Ultra-high Performance Liquid Chromatography Research Articles

Monitoring of occupational exposure to hazardous medicinal products in robotic compounding

ObjectiveThis study aims to evaluate the risk of occupational exposure to hazardous medicinal products (HMPs) when utilising robotic compounding systems for the preparation of antineoplastic sterile medications. Specifically, it assesses...

Organic Micropollutants in Waterways of a Large-Scale Water Diversion Project: Insights from Nontarget Screening and "Community" Analysis.

Large-scale water diversion projects are essential for meeting the needs of water-stressed regions, necessitating an evaluation of their impact on water quality and aquatic ecosystems. This study provides the first snapshots of organic micropollutants (OMPs) along the 1466 km Eastern Route of China's South-to-North Water Diversion Project. Using nontarget analysis with ultrahigh-performance liquid chromatography and high-resolution mass spectrometry, we identified and quantified 357 OMPs from water samples collected during the water diversion period (WDP) and the nonwater diversion period (NWDP). The OMPs included 136 household compounds, 112 agricultural compounds, 102 industrial compounds, and 7 traffic markers. Significant regional variations in OMP concentrations and compositions were observed during the NWDP due to diverse local pollution sources along the route. However, such differences were reduced during the WDP, likely due to water transfer. OMP diversity varied substantially during the NWDP but was more stable with less distance decay during the WDP. Network analysis indicated closer relationships between the OMPs during the WDP, suggesting a more consistent spatial distribution. The source water overwhelmingly influenced the water quality along the diversion route. These findings underscore the need for ongoing assessments of the impact of water diversion on regional water quality and ecosystems.

Identification of novel serum lipid metabolism potential markers and metabolic pathways for oral cancer: a population-based study

ObjectiveThis study aims to identify potential lipid biomarkers and metabolic pathways associated with oral cancer (OC). Then to establish and evaluate disease classification models capable of distinguishing OC patients from healthy controls.MethodsA total of 41 OC patients and 41 controls were recruited from a hospital in Southeast China to examine the serum lipidomics by Ultra-high Performance Liquid Chromatography Q Exactive Mass Spectrometry (UHPLC-QE-MS).ResultsThe total serum lipid profile showed that triglycerides accounted for the highest proportion of total metabolites, reaching 35.90% of the total. A total of 74 different metabolites were screened (12 up-regulated and 62 down-regulated), mainly enriched in the glycerophospholipid metabolism pathway. The three most significant changes in lipid metabolites were phosphatidylcholine (PC(18:3e/17:2)), acylcarnitine (ACar(14:2)), and glucuronosyldiacylglycerol (GlcADG(14:1/14:1)). The disease classification model, constructed using a KNN algorithm with 13 metabolites selected through LASSO screening, achieved the best performance, with an AUC of 0.978 (0.955-1.000).ConclusionLipid metabolic biomarkers identified in this study exhibit potential as candidate biomarkers for OC diagnosis. Further validation through prospective studies is required to confirm their clinical utility in early detection.

Analytical quantification of the plant specific cardiac glycosides in biological fluids in a rare case of yellow oleander (Cascabela thevetia) self-poisoning in Italy.

Yellow oleander contains digoxin-like cardiac glycosides that frequently cause intoxications in Sri Lanka and India. To date, though, no exposure has been analytically confirmed and quantitative determination of the plant specific glycosides in human biological fluids was never performed. We report an unusual case of self-poisoning in a young female in Italy who ingested 9 crushed seeds of Cascabela thevetia and was admitted to the emergency room with a 2nd degree atrio-ventricular block at the ECG. The case was managed with activated charcoal, repeated doses of anti-digoxin immune Fab and supportive care. A transient thrombocytopenia developed and resolved within a few days. Ultra-high performance liquid chromatography coupled with tandem mass spectrometry was used for the determination and quantification of thevetin A, thevetin B and peruvoside in plasma and urine. Thevetin A and thevetin B, two of the primary glycosides in the seeds, were not present in any of the samples, while detectable levels of the metabolite peruvoside were found in all biological samples on day 1 and 3. Digitoxigenin, the aglycone of thevetin B, was also identified using a semi-untargeted analysis. This is the first report of a quantitative determination of cardiac glycosides of yellow oleander in blood and urine. The finding may support very early administration of activated charcoal to possibly prevent further formation of secondary, and possibly more potent, glycosides and limit toxicity.

Optimized Enzymatic Extraction of Phenolic Compounds from Verbascum nigrum L.: A Sustainable Approach for Enhanced Extraction of Bioactive Compounds

Verbascum nigrum, commonly known as black mullein, is widely used in traditional medicine for its expectorant, mucolytic, sedative, and diuretic properties. This study aimed to develop and optimize a standardized method for extracting phenolic compounds from V. nigrum using enzymatic pretreatment followed by solvent extraction. Enzymatic treatment does not rely on harmful solvents and is a low energy-intensive process, making it a suitable green technology for the food, cosmetic, and pharmaceutical industries. The research explored the use of different lignocellulolytic enzymes, including pectinase, cellulase, α-amylase, and xylanase, to break down plant cell walls, enhancing the release and bioaccessibility of active compounds. The two-step extraction process proposed combined enzymatic pretreatment and hydroalcoholic extraction, resulting in a considerably improved yield of phenolic compounds (24 mg/g DM). Analytical characterization using a high-performance liquid chromatography (HPLC) system coupled with a diode-array-detector (DAD) and ultra-high-performance liquid chromatography (UHPLC) coupled with DAD and tandem mass spectrometry (MS/MS) revealed a higher concentration of target bioactive compounds in enzymatically treated extracts compared to traditional methods, including phenolic derivatives (e.g., caffeic acid, p-coumaric acid, and verbascoside), and flavonoids (e.g., luteolin). Up to 22 phenolic and flavonoid compounds were characterized. This study provides new insight into the potential of enzymatic extraction as a green and efficient alternative to conventional extraction methods, for the production of high-quality herbal products richer in (poly)phenolic compounds, highlighting its potential for industrial applications.

P-1264. Explore The Pharmacokinetic/pharmacodynamics Target Attainment (PTA) of cefoperazone/sulbactam

Abstract Background Cefoperazone/sulbactam (CPZ/SUL) are broad-spectrum antibiotics. Current dosage guidelines consider the renal elimination characteristics of SUL. However, with the risk of antimicrobial resistance, whether current dosage recommendation of CPZ/SUL achieves the desired probability of target attainment (PTA) is unclear. The purpose of this study is to compare the PTA between adjusted-dose (AD) CPZ/SUL and standard-dose (SD) CPZ/SUL therapy. Methods This observational study was conducted in a tertiary hospital in Taiwan during November 2023 to March 2024. Patients using CPZ/SUL (1:1 ratio) were prospectively enrolled. Two blood samples were collected during the dosing interval to measure CPZ and SUL concentrations by using ultra-high performance liquid-chromatography. Concentrations at the middle of the dosing interval (Cm) and before dosing (Ct) were calculated based on measured concentration. PTA was represented by the fraction of time the free concentration was above the minimum inhibitory concentration (fT >MIC). 50% fT >MIC was defined as Cm >MIC, and 100% fT >MIC was defined as Ct >MIC. AD group was defined as reduced CPZ/SUL doses according to the labeling, while SD group had no dose adjustment despite renal impairments. Results A total of 37 patients were enrolled (59.5% male, mean age, 75.4±16.0 years). The most common indication of CPZ/SUL was pneumonia (83.8%). There were 26 patients (70.3%) in the AD group and 11 patients (29.7%) in the SD group. The proportion of patients with 100% fT >MIC was lower in the AD group, as compared to the SD group (CPZ: 15.4% versus 54.5%, P< 0.01; SUL: 11.5% versus 72.7%, P< 0.01). Additionally, 7.7% of patients in the AD group compared to 45.5% of patients in the SD group achieved 100% fT >MIC for both CPZ and SUL (P< 0.01). Thirty patients (81.1%) received weekly vitamin K to prevent CPZ-related coagulopathy, and none had an international normalized ratio above 2. Conclusion PTA was inadequate in patients receiving CPZ/SUL based on current dosage adjustment recommendations. Future investigation is needed to determine if inadequate PTA is linked to worse clinical outcomes, especially in resistant pathogens. Disclosures All Authors: No reported disclosures

Ferula communis leaf extract: antioxidant capacity, UHPLC–MS/MS analysis, and in vivo and in silico toxicity investigations

IntroductionFerula communis has demonstrated an abundance of pharmacological and antioxidative qualities.MethodsThis study investigates the antioxidant activity of F. communis leaf aqueous extract, total polyphenol and flavonoid concentrations, and ultra-high-performance liquid chromatography (UHPLC) composition and then evaluates the toxicity of the plant’s leaves in vitro and in silico. The major compound of the studied extract, namely, p-hydroxybenzoic acid, was chosen for a molecular docking technique to discover the inhibition mechanism toward antioxidant proteins. In addition, a detailed molecular dynamics simulation was carried out to examine the thermodynamic stability of the produced intermolecular interactions. The antioxidant capacity of the extracts of F. communis was evaluated using 2,2-diphenylpicryl hydroxyl (DPPH) radical and ferric reducing antioxidant power (FRAP) procedures. Acute toxicity was tested on albino mice at doses of 200, 300, and 400 mg/kg.ResultsThe results show that the polyphenol and flavonoid contents of the extract are significant (0.257 ± 0.003 mg Eq AG/mg and 0.32 ± 0.04 mg Eq Q/mg, respectively). The antioxidant activity illustrates that the extracts have notable activity in DPPH and FRAP assays. The toxicity study revealed that the mice’s behavior, body weight, and organ weights (liver and kidneys) were unaffected by Ferula communis leaf extract administration compared to controls. UHPLC–tandem mass spectrometry (MS/MS) analysis of the extract highlights the presence of 11 compounds, the most abundant of which is p-hydroxybenzoic acid, representing 53.65%. The predicted pharmacokinetic characteristics of absorption, distribution, metabolism, excretion, and toxicity (ADMET) attest to the well-absorbed nature of the isolated compounds, with human intestinal absorption (HIA) varying from 42% for arbutin (M3) to 100% for ursolic acid (M4).ConclusionIn conclusion, the leaves of Ferula communis are a good source of natural antioxidants and phenolic compounds. Thus, this study demonstrates that this plant has a wide range of applications, including natural food preservatives, pharmaceuticals, and cosmetics, as evidenced by ongoing research.

Yunvjian decoction attenuates lipopolysaccharide-induced acute lung injury by inhibiting NF-κB/NLRP3 pathway and pyroptosis

IntroductionYunvjian (YNJ) decoction, a classic traditional Chinese medicine prescription for inflammatory diseases, has demonstrated good therapeutic effects in the clinical treatment of pneumonia. The aim of this study was to clarify the effective ingredients and mechanism of action of YNJ on lipopolysaccharide (LPS)-induced acute lung injury (ALI).MethodsThe effects of YNJ were evaluated in a mouse model of LPS-induced ALI and in LPS-treated MLE-12 murine lung epithelial cells and RAW264.7 macrophages in vitro. The mechanism of action of YNJ on these model systems was studied using RNA sequencing, immunohistochemical analysis, immunoblotting, immunofluorescence, ELISA, and polymerase chain reaction assays. Ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was applied to identify the absorbed components of YNJ.ResultsYNJ attenuated pulmonary damage in LPS-treated mice, as evidenced by reduced protein content in bronchoalveolar lavage fluid, decreased lung wet/dry weight ratio, and improved respiratory function. Analysis of pneumonia-related lung injury samples from patients in the Gene Expression Omnibus dataset GSE40012 indicated that NOD-like receptor protein 3 (NLRP3)-mediated pyroptosis was a primary mechanism in ALI. YNJ reduced the phosphorylation of nuclear factor-kappa B (NF-κB) and decreased the expression levels of lung NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), cleaved caspase-1, and interleukin-1β levels (IL-1β) in vivo. Administration of YNJ-containing mouse serum increased cell viability and decreased malondialdehyde and reactive oxidative species contents in LPS-stimulated MLE-12 cells. YNJ-containing serum also decreased the secretion of tumor necrosis factor-α, IL-6, and IL-1β in LPS-stimulated RAW264.7 macrophages, and promoted macrophage polarization toward an M2 phenotype. A total of 23 absorbed components were identified in YNJ-containing serum. Among those, network analysis and in vitro experiments indicated that diosgenin, timosaponin BII, and mangiferin are anti-inflammatory active substances.ConclusionYNJ attenuates LPS-induced ALI in mice by inhibiting pyroptosis of lung epithelial cells and macrophages via suppression of the NF-κB/NLRP3 pathway. Our findings provide novel insights into the therapeutic effects of YNJ on ALI.

Carbon nanotubes stabilized in chitosan sponge (CNT-CS) as a new alternative clean-up sorbent for pesticide multiresidue determination in eggplants using QuEChERS and UHPLC-MS/MS.

The continuous development and application of pesticides in agriculture require robust multiresidue detection methods to guarantee food safety. This study introduces a novel method for multiresidue determination of pesticides in eggplants using the QuEChERS procedure, incorporating a clean-up step using carbon nanotubes stabilized in chitosan sponge (CNT-CS) and ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) for analysis. Upon identifying the optimal extraction conditions, various sorbents were assessed for their efficacy in the dispersive solid-phase extraction (d-SPE). Among these, the biosorbent CNT-CS emerged as the most efficient and cost-effective material, showing the best recovery results from spiked blank samples. The validation demonstrated that the method was adequate for analyzing 111 pesticides in eggplant samples with practical method quantification limits from 5 to 25µgkg-1. When applied to commercial eggplant samples, 27 pesticides were detected, demonstrating the importance of the proposed method. It is noteworthy that the conducted work represents the first report on the use of the biosorbent CNT-CS in the clean-up step of the QuEChERS method. The results obtained in the validation and application steps demonstrate that the proposed method is an excellent alternative for monitoring programs.

A Comparative Study of the Biological Properties of Eugenia uniflora L. Fruits and Leaves Related to the Prevention of Cardiovascular Diseases

Cardiovascular diseases (CVDs) remain the leading cause of death globally, emphasizing the need for effective preventive strategies. Plant-based foods, rich in phytochemicals, offer a promising potential in CVD prevention. This study investigated the antioxidant, anti-inflammatory, and antihypertensive properties of two Eugenia uniflora L. varieties (orange and purple pitanga) and their leaves. Their antioxidant activity was assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation scavenging activity assays, while their antihypertensive activity was evaluated through angiotensin-converting enzyme (ACE) inhibition. Their anti-inflammatory potential was determined via protein denaturation inhibition. Both fruit varieties exhibited similar bioactivities, with the purple variety showing a slightly higher activity, except in the DPPH and ABTS assays. The leaves consistently demonstrated the lowest activities across all assays. Free polyphenols, dominated by gallic acid, were quantified using µ-QuEChERS followed by ultrahigh-performance liquid chromatography (UHPLC-PDA). The orange variety contained the highest concentration of gallic acid (13.1 mg/100 g DW). These findings highlight the potential of Eugenia uniflora L. extracts as natural antioxidant, anti-inflammatory, and antihypertensive agents, suggesting their value in food, pharmaceutical, and cosmetic applications for promoting human health and preventing CVDs.

Nutraceutical Prospects of Pumpkin Seeds: A Study on the Lipid Fraction Composition and Oxidative Stability Across Eleven Varieties

The oxidative stability of nutritive and bioactive lipids is essential for their functionality. This study evaluated the potential of lipid fractions from pumpkin seeds obtained from eleven high-performing cultivars of Cucurbita maxima Duchesne, C. pepo L., and C. moschata Duchesne cultivated in Poland, aiming to evaluate their stability for nutraceutical applications. This study investigated the intrinsic relationship between chemical composition and oxidative stability to identify cultivars with promising functional potential and commercial value. The fatty acid, sterol, and lipid antioxidant profiles were characterized using gas chromatography (GC), GC–mass spectrometry (GC-MS), and ultra-high-performance liquid chromatography (UPLC), respectively. Antiradical activity was assessed via the DPPH assay, and oxidative stability was evaluated using differential scanning calorimetry (DSC). The oils exhibited high levels of polyunsaturated fatty acids (PUFAs) (59.5–68.6%), with n-6/n-3 fatty acid ratios ranging from 66.5 to 211.6. The lipid extracts contained up to 97.1% Δ7-sterols, while key antioxidants included squalene (616.6–3092.0 mg/kg) and γ-tocopherol (54.1–423.6 mg/kg). Notably, the C. pepo cultivar ‘Moonshine’ was the least abundant in these bioactive compounds. The carotenoid content ranged from 5.7 to 19.4 mg/kg across the extracts. Among the studied cultivars, ‘Show Winner’ and ‘Pink Jumbo Banana’ (C. maxima) stood out as promising candidates for nutraceutical applications due to their elevated levels of tocopherols, carotenoids, and squalene. A moderate n-6/n-3 fatty acid ratio (100–170), coupled with balanced levels of γ-tocopherol and squalene, was found to significantly enhance the oxidative stability of pumpkin seed lipids. These lipid fractions also show potential as stabilizing additives for oils rich in α-linolenic acid but deficient in natural antioxidants.

Systematic identification of the chemical components of leaves and roots of Didymocarpus heucherifolius Hand.-Mazz. based on UHPLC Q-Exactive Orbitrap MS technology coupled with molecular networking strategy

Didymocarpus heucherifolius Hand.-Mazz. is plant of the genus Didymocarpus wall. (Gesneriaceae). Species in this genus have long been used in folk medicine to treat various illnesses, including wounds, kidney stones, inflammations, asthma, influenza, etc. However, there are few studies on chemical components. Crude plant extracts are a complex mixture of several biologically active specialized metabolites. Therefore, a rapid and accurate identification and quantification are critical in phytochemical analysis. An efficient approach based on molecular networking strategy coupled with ultra high-performance liquid chromatography coupled with Q-Exactive Orbitrap tandem mass spectrometry (UHPLC Q-Exactive Orbitrap MS) was used to analyze the chemical components systematically. The chemical was identified using retention time, accurate molecular weight, parent peaks, fragment peaks, fragmentation characteristics, comparative analysis with the literature, reference standards, and standard databases, including mzVault. A total of 108 compounds (32 phenylpropanoids, 14 phenolic acid, 6 fatty acids, 14 organic acid, 9 flavonoid, 15 amino acids, and 28 other components) were identified in the leaves and roots of D. heucherifolius in which 84 and 84 compounds were found in the leaves and roots, respectively. The results showed that 68 components were different in plant roots and leaves, and these compounds were mainly represented in organic acids. Moreover, 50 identical chemical compounds were identified in plant roots and leaves, and these compounds were mainly phenylpropanoid compounds. The UHPLC Q-Exactive Orbitrap MS method was used for the first time to accurately, quickly, and systematically identify the compounds of the roots and leaves of Didymocarpus heucherifolius Hand.-Mazz., thereby providing a theoretical basis for its quality control, in-depth research, and further development.

Anti-inflammatory and antioxidant properties of Camellia sinensis L. extract as a potential therapeutic for atopic dermatitis through NF-κB pathway inhibition

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by immune dysregulation and excessive cytokine production. This study aimed to explore the potential of Camellia sinensis L. water extract (CSE) as a treatment for AD by the impact of CSE on inflammatory responses in keratinocytes, particularly concerning the production of inflammatory cytokines and the modulation of signaling pathways relevant to AD pathogenesis. CSE was obtained via hot water extraction from Camellia sinensis L. Ultra-high-performance liquid chromatography (UPLC) analyzed catechin and caffeine content. Cell viability was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), polyphenol and flavonoid content were determined. 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay measured antioxidant activity. Enzyme-Linked Immunosorbent Assay (ELISA), western blotting, and Immunofluorescence (IF) assays examined cytokines, pathways, and protein localization, respectively. Molecular docking assessed compound binding with inflammation-related proteins. UPLC identified six CSE components including epigallocatechin (EGC) epicatechin (EC), caffeine (CF), catechin (C), epigallocatechin gallate (EGCG), and epicatechin gallate (ECG). CSE demonstrated a significant reduction in the production of inflammatory cytokines interleukin (IL)-2 and IL-6 in TNF-α/IFN-γ activated keratinocytes. Treatment with CSE inhibited the mitogen-activated protein kinase (MAPK) pathway, which resulted in decreased phosphorylation of p38, Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK). Exposure of TNF-α/IFN-γ- stimulated human keratinocytes (HaCaT) cells to CSE resulted in a 200 µg/mL dependent inhibition of p65 and signal transducer and activator of transcription 1 (STAT-1) translocation from the cytosol to the nucleus, as confirmed through immunofluorescence (IF) staining. Molecular docking simulations provided insights into the underlying mechanisms of CSE action, which supported its potential as a therapeutic agent for AD. CSE might be a potential candidate for its therapeutic efficacy for inflammatory skin conditions like AD. Thus, based on this evidence, the authors suggest that CSE should be studied further for its anti-inflammatory activities and topical application in the treatment of AD.

Simultaneous extraction of flavonoids and saponins from functional food via basic deep eutectic solvent-assisted mechanochemical extraction.

An efficient, green, and novel basic deep eutectic solvent (DES)-assisted mechanochemical extraction method, combined with ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, is proposed for simultaneous extraction and determination of target compounds from functional food. Several parameters are investigated using single-factor optimization, and response surface methodology is employed to determine the optimum extraction conditions. The conditions include a grinding time of 316.29s, 83.72μL of DES (K2CO3 and glycerol), and vortex time of 2.408min. The developed method provides good linearity (R2>0.9958), stable intraday and interday precision ranging from 0.07% to 3.31%, high sensitivity (LODs <20.83ng/mL), and acceptable accuracy with recoveries of 83.75%-102.02%. Moreover, satisfactory greenness (0.71) and practicality (72.5) are achieved in terms of the Analytical Greenness metric and the blue applicability grade index. The proposed method notably contributes to the simultaneous extraction and detection of flavonoids and saponins in astragali radix.

The Stability-Indicating Ultra High-Performance Liquid Chromatography with Diode Array Detector and Tandem Mass Spectrometry Method Applied for the Forced Degradation Study of Ritlecitinib: An Appraisal of Green and Blue Metrics.

Janus kinase inhibitors open new horizons for small-molecule drugs in treating inflammatory bowel disease, with ritlecitinib demonstrating significant efficacy in clinical trials for ulcerative colitis and Crohn's disease. Ritlecitinib, a second-generation JAK3 inhibitor, is a novel therapeutic agent for alopecia areata and other autoimmune conditions. A new stability-indicating UHPLC-DAD-MS/MS method was developed, validated, and applied for a forced degradation study of ritlecitinib under ICH guidelines. The method demonstrated high specificity, sensitivity (LOD: 0.04 µg/mL; LOQ: 0.14 µg/mL), precision (RSD ≤ 0.15%), and accuracy (99.9-100.3%). Forced degradation studies under acidic, basic, oxidative, thermal, and photolytic conditions revealed four novel degradation products. Basic degradation followed second-order kinetics, while oxidative degradation followed zero-order kinetics. The validated method reliably characterized ritlecitinib's stability and degradation products, providing essential data for optimizing formulation, determining proper storage conditions, anticipating drug-excipient interactions, and ensuring quality control. The eco-friendliness and applicability of the developed forced degradation procedure were evaluated using various green and blue metric tools. Incorporating green analytical principles underscores its potential for sustainable pharmaceutical analysis.

Detection of LGD-4033 Metabolites in Camel Urine, Plasma, and Hair Following Oral Administration for Doping Control.

LGD-4033, a selective androgen receptor modulator (SARM), is recognized for promoting muscle growth and enhancing athletic performance. Its potent anabolic effects have led to its prohibition in both human and animal sports. Although initial invitro studies have offered insights into its metabolism, an in-depth invivo analysis is necessary to fully understand its metabolic pathways. This research aims to thoroughly investigate the metabolic profile of LGD-4033 in camels to establish reliable analytical markers, thereby addressing an important gap in the doping detection in camel racing. Urine, plasma, and hair samples were collected from three healthy camels following a single oral administration of 100 mg LGD-4033 mixed with food. These biological samples were then analyzed using ultrahigh-performance liquid chromatography coupled with high-resolution Q Exactive Orbitrap mass spectrometry (UHPLC-HRMS). Fourteen metabolites were identified across all sample types. The main Phase I metabolite, M6 (dihydroxylation), appears to be a primary target analyte for urine-based doping analysis. Both dihydroxy and trihydroxy metabolites were notably abundant in urine, accompanied by glucuronic and sulfonic acid conjugates; however, sulfonic acid conjugates were absent invitro studies. Further research on hair samples, especially at higher LGD-4033 dosages, is recommended. The findings from this study will improve the rapid identification of LGD-4033 and related compounds, supporting effective doping control in camel racing. These results enhance understanding of LGD-4033 metabolism in camels and advance the development of reliable methods for detecting SARMs in this field.

Analyzing How Zhizi Baipi Decoction Regulates VEGF to Suppress RA Angiogenesis Using Network Pharmacology and Experimental Validation.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that primarily manifests with symptoms such as heat and toxin. However, the key components and molecular mechanisms of Zhizi Baipi decoction (ZBD) in the treatment of RA are still unclear. The study aimed to explore the mechanism of action of ZBD for treating RA through ingredient analysis, network pharmacology, and experimental validation. The chemical constituents of ZBD were identified by ultra-high performance liquid chromatography coupled with Q-TOF-mass spectrometry (UPLC-Q-TOF-MSE). Additionally, the active ingredients of ZBD treating RA were screened by network pharmacology and using molecular docking to verify the binding energy of the active ingredients and ZBD's targets. Then we elucidated ZBD's mechanism of action on collagen-induced arthritis (CIA) model rats. Subsequently, experimental validations were used to validate the findings of network pharmacology. A total of 84 chemical constituents was identified by UPLC-Q-TOF-MSE. The results of network pharmacology indicated that ZBD could exert its therapeutic effect on RA through the vascular endothelial growth factor (VEGF) pathway. Molecular docking revealed a strong binding capacity between the target KDR and the active ingredients. Additionally, we quantified the five active ingredients of ZBD. Invivo experiments demonstrated that ZBD inhibited synovial angiogenesis and alleviated the occurrence and progression of RA. Overall, ZBD has a significant therapeutic effect on RA. The results of qualitative analysis, network pharmacology, molecular docking, and invivo experiments indicated that the main active components of ZBD could modulate the VEGF pathway to treat RA.

Chemical Fingerprinting and Multivariate Analysis of Different Grades of Farfarae Flos Using HPLC and UPLC-ESI-Q/TOF-MS/MS.

Farfarae Flos is widely used as a traditional herbal medicine. Currently, its size has been the primary grading criterion used in market circulation. Whether this empirical criterion can accurately reflect the quality of the medicinal material has not been systematically studied. This study aimed to evaluate the quality of Farfarae Flos from different regions based on their grades. Ultra-high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q/TOF-MS/MS) were applied to study the chemical constituents of Farfarae Flos; high performance liquid chromatography (HPLC) was used to quantify the content of Farfarae Flos samples. Meanwhile, fingerprint analysis and chemometric methods, including principal component analysis (PCA) and analysis of variance (ANOVA), were used to evaluate the quality differences among 33 batches of Farfarae Flos samples of different grades. A total of 95 individual components were identified in Farfarae Flos. Fingerprint analysis revealed 23 common peaks, with fingerprint similarity among the 33 batches ranging from 0.838 to 0.995. PCA divided the 33 batches of Farfarae Flos into three categories based on their grades. ANOVA indicated significant differences in five of the 14 main active components across different grades of Farfarae Flos, with two components showing extremely significant differences. HPLC content determination showed that the content of 11 main active components was positively correlated with the grades of Farfarae Flos. This method is straightforward, efficient, and reliable, offering a valuable reference for establishing quality grading standards and ensuring the quality control of Farfarae Flos.

Simultaneous Profiling of Multiple Phosphorylated Metabolites in Typical Biological Matrices via Ion-Pair Reversed-Phase Ultrahigh-Performance Liquid Chromatography and Mass Spectrometry.

Simultaneous analysis of multiple phosphorylated metabolites (phosphorylated metabolome) in biological samples is vital to reveal their physiological and pathophysiological functions, which is extremely challenging due to their low abundance in some biological matrices, high hydrophilicity, and poor chromatographic behavior. Here, we developed a new method with ion-pair reversed-phase ultrahigh-performance liquid chromatography and mass spectrometry using BEH C18 columns modified with hybrid surface technology. This method demonstrated good performances for various phosphorylated metabolites, including phosphorylated sugars and amino acids, nucleotides, NAD-based cofactors, and acyl-CoAs in a single run using standard LC systems. Specifically, the method showed good retention (capacity factor > 2) and reproducibility (ΔtR < 0.09 min, n = 6), peak symmetry (tailing factor < 2), and sensitivity (limit-of-detection < 238 fmol-on-column with QTOFMS) for all tested analytes especially for the medium- and/or long-chain acyl-CoAs. The method demonstrated reproducible applicability across numerous biological matrices, including tissue (liver), human biofluids (urine, plasma), cells, and feces, and revealed significant molecular phenotypic differences in phosphorylated metabolite composition.

Ultra-high performance liquid chromatography ion mobility-high-resolution mass spectrometry for the assessment of raw milk traceability.

The complexity of modern food supply chains limits the effectiveness of targeted approaches to address food traceability issues. Untargeted metabolomics provides a comprehensive profile of small molecules present within biological samples. In this study, the potential of ultra-high performance liquid chromatography-ion mobility-high resolution mass spectrometry (UHPLC-IMS-HRMS) to discriminate bovine milk samples collected at individual level was evaluated for traceability purposes. For the first time, IMS coupled with UHPLC-HRMS was applied to milk analysis, increasing confidence in metabolite annotation. Supervised Partial Least Squares-Discriminant Analysis coupled to backward elimination variable selection allowed the selection of 52 and 153 features able to discriminate samples belonging to different dairy supply chains and trace samples at herd level, respectively. Amino acids, glycerolipids, and glycerophospholipids were the most represented classes, influencing the biological/technological properties of the final product. The perfect classification of samples belonging to external test sets demonstrated the reliability of the proposed approach.