Ionization Tandem Mass Spectrometry Research Articles

Preparation and characterization of ferulic oligosaccharides from different sources by cell-free GH10 and GH11 xylanases

The feruloyl oligosaccharides (FOs) produced by the decomposition of plant hemicellulose have broad potential applications in the food and biomedical areas. FOs were prepared through the specific enzymatic degradation of insoluble dietary fiber from different sources by cell-free GH10 and GH11 xylanases. The cell-free GH10 and GH11 xylanases were obtained by the heterologous expression in Escherichia coli. The enzymatic hydrolysis conditions were optimized as follows: temperature 50 °C, pH 5.5, hydrolysis time 12 h, GH10 xylanase addition 101.74 U, and GH11 xylanase addition 121.60 U. The compositions and structural characterization of wheat bran FOs (WB-FOs), corncob FOs (CC-FOs), and buckwheat straw FOs (BS-FOs) were identified by fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR), high-performance liquid chromatography (HPLC), and electrospray ionization tandem mass spectrometry (ESI-MS). Degrees of polymerization (DP) of WB-FOs, CC-FOs, and BS-FOs were 3–11, 3–7, and 3–6, respectively. Ultraviolet (UV) radiation was investigated in vitro. The results demonstrated that BS-FOs possessed excellent UV resistance and photostability, followed by effectiveness in WB-FOs and CC-FOs. These results have improved our understanding of the relationship between FOs with different structural types and their UV radiation.

Combined Metabolome and Transcriptome Analyses Reveals Anthocyanin Biosynthesis Profiles Between Purple and White Potatoes

Colored potatoes with red and purple skin or flesh possess significant nutritional value and health benefits due to their rich anthocyanin content. To investigate the genetic mechanisms underlying color formation, the high-anthocyanin-content purple-skinned and purple-fleshed potato line 15-12-16, and the white-skinned and white-fleshed Xiazhai 65 variety were used for ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) analysis, which was conducted to identify and quantify anthocyanins. RNA sequencing was performed to analyze the transcriptome. The results indicated a significant upregulation of genes within the anthocyanidin biosynthesis pathway in the purple potato, while these genes were either downregulated or absent in the white potato. The bHLH, MYB, and WRKY gene families exhibited a greater number of regulatory members, suggesting their pivotal role in color formation. Integrated analysis of the transcriptional and metabolic revealed that 12 differentially expressed genes (DEGs) related to the anthocyanidin biosynthetic had a significant correlation with 18 anthocyanin metabolites. Notably, the key gene St5GT in the anthocyanidin biosynthesis pathway was markedly upregulated in the purple skin and flesh. Furthermore, the overexpression of St5GT (PGSC0003DMG400004573) in tobacco contributed to anthocyanin accumulation. The expression of 10 DEGs was validated through quantitative real-time PCR. In conclusion, these findings provide new insights into anthocyanin biosynthesis and accumulation in purple potatoes, offering valuable candidate genes for the future breeding of colored potatoes.

Determination of Sennoside A in Rat Plasma by Liquid Chromatography/Electrospray Ionization Tandem Mass Spectrometry and Its Application to a Rat Pharmacokinetic Study.

To characterize pharmacokinetic profile of sennoside A in rats after intravenous and oral administration, a simple and sensitive liquid chromatography tandem mass spectrometry method was established and validated for quantitative determination of sennoside A in rat plasma. After prepared by protein precipitation with acetonitrile, sennoside A and internal standard were separated on a Waters ACQUITY HSS T3 (2.1 × 100 mm, 1.8 μm) column using acetonitrile and 5-mM ammonium acetate in water as mobile phase by gradient elution. The method showed excellent linearity over the range of 0.5-1000 ng/mL with acceptable intra- and inter-day precision, accuracy, matrix effect, and recovery. The stability assay indicated that sennoside A was stable in plasma during the sample collection, preparation, and analysis. Next, the method was applied to pharmacokinetic study of sennoside A in rats. After intravenous and intragastric administrated to rats, the concentrations of sennoside A in plasma at different time points were quantitated and the pharmacokinetic parameters were calculated by software of DAS 2.0. Pharmacokinetic parameters suggested that after oral administration, sennoside A was reached to the peak at 2.9-3.6 h with a Cmax value of 13.2-31.7 ng/mL. Sennoside A was eliminated slowly from the plasma with T1/2 value between 15.4 and 18.3 h. The oral absolute bioavailability was among 0.9%-1.3%, which indicated low blood exposure level.

Quantitative lipidomics reveals the effects of roasting degree on arabica coffee beans lipid profiles

Lipids in coffee beans undergo morphological changes and migration during the roasting process, but the impact of roasting intensity on lipid profiles remains unclear. The lipidomics characteristics of coffee beans with different degrees of roasting (unroasted green beans, light roast, medium roast, and dark roast) were compared using ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS). A total of 571 lipid metabolites were identified, encompassing 25 species of fatty acyls (FA), 142 species of glycerophospholipids (GP) across 12 subclasses, 42 species of sphingolipids (SP) across four subclasses, 360 species of glycerolipids (GL) across ten subclasses, and two species of prenol lipids (PR). GL content was found to be the highest, followed by FA and GP. Through multivariate analysis, 160 lipids were identified that exhibited significant differences among beans with different degrees of roasting. GP content was significantly influenced by the degree of roasting (p < 0.05), with diverse dynamic evolution observed through K-means cluster analysis. These findings have important implications for understanding lipid transformation and its effect on the flavor profile during coffee roasting.

Detection of IDH mutation in glioma by desorption electrospray ionization (DESI) tandem mass spectrometry

Desorption electrospray ionization (DESI) tandem mass spectrometry (MS) is used to assess mutation status of isocitrate dehydrogenase (IDH) in human gliomas. Due to the diffuse nature of gliomas, total gross resection is not normally achieved during surgery, leading to tumor recurrence. The mutation status of IDH has clinical significance due to better prognosis in IDH-mutant patients. The mutant IDH converts alpha-ketoglutaric acid (α-KG) into 2-hydroxyglutarate (2HG), which accumulates abnormally in cells. Immunohistochemical staining (IHC) and genetic testing, the gold standards, are incompatible with intraoperative applications but DESI tandem mass spectrometry (MS/MS) can be used to assess the mutation status of IDH enzyme from tissue intraoperatively. Here, on off-line evaluation is made of the performance of two different types of mass spectrometers in characterization of IDH mutation status. The intensity of 2HG is measured against glutamate (Glu), an intrinsic reference molecule, in both tandem MS measurements. In both cases using DESI clear separation between IDH-mutant (mut) and IDH-wildtype (wt) samples (p < 0.0001) is observed, despite the short analysis time. Due to the higher detection sensitivity, multiple reaction monitoring experiments using a triple quadrupole show slightly better performance compared to product ion MS/MS performed on a simple linear ion trap. Both DESI-MS platforms are capable of providing information on IDH mutation status, which might in future be used at the time of surgery to support decision-making on resection regions, especially at tumor margins.

Phenolic Compound Profiles, Antioxidant, and Cytoprotective Activity of Elaeagnus conferta Roxb. Fruit.

In this paper, three varieties of Elaeagnus conferta Roxb fruits prepared by ultrasonic-assisted extraction from a subtropical region southwest of China were utilized as raw materials to investigate their phenolic profiles, antioxidant activities, and protective effects on injured human umbilical vein endothelial cells (HUVECs). The ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) findings revealed that fifteen substances, including seven phenolic acids, seven flavonoids, and one gallic acid derivative, were discovered. The dihydromyricetin, ellagic acid, gallic acid were the predominant phenolic compounds in all E.conferta fruits. These E.conferta fruits extracts shown excellent antioxidant activity varied from 2.258±0.03~7.844±0.39 μM Trolox/g and protective effect on HUVECs injured by H2O2 through decrease the level of ROS, MDA, LDH and enhance the SOD level. These finding indicate that E.conferta is a valuable source of high-capacity antioxidants that might be used as an alternative material for food industries.

Composition and Biological Activity of Flavonoid-containing Fractions of an Extract from Gratiola officinalis L.

Gratiola officinalis L. (hedge hyssop), a medicinal plant of the Scrophulariaceae family, has diuretic, purgative, and vermifuge properties. It is used as a herbal tea to treat chronic gastroenteritis, renal colic, jaundice, and intestinal worms. Previously, we have found that an extract from G. officinalis is nontoxic and has antitumor, antioxidant, antimicrobial, antiinflammatory, anticachexic, and other properties. Our aims in this study were to separate the G. officinalis extract into individual fractions, to identify the most biologically active fractions, and to examine the chemical composition of these fractions and their biological activity toward A498 renal carcinoma cells. The G. officinalis extract was fractionated by reversed-phase high-performance liquid chromatography, and each fraction was tested for antitumor activity. The active fractions were characterized by UV-visible electron spectral analysis, circular dichroism analysis, Fourier transform infrared spectroscopy, high-performance liquid chromatography, electrospray ionization tandem mass spectrometry, and nuclear magnetic resonance spectroscopy. Two antitumor-active fractions of a flavonoid nature were isolated and chromatographically purified. On the basis of the nuclear magnetic resonance data, the aglycone fragment of the main component of one fraction was found to be structured as 2-(3,4-dimethoxyphenyl)-7-hydroxychroman-4-one, or 3',4'-dimethoxy-7- hydroxyflavanone. The antitumor effect of the most active fraction containing 7-O-glucoside of apigenin, glycoside 7,3'-di-O-luteolin and trace amounts of eupatilin against renal carcinoma A498 cells was manifested in its cytotoxic, cytostatic, apoptotic and autophagosomal activities. In addition, we found 3-(1-2)-glucoside of soyaspogenol B, which is a pentacyclic triterpenoid in the structure.

Efficient Simultaneous Isolation of Pinostrobin and Panduratin A from Boesenbergia rotunda Using Centrifugal Partition Chromatography.

The bioactive flavonoids pinostrobin (PN) and panduratin A (PA) from Boesenbergia rotunda are essential for research and therapeutic applications. This study introduces an innovative method utilizing ultrasound-assisted extraction with n-hexane pre-treatment, followed by one-step centrifugal partition chromatography (CPC) purification. Extraction efficiency was evaluated using ultra high-performance liquid chromatography (UHPLC), and the isolated compounds were characterized through 1H-NMR and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), adhering to AOAC validation guidelines. Optimal extraction conditions comprised a particle size of 125 μm, a solid-to-liquid ratio of 1:30 g/mL, and a 10 min extraction time, yielding a crude extract of 6.96 ± 0.07%. Using an n-hexane/MeOH/water (5/3.4/1.6, v/v) solvent system in ascending mode, PN (2.16 mg, 98.78% purity) and PA (0.4 mg, 99.69% purity) were isolated from 67 mg of crude extract within 30 min. This streamlined approach enhances purification efficiency, allowing for faster extraction and higher purity, making it a suitable method for commercial applications.

Direct aqueous injection LC−ESI-MS/MS analysis for the determination of 23 EU watch list substances in whole water samples

The Watch List (WL) monitoring program in the European Union (EU) aims to identify and prioritize emerging pollutants in the aquatic environment that may pose risks to human health and the environment. Thus, reliable methods are needed to determine these substances. This study presents the development of a simple, fast, and green method for analysing 23 WL substances (4 antibiotics, 9 azole compounds, 6 other pesticides, and 4 sunscreens), covering a range of medium to very low polarity (log Kow 2.2–6.9), in whole environmental water samples using direct aqueous injection (DAI) in combination with liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS). To process the samples, 0.5 mL of acetonitrile is added to 1 mL of raw water, followed by brief shaking. The mixture is then centrifuged to remove sedimented suspended solids before being injected into the LC instrument. A rigorous analytical greenness metric for sample preparation (AGREEprep) assigned this procedure a green score of 0.61. The method showed satisfactory performance characteristics for all analytes under investigation, including limits of quantification meeting the WL requirements, recoveries in the range of 79–120%, and intra-day precision with relative standard deviations in the range of 1.9–20%. The method is suitable for screening and monitoring purposes, as confirmed by the analysis of real samples.

Lemongrass (Cymbopogon citratus) oil nanoparticle synthesis, characteristic, and evaluation of antibacterial and antifungal effects and the influence on hardness of acrylic resin

Context: Acrylic resin is used in dentistry as a removable denture base. It can cause various pathologies when not properly cleaned. One of the pathologies is denture stomatitis caused by Candida albicans and Streptococcus mutans accumulation on the acrylic resin surface. Therefore, microbial agents such as denture cleansers are needed. Aims: To evaluate the characteristics of lemongrass (Cymbopogon citratus) nanoparticles as a better antibacterial and antifungal herbal ingredient and their relationship with acrylic hardness. Methods: C. citratus oil nanoparticles (LON) were synthesized and analyzed by transmission electron microscopy (TEM). Electrospray ionization tandem mass spectrometry (ESI-MS) analysis was used to analyze the characteristics of LON bioactive components. Minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) against C. albicans and S. mutans and mechanical hardness test of acrylic were performed. Results: The LON concentration of MIC and MBC against C. albicans and S. mutans was 25 and 100%, respectively. One-way ANOVA showed no significant difference between groups of LON with different concentrations (p=0.687). A paired t-test showed significant differences in acrylic resin hardness before and after treatment of LON with 100% (p=0.022) and 50% (p=0.021) concentration. There was no significant difference in hardness before and after treatment of other concentrations of LON and chlorhexidine as positive control. Conclusions: LON treatment on acrylic resin decreased the growth of C. albicans and S. mutans without altering the mechanical properties (hardness).

Pharmacokinetic study of iptacopan and its two acyl glucuronide metabolites in monkey plasma by liquid chromatography combined with electrospray ionization tandem mass spectrometry.

In this study, a simple and sensitive liquid chromatography tandem mass spectrometric method was developed and validated for the determination of iptacopan and two acyl glucuronidation metabolites in monkey plasma. The plasma sample was precipitated with acetonitrile and then separated on an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) using 0.1% formic acid and 5 mM ammonium acetate in water and acetonitrile as the mobile phase. The mass spectrometry (MS) detection was performed in positive multiple reactions monitoring (MRM) mode with precursor-to-production transitions. The developed assay was validated over the range of 1-2000 ng/mL for three analytes with correlation coefficient (r) more than 0.99. The validation parameters including accuracy, precision, carryover effect, matrix effect, recovery, and stability were all within the acceptable limits. The validated method has been applied to investigate the pharmacokinetics of iptacopan and its two acyl glucuronidation metabolites in monkey plasma. After intravenous administration, iptacopan showed low clearance (2.75 mL/min/kg) in monkey plasma. After oral administration, the bioavailability was 55.43%. The exposure (AUC0-t) of direct acyl glucuronide (AG) of iptacopan accounts for 9.73% of the iptacopan plasma exposure. The AUC0-t of AG of dealkylated metabolite of iptacopan was present at a lower level, accounting for 0.5% of the iptacopan plasma exposure.

Custom GC×GC configuration for the selective isolation or removal of compounds from complex samples

We developed a novel approach to selectively isolate or remove nearly any compound from complex mixtures of volatile organic compounds. This was achieved by customizing a GC×GC system with a Deans switch, a passive splitter, and a custom-made adapter for sample recollection. The new setup was evaluated with 106 standard chemicals covering a wide range of volatility (boiling points: 56 – 343 ⁰C) and polarity (log P: 0.2 – 9.4). The method was used to remove two notorious malodorous compounds from spoiled wine samples. We found that the recovery can be maximized if a custom-made adapter is attached directly on the flame ionization detector port (average recovery rate of 76 ± 7 % for the standards). Eventually, we could selectively isolate or remove chemicals with peaks separated by a minimum distance of 50 ms for the second column throughout the whole chromatographic run. The developed system is expected to mainly be used in the field of flavor and fragrance analysis (i.e., selection of flavors and odorants of interest or removal of off-flavor or malodorous compounds). At present, we can reasonably collect about 100 ng of each single compound and are currently working on sample enrichment to improve our method to isolate sufficient amounts for further chemical analysis (e.g. high sensitivity nuclear magnetic resonance or chemical ionization tandem mass spectrometry).

Investigation of the chemical composition and biological activities of Eremurus spectabilis M. Bieb through antioxidant, enzyme inhibition, COX-2 and iNOS assessment.

Eremurus spectabilis is widespread and used primarily for medicinal and culinary purposes. This study aimed to evaluate the chemical composition, antiradical and antioxidant activities, enzyme inhibitory activities, and anti-inflammatory properties of various extracts from the aerial parts of E. spectabilis. Various assays were used to investigate the antioxidant and enzyme inhibitory properties. The chemical composition of the tested extracts was analyzed using High-Performance Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry (HPLC-ESI-MS/MS). Additionally, the extracts were tested on isolated mouse colon tissue challenged with E. coli lipopolysaccharide (LPS) to replicate the inflammation and oxidative stress burden characteristic of inflammatory bowel diseases. In the chemical composition, vanillic, ferulic, 4-hydroxybenzoic acids were the prominent compounds. The greatest antioxidant activity was observed in the methanol and water extracts from the aerial parts. Enzyme inhibition tests showed that the ethyl acetate extract had the highest anti-acetylcholinesterase activity. The gene expression of pro-inflammatory cyclooxygenase-2 (COX-2) and pro-oxidant inducible nitric oxide synthase (iNOS) biomarkers were assayed. Among the extracts, the methanol extract was the most effective in blunting LPS-induced gene expression of COX-2. E. spectabilis may serve as a valuable source of phytochemicals for combating oxidative stress and inflammation-driven diseases, with a particular emphasis on colon inflammatory condition.

Online restricted access molecularly imprinted solid phase extraction coupled with electrospray ionization-tandem mass spectrometry for determination of mebendazole and albendazole in milk samples

Multifunctional materials, such as restricted access molecularly imprinted polymers covered with bovine serum albumin (RAMIP-BSA), are effective alternatives for sample preparation techniques. This material selectively adsorbs analytes while excluding macromolecules, enhancing the analysis's efficiency. Among analytical techniques, ESI-MS/MS (Electrospray Ionization-Tandem Mass Spectrometry) has successfully identified and quantified various molecules, including trace-level drugs. Therefore, we proposed, for the first time, an integrated online extraction/analysis system that combines the benefits of RAMIP-BSA and ESI-MS/MS for analyzing mebendazole (MBZ) and albendazole (ABZ) in milk samples without the need for chromatographic separation. Initially, a RAMIP selective for MBZ was synthesized using the bulk method with methacrylic acid and glycidyl methacrylate. Then, the polymer was covered with bovine serum albumin. Subsequently, this adsorbent was packed in a small column and coupled with an ESI-MS/MS instrument in an online configuration. Milli-Q water was used as the loading and reconditioning mobile phases, and a solution of formic acid in methanol (1:100 v/v) was employed as the elution phase. The system enabled simultaneous extraction and determination of MBZ and ABZ in milk samples. The method exhibited linearity between 15.0 and 125.0 μg L−1 for MBZ and 10.0 and 125.0 μg L−1 for ABZ (with a correlation coefficient exceeding 0.99). The limits of quantification were 15.0 and 10.0 μg L−1 for MBZ and ABZ, respectively. Good precision and accuracy were achieved. The developed method was used to analyze MBZ and ABZ in real milk samples and proved to be a viable alternative to conventional sample preparation and chromatographic techniques.

Insight into the Coextrusion Mechanism between Whey Protein Isolate and Cysteine.

The disulfide cross-linking sites of whey protein isolate (WPI) coextruded with dissolved cysteine (Cys) at concentrations of 0, 20, 40, 60, 80, and 100 mM were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) combined with pLink software, and the structure and gel water distribution of WPI during coextrusion (≤50 °C) were also investigated. LC/MS/MS demonstrated that α-La (6) and α-La (120) were the most active sites for intermolecular disulfide cross-linking of α-La. Meanwhile, the molecular weight of protein polymers in coextruded WPI-Cys was the largest at 100 mM Cys, and α-lactalbumin was the main reactant for polymerization from the result of SDS-PAGE and size exclusion chromatography. Additionally, the high concentration of Cys caused the secondary structure of WPI to gradually change from a highly ordered to a disordered structure during coextrusion. In addition, with an increasing concentration of Cys, the free sulfhydryl group of proteins and the binding force to immobilized water gradually increased. Therefore, this work revealed the disulfide cross-linking mechanism between WPI and Cys under low-temperature coextrusion at the molecular level, and the obtained coextruded cross-linked WPI could serve as a novel food ingredient with excellent water-holding capacity for the food industry.

Assessing the stability of daptomycin in icodextrin-based peritoneal dialysis solution.

The stability of antimicrobials in peritonitis during peritoneal dialysis (PD) solutions is a critical factor influencing treatment success. This study investigated the stability of daptomycin (DAP) when combined with icodextrin-based PD solution, by measuring DAP concentrations and observing any structural changes. A dose of DAP (350 mg) was dissolved in 7 mL of saline in a clean bench. The solution was then injected into the large compartment (1,260 mL) of NICOPELIQ® Peritoneal Dialysis Solution and thoroughly mixed. Samples were collected at intervals ranging from 0 to 336 h (7 points in total). The concentration of DAP was quantified using high-performance liquid chromatography (HPLC). The structure of any unidentified peaks was determined using HPLC coupled with electrospray ionization tandem mass spectrometry. DAP maintained 90% of the initial concentration in NICOPELIC® for 72 h at room temperature and 12 h at 37 °C. Unidentified peaks, distinct from DAP, were detected during analysis. Further investigation indicated that these peaks corresponded to anhydrated DAP. The findings from this stability study are expected to enhance the effectiveness of outpatient management and preparation for treating peritonitis using DAP.

Conversion of 5α-Androstane-3α,17β-diol to bis[(4-dimethylamino)phenyl carbamate] derivative for sensitive determination of its rat brain level by LC/ESI-MS/MS.

5α-Androstane-3α,17β-diol (3α,5α-Adiol) is a testosterone-derived neurosteroid and has anxiolytic and analgesic effects via γ-aminobutyric acid type A receptors as with the progesterone-derived neurosteroid, allopregnanolone (AP). Although the psychotropic drug-evoked changes in the brain AP concentration have been intensively studied, those in the brain 3α,5α-Adiol concentration remain poorly understood. One of the causes for this is the limited availability of a validated method for quantifying the brain 3α,5α-Adiol with a sufficient sensitivity and specificity, which is described in this study. To enhance the detectability of 3α,5α-Adiol by electrospray ionization-tandem mass spectrometry (ESI-MS/MS), derivatization with 4-dimethylaminobenzoyl azide was employed. The brain sample was purified by solid-phase extraction and the recovered 3α,5α-Adiol and the deuterated internal standard were derivatized, then measured by liquid chromatography (LC)/ESI-MS/MS with selected reaction monitoring. The derivatized 3α,5α-Adiol, i.e., the bis[(4-dimethylamino)phenyl carbamate] derivative, provided the intense doubly-protonated molecule as the precursor ion, then the specific product ion containing the 3α,5α-Adiol-skeleton by collision-induced dissociation. The detectability of 3α,5α-Adiol was eventually increased 1000-fold by derivatization. Separation of the derivatized 3α,5α-Adiol from its stereoisomers and interfering brain components was achieved using a SunShell Biphenyl column with an isopropyl alcohol-containing mobile phase. A good linearity in the sufficient concentration range, acceptable precision and accuracy, and negligible matrix effect were demonstrated by the validation tests. The animal (rat) study using this method revealed that the brain 3α,5α-Adiol levels were unaffected by the administration of fluoxetine (FLX) and clozapine (CLZ), in contrast to the significant increase of the AP levels. An LC/ESI-MS/MS method capable of quantifying 3α,5α-Adiol in the rat brain using a 20-mg tissue was developed and validated. The brain levels of 3α,5α-Adiol had an entirely different behavior from those of AP due to FLX and CLZ administration.

Rapid Analysis for α-Tocopherol and Its Oxidative Products in the Pisum sativum L. Leaf Using Supercritical Fluid Chromatography-Medium Vacuum Chemical Ionization Tandem Mass Spectrometry.

A method for the rapid determination of α-tocopherol (α-T) and its oxidative products in plant tissue has been developed using supercritical fluid extraction (SFE) coupled with supercritical fluid chromatography (SFC) and medium vacuum chemical ionization (MVCI) with tandem mass spectrometry. The method is designed to study changes in levels for α-T and its oxidative products in plant cells during photosynthesis, aiming to observe the light response curves. α-T oxidation is a non-enzymatic self-defense mechanism in plant cells. Unlike enzyme-involved reactions, it cannot be stopped, so the oxidation continues in crude extracts even after extraction. Therefore, a real-time in-situ method is essential for tracking the light response curves. To optimize the selective reaction monitoring method, the reaction mixture of α-T and singlet oxygen (1O2), generated by rose Bengal under light illumination, was used as the source of oxidative products. The relative abundance changes in α-tocopherylquinone and 8a-hydroperoxy tocopherone in Pisum sativum L. (Pea) leaves under excessive light illumination have been preliminarily analyzed as part of the light response curve study. The method archives a throughput of 10-15 minutes for analyzing duplicate leaf samples. This process includes cutting off the leaf, sectioning it, placing the sample in a frozen SFE vessel, and conducting SFE/SFC analysis. Consequently, the average throughput is approximately 5-7 minutes per sample.

Dynamic Changes in Flavonoids’ Accumulation Pattern in Tilia miqueliana Flowers at Different Developmental Stages Based on Widely Targeted Metabolomic Analysis

Tilia miqueliana is an endemic species belonging to the genus Tilia L. (Tiliaceae) in China, which is known for its fragrant flowers and nectar, but the dynamic changes in metabolites during its growth and development are still unclear. In this study, the metabolic profiles from T. miqueliana flowers at three developmental stages were detected by performing an ultra-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC-ESI-MS/MS)-based widely targeted metabolomic analysis. A total of 1138 metabolites were detected, with 288 Differentially Accumulated Metabolites (DAMs) determined, flavonoids accounting for the largest proportion. The trend analysis showed that DAMs present seven distinctive patterns, and subclass 5 obtained the largest amount with continuously increased relative content during flower development. The Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation and enrichment analysis of DAMs showed different overlap and variability in metabolic pathways, indicating different directions of flavonoids’ metabolic flux in the three developmental stages. A correlation network analysis further revealed five core metabolites that played essential roles in flavonoid biosynthesis. This research provides comprehensive insights into the exploitation and utilization of T. miqueliana as well as a scientific basis for phylogenetic studies of the genus Tilia.

Polyphenol composition and antioxidant activity of fermentation combined with enzymatic hydrolysis modified Astragalus membranaceus stems

Astragalus membranaceus (AM) roots are a well-known homologous medicine and food in China. AM stems, which are discarded and not used effectively, also contain many active compounds and exhibit beneficial effects. It has the potential to be explored as antibiotic alternative. Fermentation combined with enzymatic hydrolysis (FEH) is an effective strategy for extracting polyphenol and improving the usage of AM stems. Therefore, in this study, the conditions of FEH and extraction for polyphenol in AM stems were screened. The antioxidant activity of extract from AM stems without and with FEH (AMSE and FAMSE) was evaluated. The metabolite profiles of phenolic acids and flavonoids in AMSE and FAMSE were characterized by ultra-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC–ESI-MS/MS). The results showed that the highest polyphenol content from AM stems was obtained with cellulase and pectinase (1:1, 2000 U/g), moisture content 43%, fermentation temperature 30 °C, and fermentation time 7 days. Selected extraction conditions of polyphenol were ethanol concentration 50%, ultrasonic power 500 W, extraction temperature 35 °C, and extraction time 40 min. On this condition, compared with AMSE, the polyphenol and flavonoid contents in FAMSE were significantly higher. FAMSE exhibited stronger DPPH, hydroxyl radical scavenging rate and reducing power than AMSE. The relative content of 3-(4-hydroxyphenyl)-propionic acid, dihydroferulic acid, isoferulic acid, 4-hydroxybenzoic acid, 4-hydroxyphenyllactic acid, ferulic acid, vanillic acid, syringic acid, gentisic acid, sinapic acid, apigenin, tricin, acacetin, daidzein, genistein, formononetin, prunetin, pratensein, rhamnocitrin and galangin were significantly upregulated in FAMSE.Graphical