Ultra-high-performance Liquid Chromatography-high-resolution Mass Spectrometry Research Articles

Surface multi-walled carbon nanotube modified quaternary amine-functionalized polymers for purification and determination of glyphosate and its four metabolites in plasma samples

The present study focused on the pretreatment and detection of GLY and its four metabolites AMPA, N-acetyl AMPA, N-methyl GLY and N-acetyl GLY in plasma samples. Multi-walled carbon nanotube-modified quaternary amine-functionalized polymers (QA-PDNV@MWCNTs) were synthesized in a controlled manner by self-assembly, and its morphology and composition were extensively characterized. The QA-PDNV@MWCNTs microspheres were then used as an SPE adsorbent for the preparation and rapid determination of GLY and its four metabolites in plasma samples combined with ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). The SPE conditions based on QA-PDNV@MWCNTs were optimized for GLY and its metabolites to obtain the best purification efficiency. The experimental results show that when the adsorbent contains 8% MWCNTs, it can balance the adsorption of target analytes and the purification performance of the adsorbent for impurities. In addition, this study compared the QA-PDNV@MWCNTs based SPE method with the commercial Waters Oasis MAX SPE cartridge and the results showed that the developed method in this study has better resistance to matrix interference. Under optimal conditions, the recoveries of GLY and its metabolites spiked in plasma were 82.6-99.4% with relative standard deviations (RSDs) of 1.0-7.8%. The limits of detection (LODs, S/N≥3) and limits of quantification (LOQs, S/N≥9) of the method were 0.05-0.33 μg/L and 0.15-1.00 μg/L, respectively. Finally, the developed QA-PDNV@MWCNTs based SPE-UPLC-HRMS method was used to confirm GLY poisoning not only on the basis of the detection of the GLY prototype, but also on the basis of its four metabolites.

Rapid Determination of Dapagliflozin in Rat Plasma by UHPLC-Q-Orbitrap MS and Its Application to a Pharmacokinetic Study.

Dapagliflozin (DAPA), sodium-glucose co-transporter 2 (SGLT-2) inhibitor, is used to treat Type 2 diabetes. In this study, a highly sensitive and selective analytical method based on ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was established and validated for the determination of DAPA in rat plasma. The separation of DAPA and internal standard (DAPA-d5) were performed on a reversed-phase ACQUITY UPLC® BEH C18 column (100 × 3.0 mm, 1.7 µm). The mobile phase is composed of 0.1% formic acid in water (solvent A) and methanol (solvent B) in gradient elution. Under the negative ion mode, full MS/dd-MS2 was adopted to collect data via Q-Orbitrap. DAPA was effectively separated from matrix backgrounds within 10 min, and DAPA in plasma showed a good linear relationship in the range of 10-10000 µg/L. The determination coefficient (R2) was 0.9987, and the lower limit of quantification (LLOQ) was 10 µg/L. The precision and accuracy were all less than 10%, and the extraction recovery of DAPA was 86.16-96.06% from plasma. This study offered an efficient separation and quantification method for DAPA. The improved and validated method succeeded in evaluating the pharmacokinetics of DAPA in rat plasma samples after a single oral administration of 1 mg/kg.

Metabolism investigation of the peptide-drug conjugate LN005 in rats using UHPLC[sbnd]HRMS

LN005, as a peptide-drug conjugate (PDC), is a conjugate of the homing peptide VAP and doxorubicin (DOX). The exceptional targeting ability of the homing peptide VAP is directed toward glucose-regulated protein (GRP78), a highly expressed protein primarily found in the endoplasmic reticulum of various solid tumors. However, there are limited reports regarding the metabolism of peptide-drug conjugates (PDCs), and the in vivo metabolism of LN005 has yet to be investigated. After intravenous injection of 18 mg/kg LN005 in SD rats, biological samples including plasma, urine, fecal, and bile samples, were collected and analyzed by ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). A total of 11 possible metabolites of LN005 were identified. Unchanged LN005 was found to be the main component in rat blood and urine, accounting for 46.46% and 63.79% of the total peak areas, respectively. M1057 was the most abundant metabolite in feces, accounting for 57.65% of the total peak area. Only one metabolite, M398, was identified in rat bile. The metabolism of LN005 is closely related to DOX, and the primary metabolic pathways involved oxidative deamination or hydrolysis, reductive glycosidic cleavage, hydrolytic glycosidic cleavage, and dehydrogenation.

Phenolic Profile and Bioactivity Changes of Lotus Seedpod and Litchi Pericarp Procyanidins: Effect of Probiotic Bacteria Biotransformation.

Theoretically, lactic acid bacteria (LABs) could degrade polyphenols into small molecular compounds. In this study, the biotransformation of lotus seedpod and litchi pericarp procyanidins by Lactobacillus plantarum 90 (Lp90), Streptococcus thermophilus 81 (ST81), Lactobacillus rhamnosus HN001 (HN001), and Pediococcus pentosus 06 (PP06) were analysed. The growth curve results indicated that procyanidins did not significantly inhibit the proliferation of LABs. Ultra-high-performance liquid chromatography high-resolution mass spectrometry (UPLC-HRMS) revealed that procyanidin B2 and procyanidin B3 in lotus seedpod decreased by 62.85% and 25.45%, respectively, with ST81 metabolised, while kaempferol and syringetin 3-O-glucoside content increased. Although bioconversion did not increase the inhibitory function of procyanidins against glycosylation end-products in vitro, the 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) free radical scavenging capacity and ferric reducing antioxidant power of litchi pericarp procyanidins increased by 157.34% and 6.8%, respectively, after ST81 biotransformation. These findings may inspire further studies of biological metabolism of other polyphenols and their effects on biological activity.

A classical herbal formula alleviates high-fat diet induced nonalcoholic steatohepatitis (NASH) via targeting mitophagy to rehabilitate dysfunctional mitochondria, validated by UPLC-HRMS identification combined with in vivo experiment

BackgroundNonalcoholic steatohepatitis (NASH) has caused a significant burden on public health care systems, the economy and society. However, there has still been no officially approved pharmacotherapy for NASH. It has been suggested that oxidative stress and mitochondrial dysfunction play vital roles in NASH pathological progression. Shugan Xiaozhi (SG) formula, as a kind of classical herbal formula, was shown to attenuate NASH. PurposeThis study aimed to explore the potential mechanisms of SG formula treating NASH. Study design and methodsUltra-high-performance liquid chromatography-high resolution mass spectrometry combined with bioinformatics analysis was applied to explore the therapeutic targets and main components of SG formula. Moreover, in vivo NASH model was utilized to confirmed the therapeutic effects of SG formula. Molecular docking analysis and further validation experiments were conducted to verify the results of bioinformatics analysis. ResultsThe in vivo experiments confirmed SG formula significantly attenuated hepatic pathological progression and relieved oxidative stress in high-fat diet (HFD) induced - NASH model. Ultra-high-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) combined with bioinformatics analysis expounded the components of SG formula and revealed the mitochondrial regulation mechanism of SG formula treating NASH. Further in vivo experiments validated that SG formula could alleviate oxidative stress by rehabilitating the structure and function of mitochondria, which was strongly related to regulating mitophagy. ConclusionIn summary, this study demonstrated that SG formula, which could attenuate NASH by regulating mitochondria and might be a potential pharmacotherapy for NASH.

Liquid chromatography-high resolution mass spectrometry analysis of 300 illegally added drugs and their analogues in functional milk powder for the elderly

Various types of milk powder purportedly providing diverse health functions have emerged with the growth of the country's elderly population. Some manufacturers illegally add chemical drugs to their products to achieve their reported benefits, which poses a threat to consumer health. The existing standard methods are inapplicable to such complex sample matrices and require testing based on functional claims and classification. These limitations not only consume manpower and resources but also seriously impede daily regulatory efforts to detect unknown risk substances. In this study, a high-throughput method for the screening and quantitative analysis of 300 illegally added chemical drugs in functional milk powder and an identification strategy for unknown structural analogues were established using Zeno SWATH® data-independent acquisition (DIA) ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) technology combined with a QuEChERS sample purification method. The QuEChERS purification process was developed according to the characteristics of milk powder matrix. The supernatant was separated on a Kinetex F5 column (100 mm×3.0 mm, 2.6 μm) by gradient elution using 5 mmol/L ammonium formate aqueous solution (0.1% (v/v) formic acid, ) and methanol-acetonitrile (1∶1, v/v) as mobile phases. The method was validated in terms of selectivity, linearity, limits of detection and quantification (LODs and LOQs, respectively), matrix effect, accuracy, and precision. Based on a screening database for the 300 target substances, electron-activated dissociation (EAD) fragmentation was applied to obtain rich secondary MS fragmentation information, and unknown structural analogues were identified and confirmed through fragment attribution analysis. The results indicated that all compounds had good linear relationships in certain ranges with correlation coefficients >0.99. The LODs and LOQs were 0.04-2.7 and 0.2-8.0 μg/kg, respectively. The average recoveries at three spiked levels were in the range of 73.1%-125.2%, and the relative standard deviations were ≤14.8% (n=6). When the developed method was applied to detect illegally added chemicals in 60 functional milk powder samples, it detected benzoguanidine and sildenafil and successfully identified ethylphenidate, which is the structural analogue of an amphetamine. The proposed method is simple, sensitive, and accurate; thus, it may have practical application value for the daily supervision and law enforcement of milk powders with reported health functions.

Stable Isotope Labeling-Based Nontargeted Strategy for Characterization of the In Vitro Metabolic Profile of a Novel Doping BPC-157 in Doping Control by UHPLC-HRMS

Traditional strategies for the metabolic profiling of doping are limited by the unpredictable metabolic pathways and the numerous proportions of background and chemical noise that lead to inadequate metabolism knowledge, thereby affecting the selection of optimal detection targets. Thus, a stable isotope labeling-based nontargeted strategy combined with ultra-high-performance liquid chromatography–high-resolution mass spectrometry (UHPLC-HRMS) was first proposed for the effective and rapid metabolism analysis of small-molecule doping agents and demonstrated via its application to a novel doping BPC-157. Using 13C/15N-labeled BPC-157, a complete workflow including automatic 13C0,15N0-13C6,15N2 m/z pair picking based on the characteristic behaviors of isotope pairs was constructed, and one metabolite produced by a novel metabolic pathway plus eight metabolites produced by the conventional amide-bond breaking metabolic pathway were successfully discovered from two incubation models. Furthermore, a specific method for the detection of BPC-157 and the five main metabolites in human urine was developed and validated with satisfactory detection limits (0.01~0.11 ng/mL) and excellent quantitative ability (linearity: 0.02~50 ng/mL with R2 > 0.999; relative error (RE)% < 10% and relative standard deviation (RSD)% < 5%; recovery > 90%). The novel metabolic pathway and the in vitro metabolic profile could provide new insights into the biotransformation of BPC-157 and improved targets for doping control.

Study on the Common Molecular Mechanism of Metabolic Acidosis and Myocardial Damage Complicated by Neonatal Pneumonia.

Pneumonia is a common clinical disease in the neonatal period and poses a serious risk to infant health. Therefore, the understanding of molecular mechanisms is of great importance for the development of methods for the rapid and accurate identification, classification and staging, and even disease diagnosis and therapy of pneumonia. In this study, a nontargeted metabonomic method was developed and applied for the analysis of serum samples collected from 20 cases in the pneumonia control group (PN) and 20 and 10 cases of pneumonia patients with metabolic acidosis (MA) and myocardial damage (MD), respectively, with the help of ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). The results showed that compared with the pneumonia group, 23 and 21 differential metabolites were identified in pneumonia with two complications. They showed high sensitivity and specificity, with the area under the curve (ROC) of the receiver operating characteristic curve (ROC) larger than 0.7 for each differential molecule. There were 14 metabolites and three metabolic pathways of sphingolipid metabolism, porphyrin and chlorophyll metabolism, and glycerophospholipid metabolism existing in both groups of PN and MA, and PN and MD, all involving significant changes in pathways closely related to amino acid metabolism disorders, abnormal cell apoptosis, and inflammatory responses. These findings of molecular mechanisms should help a lot to fully understand and even treat the complications of pneumonia in infants.

Detection of three metabolites of xylene in urine samples by solid-phase extraction coupled with liquid chromatography-mass spectrometry

Objective: To establish a method for the rapid determination of the three metabolites of xylene, 2-methylmarmaluronic acid, 3-methylmarmaluronic acid and 4-methylmarmaluronic acid, in urine of occupationally exposed workers by ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) . Methods: In July 2022, urine samples were diluted and extracted with pH=6.86 phosphate cache solution, cleaned up by a MAX solid-phase extraction (SPE) column and separated by an Accucore Ph/Hexyl column (100 mm×2.1 mm, 2.6 μm) with a gradient of 5 mmol/L ammonium formate-0.1% formic acid aqueous solution and methanol as mobile phases. The analysis was carried out in electrospray ionization mode and full mass-data dependent secondary mass spectrometry mode, and quantified by external standard method. The characteristics of each index of this method were analyzed. Results: A good linearity was obtained in the concentration range of 1.0-200.0 μg/L for 2-methylmuramic acid, 3-methylmuramic acid and 4-methylmuramic acid with the correlation coefficients of 0.9979-0.9993. The limits of detection of the method were 0.18-0.24 μg/L. While the spiked recoveries at the three concentrations (1.0 μg/L, 100.0 μg/L, and 180.0 μg/L) were in the range of 83.0%-93.7%, with the relative standard deviations of 2.2%-7.9%. Conclusion: The UPLC-HRMS method is simple, rapid, sensitive and accurate, and is suitable for the simultaneous determination of the three metabolites of xylene in the urine of occupationally exposed workers.

Application of Liquid- and Supercritical Fluid Chromatography Coupled with High-Resolution Mass Spectrometry for the Analysis of Short-, Medium-, and Long-Chain Chlorinated Paraffins in Dietary Supplements

Chlorinated paraffins (CPs) are an emerging and ubiquitous group of environmental pollutants associated with adverse effects on human health, including endocrine disruption and possible carcinogenicity. In this study, supercritical fluid chromatography (SFC) and ultrahigh-performance liquid chromatography (UHPLC)—both coupled with high-resolution mass spectrometry (HRMS)—methods for the analysis of short-, medium-, and long-chain CPs in fish oil-based dietary supplements were developed and validated at concentration levels of 0.6 and 3.0 μg/g lipid weight (lw). The recoveries were in the range of 80–96% and repeatabilities, expressed as relative standard deviations, were <19%. The limits of detection for the UHPLC–HRMS method (from 0.03 to 0.05 μg/g lw) were 5 to 10 times lower than those obtained by SFC–HRMS (from 0.13 to 0.50 μg/lw).

Comprehensive Metabolite Identification of Genipin in Rats Using Ultra-High-Performance Liquid Chromatography Coupled with High Resolution Mass Spectrometry.

Genipin, an aglycone of geniposide, is a rich iridoid component in the fruit of Gardenia jasminoides Ellis and has numerous biological activities. However, its metabolic profiles in vivo and vitro remain unclear. In this study, an effective analytical strategy based on ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) in positive and negative ion modes was developed to analyze and identify genipin metabolites in rat urine, blood, feces, and fecal fermentation in combination with many methods including post-collection data mining methods, high-resolution extracted ion chromatography (HREIC), and multiple mass defect filtering (MMDF). Simultaneously, the metabolites of genipin in vivo were verified by fecal fermentation of SD rats at different times. Finally, based on information such as reference substances, chromatographic retention behavior, and accurate mass determination, a total of 50 metabolites (including prototypes) were identified in vivo. Among them, 7, 31 and 28 metabolites in vivo were identified in blood, urine, and feces, respectively. Our results showed that genipin could generate different metabolites that underwent multiple metabolic reactions in vivo including methylation, hydroxylation, dehydroxylation, hydrogenation, sulfonation, glucuronidation, demethylation, and their superimposed reactions. Forty-six metabolites were verified in vitro. Meanwhile, 2 and 19 metabolites identified in blood and urine were also verified in fecal fermentation at different times. These results demonstrated that metabolites were produced in feces and reabsorbed into the body. In conclusion, the newly discovered metabolites of genipin can provide a new perspective for understanding its pharmacological effects and build the foundation for thee toxicity and safety evaluations of genipin.

Non-targeted lipidomics analysis of lipid distribution and variation of Eucommia ulmoides oliver by ultra-high performance liquid chromatography

Eucommia ulmoides Oliver (EU) is an important natural polymer source to relieve the serious shortage of natural rubber (NR), and the lipids of EU as potential non-rubber component have inherently a strong intramolecular connection to rubber biosynthesis. In this paper, Non-target lipidomics analysis platform based on ultra-high performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) system was used to determine the lipids in EU. The orthogonal partial least square discriminant analysis (OPLS-DA) and a statistical analysis approach were used to compare the differences of lipids in EU. Total 6 categories, 26 classes and 785 species of lipids were identified in leaves, seed kernels, seed cases and barks of EU. The contents of EU lipids with time in female and male leaves were compared. The lipids contents in female EU leaves were more abundant than that in male EU leaves, and the content of most phospholipids in the female and male leaves both increased obviously after September. The content of lipids between seed cases and leaves were compared, and further screened for different glycerophospholipids between them. There were 53 phospholipids in the seed cases and fresh leaves samples of EU that had significantly different contents. Therefore, this study provides a theoretical basis for the identification of lipids in different EU organs and also helps further explore the relationship between lipids with rubber in EU.

Improved metabolomic approach for evaluation of phytochemicals in mustard, kale, and broccoli microgreens under different controlled environment agriculture conditions

The fast-growing field of controlled environment agriculture (CEA) offers unprecedented opportunities for targeted improvement in concentrations of bioactive compounds in fresh produce achieved through precise modulation of production conditions. To gain full sight of the phytochemical profiles of vegetables grown under different conditions, a rapid analytical strategy is needed for the evaluation of different CEA growing conditions. In this study, Brassica microgreens including ruby streaks mustard (B. juncea), red kale (B. oleracea), and broccoli (B. oleracea) were used as model plants for the evaluation of CEA conditions. Analysis of two first leaves (cotyledons) in microgreens with minimum sample extraction and ultra-high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) based metabolomic approach was applied for phytochemical analysis for evaluation of the brassica microgreens grown under four different light sources, namely white (W), dark (D), white and far-red (WF) and far-red (F) and fertilizer conditions (CaCl2 and K2SO4). An image-based normalization method using leaf area coupled with chemometrics-based strategies including principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) was performed for the post-acquisition data analysis. The method successfully distinguished between Brassica microgreens grown under different CEA settings in a shortened cycle with less organic solvent and labor, which is more environmentally friendly and sustainable. Marker compounds that are responsible for differentiating the Brassica microgreens under various CEA conditions were tentatively identified. Among the tentatively identified marker compounds, gingerglycolipid A was first reported in red kale and broccoli. The results from the present study may serve as a scientific foundation for the rapid and simple assessment to optimize CEA conditions.

Disturbance of OH-PAH metabolites in urine induced by single PAH lab exposure.

Due to the high exposure toxicity and individual variability of polycyclic aromatic hydrocarbons (PAHs), it is difficult to accurately characterize the actual exposure of exposed individuals through external exposure detection. In this study, the monohydroxyl metabolites of naphthalene, phenanthrene, pyrene, and 9-fluorenone were identified in the urine of low-dose PAH-exposed individuals based on ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS), and their concentrations were monitored for 15 consecutive days after exposure. The results showed that the metabolite concentrations of naphthalene, phenanthrene, and pyrene were basically the same, and all of them reached the maximum value at day 8. In contrast, the metabolite of 9-fluorenone reached its maximum value on day 2. This study showed that the four metabolites were strongly correlated with their parent PAH exposure, with a wide detection window, and their assays were specific, sensitive, and reliable, while the sampling difficulty was low, so the four hydroxylated PAHs may be potential low-dose biomarkers of PAH internal exposure. This study will provide methodological and data support for further health risk studies involving internal exposure to organic pollutants such as PAHs.

Geographical discrimination of Flos Trollii by GC-MS and UHPLC-HRMS-based untargeted metabolomics combined with chemometrics

For centuries, Flos Trollii has been consumed as functional tea and a folk medicine in China's north and northwest zones. The quality of Flos Trollii highly depends on the producing zones. Unfortunately, few studies have been reported on the geographical discrimination of Flos Trollii. This work comprehensively investigated Flos Trollii compounds with an integration strategy combining gas chromatography-mass spectrometry (GC-MS) and ultrahigh-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) with chemometrics to explore the differences between Flos Trollii obtained from various origins of China. About 71 volatile and 22 involatile markers were identified with GC-MS and UHPLC-HRMS, respectively. Geographical discrimination models were synthetically investigated based on the identified markers. The results indicated that the UHPLC-HRMS coupled with the fisher discrimination model provided the best prediction capability (>97%). This study provides a new solution for Flos Trollii discrimination.

Organophosphate esters (OPEs) in plastic food packaging: non-target recognition, and migration behavior assessment

Organophosphate esters (OPEs) are widely used as plasticizers in plastic food packaging; however, the migration of OPEs from plastic to food is largely unstudied. We do not even know the specific number of OPEs that exist in the plastic food packaging. Herein, an integrated target, suspect, and nontarget strategy for screening OPEs was optimized using ultrahigh-performance liquid chromatography–high-resolution mass spectrometry (UHPLC–HRMS). The strategy was used to analyze 106 samples of plastic food packaging collected in Nanjing city, China, in 2020. HRMS allowed full or tentative identification of 42 OPEs, of which seven were reported for the first time. Further, oxidation products of bis(2,4-di-tert-butylphenyl) pentaerythritol diphosphite (AO626) in plastics were identified, implying that the oxidation of organophosphite antioxidants (OPAs) could be an important indirect source of OPEs in plastics. The migration of OPEs was examined with four simulated foods. Twenty-six out of 42 OPEs were detected in at least one of the four simulants, particularly isooctane, in which diverse OPEs were detected at elevated concentrations. Overall, the study supplements the list of OPEs that humans could ingest as well as provides essential information regarding the migration of OPEs from plastic food packaging to food.

Mass spectrometry-based metabolomics reveal Dendrobium huoshanense polysaccharide effects and potential mechanism of N-methyl-N'-nitro-N-nitrosoguanidine -induced damage in GES-1cells.

Dendrobium huoshanense C. Z. Tang et S. J. Cheng is an important edible medicinal plant that thickens the stomach and intestines, and its active ingredient, polysaccharide, can have anti-inflammatory, immunoregulatory, and antitumor effects. However, the gastroprotective effects and potential mechanisms of Dendrobium huoshanense polysaccharides (DHP) remain unclear. An N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induced human gastric mucosal epithelial cells (GES-1) damage model was used in this research, aiming to investigate whether DHP has a protective effect on MNNG-induced GES-1cell injury and its underlying mechanism based on the combination of multiple methods. DHP was extracted using water extraction and alcohol precipitation methods, and the proteins were removed using the Sevag method. The morphology was observed using scanning electron microscopy. A MNNG-induced GES-1cell damage model was developed. Cell viability and proliferation of the experimental cells were investigated using a cell counting kit-8 (CCK-8). Cell nuclear morphology was detected using the fluorescent dye Hoechst 33342. Cell scratch wounds and migration were detected using a Transwell chamber. The expression levels of apoptosis proteins (Bcl-2, Bax, Caspase-3) in the experimental cells were detected by Western blotting. Ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was performed to investigate the potential mechanism of action of DHP. The CCK-8 kit analysis showed that DHP increased GES-1cell viability and ameliorated GES-1cell injury by MNNG. In addition, scratch assay and Transwell chambers results suggested that DHP improved the MNNG-induced motility and migration ability of GES-1cells. Likewise, the results of the apoptotic protein assay indicated that DHP had a protective effect against gastric mucosal epithelial cell injury. To further investigate the potential mechanism of action of DHP, we analyzed the metabolite differences between GES-1cells, GES-1cells with MNNG-induced injury, and DHP+MMNG-treated cells using UHPLC-HRMS. The results indicated that DHP upregulated 1-methylnicotinamide, famotidine, N4-acetylsulfamethoxazole, acetyl-L-carnitine, choline and cer (d18:1/19:0) metabolites and significantly down-regulated 6-O-desmethyldonepezil, valet hamate, L-cystine, propoxur, and oleic acid. DHP may protect against gastric mucosal cell injury through nicotinamide and energy metabolism-related pathways. This research may provide a useful reference for further in-depth studies on the treatment of gastric cancer, precancerous lesions, and other gastric diseases.

Characterization of Thymoma by Ultra-Performance Liquid Chromatography—High Resolution Mass Spectrometry (UPLC-HRMS) and Metabolomics of Serum

Thymoma is a rare thymic tumor but is the most common anterior mediastinal tumor. Currently, there are still limitations regarding to its screening and the pathology remains elusive. In this study, nontargeted metabolomic analysis of serum was performed to study the metabolic characteristics of thymoma using ultra-performance liquid chromatography—high resolution mass spectrometry (UPLC-HRMS) in positive and negative ionization modes. The metabolites that were differentially abundant in thymoma were identified by MS/MS and matching to chemical standards. Forty-one metabolic features differentially abundant in thymoma were identified in the corresponding age groups, including uridine, dipeptides, arachidonic acid (AA)/8,11,14,17-eicosatetraenoic acid, lactic acid and its dimer, acylcarnitines, lysophosphatidylcholines (LPCs), and ceramide 1-phosphate (Cer1P) (d18:0/3:0). Among these metabolites, lactic acid and 9-decenoylcarnitine also had significant abundance changes between thymoma patients with and without myasthenia gravis (MG). Uridine, lactic acid and Cer1P (d18:0/3:0) may also be associated with the aberrant immune status in thymoma. Orthogonal partial least squares—discriminant analysis (oPLS-DA) showed that classification between thymoma and thymic hyperplasia was possible with 94.7% sensitivity, 100% specificity and 96.8% accuracy for all patient samples. The discriminant feature panel included uridine, AA/8,11,14,17-eicosatetraenoic acid, lactic acid dimer, and N-(1-deoxy-1-fructosyl)tryptophan. The distinct serum metabolic characteristics of thymoma identified in this study may offer insights into improvement of the diagnosis and discovery of new therapeutic strategies for thymoma, in addition to providing information for study of the underlying molecular mechanism of thymoma pathogenesis.

UHPLC-HRMS-based metabolomic and lipidomic characterization of glioma cells in response to anlotinib

Anlotinib, as a promising oral small-molecule antitumor drug, its role in glioma has been only reported in a small number of case reports. Therefore, anlotinib has been considered as a promising candidate in glioma. The aim of this study was to investigate the metabolic network of C6 cells after exposure to anlotinib and to identify anti-glioma mechanism from the perspective of metabolic reprogramming. Firstly, CCK8 method was used to evaluate the effects of anlotinib on cell proliferation and apoptosis. Secondly, ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS)-based metabolomic and lipidomic were developed to characterize the metabolite and lipid changes in cell and cell culture medium (CCM) caused by anlotinib in the treatment of glioma. As a result, anlotinib had concentration-dependent inhibitory effect with the concentration range. In total, twenty-four and twenty-three disturbed metabolites in cell and CCM responsible for the intervention effect of anlotinib were screened and annotated using UHPLC-HRMS. Altogether, seventeen differential lipids in cell were identified between anlotinib exposure and untreated groups. Metabolic pathways, including amino acid metabolism, energy metabolism, ceramide metabolism, and glycerophospholipid metabolism, were modulated by anlotinib in glioma cell. Overall, anlotinib has an effective treatment against the development and progression of glioma, and these remarkable pathways can generate the key molecular events in cells treated with anlotinib. Future research into the mechanisms underlying the metabolic changes is expected to provide new strategies for treating glioma.

Removal of multiple lipids from human plasma using a hydroxyl-functionalized covalent organic framework aerogel as a new sorbent.

A hydroxyl-functionalized covalent organic framework aerogel COFTHB-TAPB-aerogel was designed and prepared as an adsorbent for the removal of multiple lipids from human plasma. The applications of 1,3,5-tris(4'-hydroxy-5'-formylphenyl)benzene (THB) and 1,3,5-tris(4-aminophenyl)benzene (TAPB) as monomers, DMSO/mesitylene (v/v, 4/1) as reaction solvent, and n-propylamine as reaction regulator endow COFTHB-TAPB-aerogel with good adsorption performance for multiple lipids. The morphology, phase purity, specific surface area, pore size, surface charge, and stability of COFTHB-TAPB-aerogel were characterized. Adsorption thermodynamics and adsorption kinetics studies showed that COFTHB-TAPB-aerogel had high equilibrium adsorption capacities (> 15913mgg-1) and fast adsorption equilibrium (≤ 10s) for the four model lipidstested. COFTHB-TAPB-aerogel had good reusability with the removal of the model lipids being still more than 91% after 10 use cycles. The sample pretreatment conditions and adsorbent amounts used in lipids removal experiments were optimized. Under the optimized conditions, the method of ultra-high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) using COFTHB-TAPB-aerogel as solid-phase extraction sorbent was validated with negligible matrix effects (0.4-3.0%) and good accuracy (86.7-110%) and was applied to determine 20 amino acids in human plasma samples from healthy individuals and gastric adenocarcinoma (GA) patients. The established method has beenproved to have good application potential for the removal of multiple lipids in human plasma to reduce the matrix effects and improve the accuracy of clinical LC-MS analysis.