-tumor, antiviral, and antibacterial activities. However, its water extracts' chemical components and metabolites are inadequately understood, limiting further research. In this study, the chemical components and metabolism processes of Senecio scandens, both in vivo (plasma, feces, urine, and bile) and in vitro (gut microbiota and liver microsomes), were characterized based on ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap high-resolution mass spectrometry. Additionally, metabolites detectable in fecal samples and intestinal microbiota incubated but absent in liver microsomes were identified as characteristic metabolites of intestinal microbiota. The targets of the characteristic metabolites of intestinal microbiota were collected, followed by exploration of potential pathways through KEGG enrichment analysis. As a result, a total of 133 chemical components were preliminarily identified, including 35 organic acids, 21 alkaloids, 19 flavonoids and their glycosides, 17 phenylpropanoids, 10 jacaranda ketones, and 31 other compounds. Notably, 12 of these were potentially novel compounds. In addition, 39 prototype components in rats and 109 metabolites were identified and characterized, including 102 in vivo and 52 metabolites in vitro (51 in rat gut microbiota and 24 in rat liver microsomes). The main metabolic pathways include oxidation, reduction, hydrolysis, methylation, glucuronidation, sulfonation, and acetylation reactions. Furthermore, KEGG enrichment analysis revealed that the characteristic metabolites of intestinal microbiota may be related to the ErbB, FoxO, mTOR, and MAPK signaling pathways, exhibiting anti-inflammatory and anti-tumor effects. In summary, the chemical components and metabolites of Senecio scandens were comprehensively identified using a rapid and accurate method, providing a scientific basis for the in-depth study of the material basis and its clinical application of Senecio scandens.