Influence of Software Settings on the Identification Rate, Quantification Results, and Reproducibility in Profiling Post-Translational Modifications by Microflow Liquid Chromatography-Ion Mobility-Quadrupole Time-Of-Flight Analysis Using PEAKS Software.

The influence of data evaluation parameters on qualitative and quantitative results of untargeted shotgun profiling of enzymatic and nonenzymatic post-translational modifications (PTMs) was investigated in a model of bovine whey protein α-lactalbumin heated with lactose. Based on the same raw data, individual adjustments to the protein database and enzyme settings of PEAKS studio software increased the identification rate from 27 unmodified peptides to 48 and from 322 peptides in total to 535. The qualitative and quantitative reproducibility was also assessed based on 18 measurements of one sample across three batches. A total of 570 peptides were detected. While 89 peptides were identified in all measurements, the majority of peptides (161) were detected only once and mostly based on nonindicative spectra. The reproducibility of label-free quantification (LFQ) in six measurements of the same sample was similar after processing the data by either the PTM algorithm or the LFQ algorithm. In both cases, about one-third of the peptides showed a coefficient of variation of above 20%. However, the LFQ algorithm increased the number of quantified peptides from 75 to 179. Data are available at the PRIDE Archive with the data set identifier PXD050363.

Reinforcing the Evidence of Mitochondrial Dysfunction in Long COVID Patients Using a Multiplatform Mass Spectrometry-Based Metabolomics Approach.

Despite the recent and increasing knowledge surrounding COVID-19 infection, the underlying mechanisms of the persistence of symptoms for a long time after the acute infection are still not completely understood. Here, a multiplatform mass spectrometry-based approach was used for metabolomic and lipidomic profiling of human plasma samples from Long COVID patients (n = 40) to reveal mitochondrial dysfunction when compared with individuals fully recovered from acute mild COVID-19 (n = 40). Untargeted metabolomic analysis using CE-ESI(+/-)-TOF-MS and GC-Q-MS was performed. Additionally, a lipidomic analysis using LC-ESI(+/-)-QTOF-MS based on an in-house library revealed 447 lipid species identified with a high confidence annotation level. The integration of complementary analytical platforms has allowed a comprehensive metabolic and lipidomic characterization of plasma alterations in Long COVID disease that found 46 relevant metabolites which allowed to discriminate between Long COVID and fully recovered patients. We report specific metabolites altered in Long COVID, mainly related to a decrease in the amino acid metabolism and ceramide plasma levels and an increase in the tricarboxylic acid (TCA) cycle, reinforcing the evidence of an impaired mitochondrial function. The most relevant alterations shown in this study will help to better understand the insights of Long COVID syndrome by providing a deeper knowledge of the metabolomic basis of the pathology.

Anti-inflammatory potential of aspergillus unguis SP51-EGY: TLR4-dependent effects & chemical diversity via Q-TOF LC-HRMS.

Inflammation serves as an intricate defense mechanism for tissue repair. However, overactivation of TLR4-mediated inflammation by lipopolysaccharide (LPS) can lead to detrimental outcomes such as sepsis, acute lung injury, and chronic inflammation, often associated with cancer and autoimmune diseases. This study delves into the anti-inflammatory properties of "Aspergillus unguis isolate SP51-EGY" on LPS-stimulated RAW 264.7 macrophages. Through real-time qPCR, we assessed the expression levels of pivotal inflammatory genes, including iNOS, COX-2, TNF-α, and IL-6. Remarkably, our fungal extracts significantly diminished NO production and showed noteworthy reductions in the mRNA expression levels of the aforementioned genes. Furthermore, while Nrf2 is typically associated with modulating inflammatory responses, our findings indicate that the anti-inflammatory effects of our extracts are not Nrf2-dependent. Moreover, the chemical diversity of the potent extract (B Sh F) was elucidated using Q-TOF LC-HRMS, identifying 54 compounds, some of which played vital roles in suppressing inflammation. Most notably, compounds like granisetron, fenofibrate, and umbelliprenin were found to downregulate TNF-α, IL-1ß, and IL-6 through the NF-κB signaling pathway. In conclusion, "Aspergillus unguis isolate SP51-EGY", isolated from the Red Sea, Egypt, has been unveiled as a promising TLR4 inhibitor with significant anti-inflammatory potentials, presenting novel insights for their potential therapeutic use in inflammation.

Eight key rules for successful data-dependent acquisition in mass spectrometry-based metabolomics.

In recent years, metabolomics has emerged as a pivotal approach for the holistic analysis of metabolites in biological systems. The rapid progress in analytical equipment, coupled to the rise of powerful data processing tools, now provides unprecedented opportunities to deepen our understanding of the relationships between biochemical processes and physiological or phenotypic conditions in living organisms. However, to obtain unbiased data coverage of hundreds or thousands of metabolites remains a challenging task. Among the panel of available analytical methods, targeted and untargeted mass spectrometry approaches are among the most commonly used. While targeted metabolomics usually relies on multiple-reaction monitoring acquisition, untargeted metabolomics use either data-independent acquisition (DIA) or data-dependent acquisition (DDA) methods. Unlike DIA, DDA offers the possibility to get real, selective MS/MS spectra and thus to improve metabolite assignment when performing untargeted metabolomics. Yet, DDA settings are more complex to establish than DIA settings, and as a result, DDA is more prone to errors in method development and application. Here, we present a tutorial which provides guidelines on how to optimize the technical parameters essential for proper DDA experiments in metabolomics applications. This tutorial is organized as a series of rules describing the impact of the different parameters on data acquisition and data quality. It is primarily intended to metabolomics users and mass spectrometrists that wish to acquire both theoretical background and practical tips for developing effective DDA methods.

Temporal changes in plasma metabolic signatures to predict immune response of antiretroviral therapy among people living with HIV.

Antiretroviral therapy (ART) is an effective treatment for people living with HIV (PLHIVs), requiring an extended period to achieve immune reconstitution. Metabolic alterations induced by ART are crucial for predicting long-term therapeutic responses, yet comprehensive investigation through large-scale clinical studies is still lacking. Here, we collected plasma samples from 108 PLHIVs to the untargeted plasma metabolomics study, based on the longitudinal metabolomics design. Cross-sectional analyzes were performed at pre- and post-ART to explore the metabolic transformation induced by the therapy. Subsequently, delta values between pre- and post-ART measurements were calculated to quantify metabolic alterations. Then, the optimal set of metabolic traits and clinical signatures were further identified and applied to construct random forest model for predicting the future therapeutic responses to ART. We found distinct ART-induced metabolic transformation among PLHIVs. After confounder-adjustments, five metabolites exhibited significant associations with future immune response: tetracosatetraenoic acid (24:4n-6) (pre-ART) (odds ratio [OR]: 0.978, 95% confidence interval [CI]: 0.955~0.997), 1-(3,4-dihydroxyphenyl)-5-hydroxy-3-decanone (pre-ART) (OR: 1.298, 95% CI: 1.061~1.727), beta-PC-M6 (change) (OR: 0.967, 95% CI: 0.938~0.993), d-Galactaro-1,4-lactone (change) (OR: 1.032, 95% CI: 1.007~1.063), Annuionone C (change) (OR: 1.100, 95% CI: 1.030~1.190). The addition of plasma metabolites to clinical markers accurately predicted immune response to ART with an area under curve of 0.91. Notably, most disrupted metabolites were significantly correlated with blood lipids, suggesting that metabolic transformation might contribute to dyslipidemia among PLHIVs. This study highlights the distinct metabolic transformation post-ART among PLHIVs and reveals the potential role of metabolic transformation as key determinants of ART efficacy.

An untargeted metabolomics approach to study changes of the medium during human cornea culture.

INTRODUCTION: Two main approaches (organ culture and hypothermia) for the preservation and storage of human donor corneas are globally adopted for corneal preservation before the transplant. Hypothermia is a hypothermic storage which slows down cellular metabolism while organ culture, a corneal culture performed at 28-37 °C, maintains an active corneal metabolism. Researchers, till now, have just studied the impact of organ culture on human cornea after manipulating and disrupting tissues. OBJECTIVES: The aim of the current work was to optimize an analytical procedure which can be useful for discovering biomarkers capable of predicting tissue health status. For the first time, this research proposed a preliminary metabolomics study on medium for organ culture without manipulating and disrupting the valuable human tissues which could be still used for transplantation. METHODS: In particular, the present research proposed a method for investigating changes in the medium, over a storage period of 20 days, in presence and absence of a human donor cornea. An untargeted metabolomics approach using UHPLC-QTOF was developed to deeply investigate the differences on metabolites and metabolic pathways and the influence of the presence of the cornea inside the medium. RESULTS: Differences in the expression of some compounds emerged from this preliminary metabolomics approach, in particular in medium maintained for 10 and 20 days in presence but also in the absence of cornea. A total of 173 metabolites have been annotated and 36 pathways were enriched by pathway analysis. CONCLUSION: The results revealed a valuable untargeted metabolomics approach which can be applied in organ culture metabolomics.

Detection of dynorphin 1-17 biotransformation fragments in human nasal polyps by UPLC-QTOF-MS.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a persistent inflammation of the sinonasal mucosa. CRSwNP treatments are associated with inconsistent efficacy and recurrence of symptoms. Dynorphin 1-17 (DYN 1-17) and its fragments have been shown to modulate the immune response in various inflammatory conditions. This study aimed to investigate the effect of different pH and degrees of inflammation on DYN 1-17 metabolism in human CRSwNP tissues. DYN 1-17 was incubated with grade 3 and grade 4 inflamed tissues of CRSwNP patients at pH 5.5 and pH 7.4 over a range of incubation periods. The resulting fragments were identified using an ultra-performance liquid chromatography (UPLC) system coupled to quadrupole-time of flight (QTOF) mass spectrometry based on their accurate mass. The rate of DYN 1-17 fragmentation was slower at pH 5.5 in comparison to pH 7.4. The extent and rate of metabolism of DYN 1-17 were much lower in grade 3 inflamed tissue (31-32 fragments) than in grade 4 (34-41 fragments). N-Terminal fragments (DYN 1-15, 1-11, 1-10, and 1-6) were metabolized slower at pH 5.5 as compared to pH 7.4. DYN 1-12, 1-8, 2-10, 4-10, 5-10, and 8-14 were only observed under the inflammatory pH while DYN 5-17 and 6-17 were only identified upon incubation with grade 4 CRSwNP tissues. DYN 1-17 metabolism was significantly affected by the pH level and the severity of the inflammation of CRSwNP tissues, indicating the potential roles of DYN 1-17 and its fragments in modulating the inflammatory response and their avenue as therapeutics in future studies.

Comparison of high-resolution mass spectrometry acquisition methods for the simultaneous quantification and identification of per- and polyfluoroalkyl substances (PFAS).

Simultaneous identification and quantification of per- and polyfluoroalkyl substances (PFAS) were evaluated for three quadrupole time-of-flight mass spectrometry (QTOF) acquisition methods. The acquisition methods investigated were MS-Only, all ion fragmentation (All-Ions), and automated tandem mass spectrometry (Auto-MS/MS). Target analytes were the 25 PFAS of US EPA Method 533 and the acquisition methods were evaluated by analyte response, limit of quantification (LOQ), accuracy, precision, and target-suspect screening identification limit (IL). PFAS LOQs were consistent across acquisition methods, with individual PFAS LOQs within an order of magnitude. The mean and range for MS-Only, All-Ions, and Auto-MS/MS are 1.3 (0.34-5.1), 2.1 (0.49-5.1), and 1.5 (0.20-5.1) pg on column. For fast data processing and tentative identification with lower confidence, MS-Only is recommended; however, this can lead to false-positives. Where high-confidence identification, structural characterisation, and quantification are desired, Auto-MS/MS is recommended; however, cycle time should be considered where many compounds are anticipated to be present. For comprehensive screening workflows and sample archiving, All-Ions is recommended, facilitating both quantification and retrospective analysis. This study validated HRMS acquisition approaches for quantification (based upon precursor data) and exploration of identification workflows for a range of PFAS compounds.

Comprehensive metabolome characterization and comparison between two sources of Dragon's blood by integrating liquid chromatography/mass spectrometry and chemometrics.

Dragon's Blood (DB) serves as a precious Chinese medicine facilitating blood circulation and stasis dispersion. Daemonorops draco (D. draco; Qi-Lin-Jie) and Dracaena cochinchinensis (D. cochinchinenesis; Long-Xue-Jie) are two reputable plant sources for preparing DB. This work was designed to comprehensively characterize and compare the metabolome differences between D. draco and D. cochinchinenesis, by integrating liquid chromatography/mass spectrometry and untargeted metabolomics analysis. Offline two-dimensional liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (2D-LC/IM-QTOF-MS), by utilizing a powerful hybrid scan approach, was elaborated for multicomponent characterization. Configuration of an XBridge Amide column and an HSS T3 column in offline mode exhibited high orthogonality (A0 0.80) in separating the complex components in DB. Particularly, the hybrid high-definition MSE-high definition data-dependent acquisition (HDMSE-HDDDA) in both positive and negative ion modes was applied for data acquisition. Streamlined intelligent data processing facilitated by the UNIFI™ (Waters) bioinformatics platform and searching against an in-house chemical library (recording 223 known compounds) enabled efficient structural elucidation. We could characterize 285 components, including 143 from D. draco and 174 from D. cochinchinensis. Holistic comparison of the metabolomes among 21 batches of DB samples by the untargeted metabolomics workflows unveiled 43 significantly differential components. Separately, four and three components were considered as the marker compounds for identifying D. draco and D. cochinchinenesis, respectively. Conclusively, the chemical composition and metabolomic differences of two DB resources were investigated by a dimension-enhanced analytical approach, with the results being beneficial to quality control and the differentiated clinical application of DB.

Characteristic alkaloids from Stemona sessilifolia with lung protective effects.

In the research on lung protective effects from the roots of Stemona sessilifolia, twenty-five Stemona alkaloids have been isolated, including four undescribed components (1, 3-5), a new natural product (2) and 20 known alkaloids (6-25). Their structures were analyzed by NMR spectra, high-resolution mass spectrum data, and other chemical methods. UPLC-QTOF/MS method was used to identify the Stemona alkaloids and summarize the fragmentation patterns of mass spectrometry. The lung-protective effects of these compounds were evaluated using MLE-12 cells induced by NNK and nm SiO2. The results showed that compounds 3, 5, 8, 10-11, 17-21 and 23 exhibited protective effects on NNK-induced cell injury. Compounds 2, 8-11, 14, 17-19 and 22 showed improvement in nm SiO2-induced lung epithelial cell injury. Compound 10 (tuberostemonine D), a representative alkaloid with a high content in Stemona sessilifolia, significantly protected C57BL/6 lung injury mice induced by nm SiO2, suggesting it a key component of Stemona alkaloids that play a protective role in lung injury. The results of in vivo activity showed that compound 10 could improve the lung injury of mice, reduce ROS content, and recover the levels of SOD and MDA in serum. Its protective effect on lung injury might be related to Nrf2 activation.

Enhancement of per- and Polyfluoroalkyl Substances (PFAS) quantification on surface waters from marinas in the douro river, Portugal.

PFAS, known as "forever" compounds, are prevalent in various environments, including soils and aquatic systems, due to extensive usage. Surface waters in several European countries, especially marinas and ports with high boat traffic, require further study as potential contamination sources. Reliable methods for the extraction and quantification of these emergent compounds are essential. This study aimed to improve an existent solid phase extraction method to analyse marinas and ports' surface waters with variable salinities (2, 9 and 17 PSU). The objectives were to: 1) optimise the solid phase extraction method, considering matrix salinity effects and cross-contaminations, 2) validate the extraction and quantification method of 18 EPA 537.1 PFAS in estuarine surface waters, using the Ultra-High Performance Liquid Chromatography - Quadrupole Time - Of - Flight - Tandem Mass spectrometry, and 3) apply the optimised method for PFAS quantification in three Portuguese marinas. All ICH criteria were successfully validated considering 9 PSU. Limits of quantification ranged from 117.80 ng/L to 385 ng/L, except for PFHpA (645.85 ng/L). PFAS levels (PFOA, HFPO-DA, PFBS, PFHxS and PFOS) were relatively low, reaching a maximum of 0.32 ng/L only for the PFOA. In Freixo marina, total average concentrations were slightly higher (∑PFAS = 1.02 ng/L) when compared to the ones found in Cais da Ribeira Port (∑PFAS = 0.94 ng/L) and Afurada marina (∑PFAS = 0.81 ng/L). PFOS concentrations are below the limit values set by the Environmental Quality Standards (36000 ng/L of PFOS for inland surface water, respectively), similar to other Portuguese river studies. This study enabled the development of a precise and reliable extraction and quantification method to quantify PFAS in estuarine surface waters, particularly from marinas. This method can be readily applied to analyse PFAS in other estuarine samples.

Development and Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method for Screening Potential Citrate Lyase Inhibitors from a Library of Marine Compounds.

Tuberculosis, a persistent illness caused by Mycobacterium tuberculosis, remains a significant global public health challenge. The widespread use of anti-tuberculosis drugs has resulted in the emergence of drug-resistant strains, which complicates treatment efforts. Addressing this issue is crucial and hinges on the development of new drugs that can effectively target the disease. This involves identifying novel therapeutic targets that can disrupt the bacterium's survival mechanisms in various environments such as granulomas and lesions. Citrate lyase, essential for the survival of Mycobacterium species at lesion sites and in granulomatous conditions, is a potential target for the treatment of tuberculosis. This manuscript aimed to construct an efficient enzyme inhibitor screening platform using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF MS). This system can accurately identify compounds with enzyme inhibitory activity from a library of marine terpenoids and phenolic compounds. Utilizing the screened herbal enzyme inhibitors as a starting point, we analyzed their chemical structures and skillfully built a library of marine compounds based on these structures. The results showed that all of the tested compounds from the phenolics library inhibited citrate lyase by more than 50%, and a significant portion of terpenoids also demonstrated inhibition, with these active terpenoids comprising over half of the terpenoids tested. The study underscores the potential of marine-derived phenolic and terpenoid compounds as potent inhibitors of citrate lyase, indicating a promising direction for future investigations in treating tuberculosis and associated disorders.

An UHPLC-QTOF-MS-based strategy for systematic profiling of chemical constituents and associated in vivo metabolites of a famous traditional Chinese medicine formula, Yinchenhao decoction.

Yinchenhao decoction (YCHD), a famous traditional Chinese medicine formula, has been applied for relieving jaundice in China for more than 1800 years. However, the material basis for YCHD is still unclear, and the chemical composition and metabolism characteristic in vivo are undefined, making the potential effective constituents and mechanism of action unclear. Herein, an ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS)-based strategy was applied for the chemical profiling of YCHD, as well as their in vivo prototypes and global metabolites that defined the metabolome. Our results showed that a total of 139 chemicals were identified in YCHD, including 28 organic acids, 12 monoterpenoids, five diterpenes, three triterpenoids, 17 iridoids, 23 anthraquinones, 26 flavonoids, four coumarins and 21 other types. Moreover, 58 prototypes and 175 metabolites were found in rat biological samples after oral administration of YCHD; those distributed in plasma, liver, intestine and feces were suggested to be potentially effective substances. Oxidation, hydrogenation, decarboxylation and conjugations with methyl, sulfate and glucuronate were considered as the predominant metabolic pathways in vivo. In conclusion, this is a systemic study of chemical constituents and in vivo metabolome profiles of YCHD, contributing to the material basis understanding and further mechanism research.

UPLC-quadrupole time-of-flight-tandem mass spectrometry combined with chemometrics and network pharmacology to differentiate Coreopsis tinctoria Nutt.

Coreopsis tinctoria Nutt. (C. tinctoria) is a traditional medicinal plant, primarily found in plateau areas with altitudes exceeding 3000 m. The efficacy of C. tinctoria appears to be intricately tied to its quality. However, there is a scarcity of studies focused on evaluating the quality of C. tinctoria from diverse geographical locations. In this study, we used ultra-performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometry to analyze and identify the prevalent chemical components in 12 batches of C. tinctoria sourced from Xinjiang, Qinghai, Tibet, and Yunnan provinces in China. By using cluster analysis and discriminant analysis of partial least squares, we assessed the similarity and identified varying components in the 12 batches of C. tinctoria. Subsequently, their quality was further evaluated. Utilizing network pharmacology, we identified potential active components for the treatment of diabetes mellitus. The findings revealed the presence of 16 flavonoids, 3 phenylpropanes, 2 sugars, 2 amino acids, and 7 hydrocarbons in the analyzed samples. Through variable importance screening, 17 constituents were identified as quality difference markers. Marein and flavanomarein emerged as pivotal markers, crucial for distinguishing variations in C. tinctoria. In addition, network pharmacology predicted 187 targets for 9 common active components, including marein and flavanomarein. Simultaneously, 1747 targets related to diabetes mellitus were identified. The drug-component-disease target network comprised 91 nodes and 179 edges, encompassing 1 drug node, 9 component nodes, and 81 target nodes. In summary, marein and flavanomarein stand out as key biomarkers for assessing the quality of C. tinctoria, offering a scientific foundation for the quality evaluation of C. tinctoria Nutt.

Screening immunomodulatory Q-markers in Astragali Radix based on UHPLC-QTOF-MS analysis and spectrum-effect relationship.

Astragali Radix (AR) is one of the famous traditional Chinese medicines (TCMs) for boosting immunity, whereas the quality markers (Q-markers) of AR have not been clearly researched. The immunomodulatory activities of the bioactive extractions and components were evaluated by NO inhibition rate; phagocytic index; IL-10, TNF-α, IL-1ß, and IL-6 cytokines in RAW264.7 cells; and the relative proliferation rate of spleen cells. The total saponins (TS) and the grade 2 (Xiaoxuan, XX) of AR showed the strongest immunomodulatory activities. At the concentration of 40 µg/mL, the TS increased spleen cells proliferation by 48.0% and upregulated the level of IL-1ß and IL-6. Cytokines in the XX-treated group were at least 1.6 times higher than the control group. A total of 190 common peaks were detected in AR by ultrahigh-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF-MS). The multivariate statistical analyses revealed that 41 compounds were positively correlated with immune responses, and bioactive compounds were verified by using RAW264.7 cell assay. Subsequently, the contents of six compounds in different commercial grades were determined, and the results showed the same trend in contents and activities. Finally, calycosin-7-O-ß-D-glucoside, astragaloside IV, astragaloside II, astragaloside I, isomucronulatol-7-O-glucoside, and 9,10-dimethoxypterocarpan-3-O-glucoside were screened out as immunomodulatory Q-markers of AR.

Identification and quantification of the antioxidants in Ginkgo biloba leaf.

The antioxidant activity of Ginkgo biloba leaf (GBL) extract is closely related to its efficacy against various diseases; however, the antioxidant activities of the specific constituents of GBL remain unclear. In this study, 194 GBL constituents were identified using ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry, including 97 flavonoids, 37 terpenoids, 29 lignans, 19 carboxylic acids, 5 alkylphenolic acids, 5 alkylphenols, and 2 other compounds. The cleavage rules of the main constituents of GBL were dissected in detail. The 36 GBL constituents with high antioxidant activity were subsequently discovered using the oxygen radical absorbance capacity assay, including 30 flavonoids and six carboxylic acids. Finally, an HPLC analysis method was established to determine the content of the nine major antioxidants in the three batches of GBL. Among them, kaempferol 3-O-ß-D-(6″-p-coumaroyl) glucopyranosyl-(1-2)-α-L-rhamnopyranoside, kaempferol-3-O-rutinoside, and rutin exhibited high antioxidant activity and were found in significant amounts in GBL, with concentrations greater than 0.7 mg/g. These results provide an important reference for the development of pharmaceuticals and health products containing GBL.

Screening and characterization of cosmetic efficacy components of Terminalia chebula based on biological activity-guided methodology.

Terminalia chebula exhibits a high level of antioxidant capacity and is highly valued in medicine and cosmetics. However, its main efficacy and active ingredients related to antioxidant, whitening, and anti-aging are still unclear. In this study, the active site responsible for its cosmetic efficacy was specified by the biological activity-guided method and further characterized by using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS/MS). T. chebula was ultrasonically extracted by five solvents, and 30% ethanol extract was screened out for subsequent purification by 1,1-D-iphenyl-2-picrylhydrazyl radical (DPPH), 2,2'-Azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS), hydroxyl, and superoxide anion free radical scavenging assays. Five elution fractions were obtained by column chromatography on D101 macroporous adsorbent resin eluted by an increased proportion of ethanol. The 30% ethanol elution fraction was specified as the enrichment site of active ingredients showing good antioxidant capacity and potent inhibitory activity against tyrosinase and elastase. A total of 30 compounds were identified by UHPLC-QTOF-MS/MS in the 30% ethanol elution fraction, including 11 gallotannins, 14 ellagitannins, and 5 other compounds, and these compounds may be the key ingredients in cosmetics beneficial for the skin. Such a biological activity-guided method has provided a simple and rapid venue for specifying the components of medicinal herbs responsible for cosmetic efficacy.

Serum and urine metabolomics based on UPLC-QTOF/MS reveal the effect and potential mechanism of "schisandra-evodia" herb pair in the treatment of Alzheimer's disease.

The "schisandra-evodia" herb pair (S-E) is a herbal preparation to treat Alzheimer's disease (AD). This study aims to investigate the therapeutic efficacy and potential mechanism of S-E in AD rats, utilizing pharmacodynamic assessments and serum- and urine-based metabolomic analyses. Pharmacodynamic assessments included Morris water maze test, hematoxylin-eosin staining and immunohistochemistry experiments. The results of the study showed that the AD model was successful; the S-E significantly enhanced long-term memory and spatial learning in AD rats. Meanwhile, S-E notably ameliorated Aß25-35-induced cognitive impairment, improved hippocampal neuron morphology, decreased Aß deposition in the hippocampus and mitigated inflammatory damage. We then analyzed serum and urine samples using UPLC-MS/MS to identify potential biomarkers and metabolic pathways. Metabolomic analysis revealed alterations in 40 serum metabolites and 38 urine metabolites following S-E treatment, predominantly affecting pathways related to taurine and hypotaurine metabolism, linoleic acid metabolism, α-linolenic acid metabolism, glycerophospholipid metabolism and arachidonic acid metabolism. This study elucidates the biochemical mechanism underlying AD and the metabolic pathway influenced by S-E, laying the groundwork for future clinical applications.

A novel standard-free detection of adulteration method for sildenafil derivatives in dietary supplements.

The rapid and accurate detection of illegal adulteration of chemical drugs into dietary supplements is a big challenge in the food chemistry field. Detection of compounds without a standard reference is even more difficult; however, this is a common situation. Here in this study, a novel "standard-free detection of adulteration" (SFDA) method was proposed and phosphodiesterase-5 inhibitor derivatives were used as an example to figure out the possibility and reliability of this SFDA method. After analysis by quadrupole coupled time of flight-tandem mass spectrometry detection and multivariable statistics, six common fragment ions were chosen to indicate whether adulteration was present or not, while 20 characteristic fragment ions indicated whether adulteration was by nitrogen-containing heterocycles or by anilines. Furthermore, the quantitative methods were conducted by high-performance liquid chromatography-tandem mass spectrometry. In a word, this strategy allows for a quick determination of dietary supplement adulteration without any need for standard materials, improving the efficacy of food safety testing.

A novel liquid chromatography with quadrupole time-of-flight-tandem mass spectroscopy method for ultra-trace level identification and quantification of the genotoxic impurity 2,6-diamino-5-nitropyrimidin-4(3H)-one in valganciclovir hydrochloride.

In the present study, the main objective is to develop an analytical method for ultra-trace level measurement of 2,6-diamino-5-nitropyrimidin-4(3H)-one (DMNP) in valganciclovir hydrochloride (VAL) using liquid chromatography-quadrupole time-of-flight-tandem mass spectroscopy (LC-QTOF-MS/MS). In the early stages of guanine synthesis, DMNP is formed, and guanine is known to be the key starting material for the synthesis of VAL. Taking into consideration DMNP potential genotoxicity, this analytical method has been developed. This method is time saving and suitable for confirming the masses of parent and fragment ions by MS and MS/MS further fragmentation. An isocratic program and Acquity UPLC HSS cyano column (100 × 2.1 mm × 1.8 µm) were used to achieve optimal separation between VAL and the DMNP impurity. A 0.1% ammonia solution in Milli-Q water was used as mobile phase A, and methanol was used as mobile phase B in the ratio 90:10 v/v in isocratic mode. In accordance with the International Conference on Harmonization's requirements, the developed method was validated. The detection and quantification levels were found to be 0.028 and 0.083 ppm respectively. The DMNP impurity is linear from 0.083 to 1.245 ppm levels with correlation coefficient (R2 ) of 0.9960. The recoveries were found to be 97.0-107.9%.