Chromatography-electrospray Ionization-tandem Mass Spectrometry Research Articles

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.

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.

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.

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.

Selenium speciation analysis of selenium-enriched Shiitake mushrooms (Lentinula edodes) by HPLC-ESI-MS

Selenium (Se) is an essential component of critical enzymes and is intricately linked to human health. Shiitake mushrooms (Lentinula edodes) are considered as good dietary supplements for producing Se-enriched foods due to their high protein content and ability to absorb minerals from growth substrates. In this study, the distribution of soluble Se in Se-enriched L. edodes was analyzed by microwave digestion and hydride generation-atomic fluorescence spectrometry (HG-AFS). In order to evaluate the absorption and conversion effect of Se by L. edodes, the Se-containing compounds in selenized L. edodes were quantitatively and qualitatively analyzed by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS). It can be shown that L. edodes possessed a good biological transformation ability of organic Se enrichment though the quantitative analysis of three seleno-amino acids, selenocystine (Se-Cys2), methylselenocysteine (Se-MeSeCys) and selenomethionine (Se-Met), in selenized L. edodes. The structures of three unknown Se-containing compounds were identified by qualitative analysis, the results indicated that the possible structures were selenomethylmethionine (C6H14NO2Se), N-Acetyl-selenomethionine (C7H13NO3Se) and Se-methyl-5-selenoadenosinese-oxide (C12H18N5O3Se). The discovery of these Se compounds filled the research gap and provided a theoretical basis for molecular structure identification and mechanism study of organic Se in Se-enriched L. edodes.

(S)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidine-2-carboxylate derivatization combined with UPLC/ESI-MS/MS as a powerful method for dipeptide determination in foods: An application example in fermented cocoa beans

Dipeptides play a crucial role in food flavor, as well as in physiological and nutritional functions. However, as comprehensive methods for determining dipeptides are still under development, the dipeptide composition of foods remains largely unexplored. This study proposed a novel analytical method for dipeptides by applying an amino acid determination technique we previously developed: (S)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidine-2-carboxylate (DMT-(S)-Pro-OSu) derivatization coupled with ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS). The DMT-(S)-Pro-OSu derivatization of dipeptides improved both separation in LC and detection sensitivity in MS, allowing for the optimized acquisition of m/z and elution times for 313 available dipeptides, and enabling the absolute quantification of 126 dipeptides in a simultaneous analysis. Subsequent analysis of fermented cocoa beans detected and quantified 42 dipeptides, revealing distinct dipeptide profiles depending on the origin of the beans. Additionally, by conducting in silico analysis of the elution times for 313 dipeptides, a machine learning model was constructed that accurately predicts dipeptide elution times. The DMT-(S)-Pro-OSu-UPLC/MS/MS method and the elution time prediction model developed in this study are expected to contribute to the understanding of dipeptide composition in foods.

Comprehensive Secondary Metabolite Profiling and Antioxidant Activity of Aqueous and Ethanol Extracts of Neolamarckia cadamba (Roxb.) Bosser Fruits.

Neolamarckia cadamba (Rubiaceae) is a well-recognized medicinal plant with recorded therapeutical attributes. However, a thorough assessment of active compounds in its fruits is lacking, limiting their use and valorization in pharmacological industries. Thus, this study investigated variations in the fruits' secondary metabolite (SM) profiles, as well as antioxidant activities in aqueous (WA) and ethanol (ET) extracts. Liquid chromatography-electrospray ionization tandem mass spectrometry identified 541 SMs, of which 14 and 1 (di-O-glucosylquinic acid) were specifically detected in ET and WA, respectively. Phenolic acids (36.97%), flavonoids (28.10%), terpenoids (12.20%), and alkaloids (9.98%) were the dominant SMs. The SM profiles of the fruits in WA and ET were quite different. We revealed 198 differentially extracted (DE) metabolites between WA and ET, including 62 flavonoids, 57 phenolic acids, 45 terpenoids, 14 alkaloids, etc. Most DE flavones (36 out of 40), terpenoids (45 out of 45), and alkaloids (12 out of 14) had higher content in ET. Catechin and its derivatives, procyanidins, and tannins had higher content in WA. ABTS and DPPH assays showed that the antioxidant activity of ET was significantly higher than that of WA. Our findings will facilitate the efficient extraction and evaluation of specific active compounds in N. cadamba.

Comprehensive analysis of Moringa oleifera leaves’ antioxidant properties in ovine cheese

This study aimed to enhance “Pecorino” type ovine cheese by adding Moringa oleifera leaves powder (MOLP). Cheese-making trials, conducted at industrial level, used raw ewes' milk and two selected Lactococcus lactis strains. The experimental plan included a control production (CTR), and two experimental productions with 1% or 2% MOLP addition (1-MOLP and 2-MOLP, respectively). MOLP did not hinder starters development, which reached about 8.0 Log CFU/g in 2-month ripened cheeses. Illumina results highlighted lactococci dominance in all trials [45.98%–62.48% of relative abundance (RA)]. Physicochemical analysis showed that MOLP-enriched cheeses had higher protein content and lower secondary lipid oxidation. The addition of MOLP increased total phenolic compounds in cheese, reaching 3.64 mg GAE/g in the 2-MOLP sample. MOLP-enriched cheeses showed significantly higher radical scavenging activity than CTR production (p < 0.0001). Ultrahigh-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) revealed increased levels of chlorogenic acid, protocatechuic acid, caffeic acid, and ferulic acid due to MOLP enrichment. In the presence of MOLP, cheese volatile organic compounds were affected by compounds like 2-octanone, 3-hexen-2-one, heptane, nonanol, and linalool. 1-MOLP cheese was comparable to CTR production in overall satisfaction (sensory evaluation). Including MOLP in cheese production offers exciting opportunities for functional Sicilian ewes’ milk products.

Mercaptans in malodorants break disulfide bridges in human serum albumin and form adducts suitable as biomarkers of exposure in vitro.

Malodorants comprise notoriously smelling mercaptans and might be applied for crowd control. Because exposure to malodorants may lead to irritation of the respiratory system, choking, and coma, bioanalytical verification of poisoning might be required in a medical and forensic context. We herein present the detection and identification of novel biomarkers of exposure to ethyl mercaptan, n-butyl mercaptan, tert-butyl mercaptan, and iso-amyl mercaptan. These alkyl thiol compounds were found to form disulfide adducts in human serum albumin (HSA) in plasma in vitro with the only non-disulfide-bridged Cys34 residue and with other residues being part of the disulfide-bridged pattern in HSA. After proteinase K-catalyzed proteolysis, adducts of all mercaptans were detected simultaneously as the tripeptide Cys34*ProPhe and the dipeptides Cys369*Tyr, ValCys316*, and Cysx*Ala (x denominates either Positions 91, 200, 253, 361, and/or 448) by a sensitive micro-liquid chromatography-electrospray ionization tandem mass spectrometry (μLC-ESI MS/MS) method working in the scheduled multiple reaction monitoring (sMRM) mode. Time- and concentration-dependent adduct formations while exposure and proteolysis were investigated and the suitability of adducts as biomarkers of exposure was elaborated. Adducts at Cys34 showed the lowest limits of identification (LOIs, 6nM to 1.2μM mercaptan in plasma) and superior stability in plasma at 37°C. Therefore, Cys34*ProPhe appears as the most promising target to prove exposure to mercaptans at least in vitro.

Characterization of the covalent binding of cyanidin-3-glucoside to bovine serum albumin and its inhibition mechanism for advanced nonenzymatic glycosylation reactions.

Nonenzymatic glycosylation of proteins can generate advanced glycosylation end products, which are closely associated with the pathogenesis of certain chronic physiological diseases and aging. In this study, we characterized the covalent binding of cyanidin-3-glucoside (C3G) to bovine serum albumin (BSA) and investigated the mechanism by which this covalent binding inhibits the nonenzymatic glycosylation of BSA. The results indicated that the covalent interaction between C3G and BSA stabilized the protein's secondary structure. Through liquid chromatography-electrospray ionization tandem mass spectrometry analysis, we identified the covalent binding sites of C3G on BSA as lysine, arginine, asparagine, glutamine, and cysteine residues. This covalent interaction significantly suppressed the nonenzymatic glycosylation of BSA, consequently reducing the formation of nonenzymatic glycosylation products. C3G competitively binds to nonenzymatic glycosylation sites (e.g., lysine and arginine) on BSA, thereby impeding the glycosylation process and preventing the misfolding and structural alterations of BSA induced by fructose. Furthermore, the covalent attachment of C3G to BSA preserves the secondary structure of BSA and hinders subsequent nonenzymatic glycosylation events.

Simultaneous Detection Method of 11 Respiratory Drug Substances Including Theobromine, Oxymetazoline, etc. in Adulterated Dietary Supplements Using Liquid Chromatography–Electrospray Ionization–Tandem Mass Spectrometry and Liquid Chromatography–Quadrupole Time-of-Flight Mass Spectrometry Analysis

Abstract Recently, the demand for respiratory disease-related products has surged due to the influence of coronavirus disease 2019, prompting warnings about illegal dietary supplements containing unauthorized substances. Additionally, adulterated dietary supplements are continuously detected in open markets, posing significant public health safety problem. In this study, we developed and validated an analytical method for 11 respiratory drug substances using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) and proposed optimal conditions for LC–quadrupole time-of-flight MS (LC–QTOF-MS) to determine the fragmentation patterns of each substance. This method underwent thorough validation considering specificity, linearity, limits of detection and quantification, accuracy, precision, matrix effect, stability, etc. All results met international guidelines. These validated methods were applied to 52 dietary supplements advertised for treating respiratory diseases and enhancing respiratory function, among which one sample was found to contain 313.7 mg/g of theobromine. This determination was made by comparing the product ion ratios with the standards and subsequent quantification. To re-confirm the detected substances, their fragmentation patterns were compared with those of the standards using LC–QTOF-MS. In conclusion, the mass-based information, coupled with the LC–ESI–MS/MS method development, can be successfully applied to rapidly identify 11 respiratory drug substances in illegal dietary supplements used for respiratory disease treatment. The developed simultaneous detection method contributes to public health and safety improvements.

Effect of saponins in Panax notoginseng (Burkill) F. H. Chen on the steroid hormone levels in the chronic unpredictable mild stress model of depression in rats

Recent research has indicated that Panax notoginseng saponins (PNS) extracted from the radix of Panax notoginseng (Burkill) F. H. Chen exert antidepressant effects. This study aimed to assess the antidepressive effects of ginsenoside Rg1 and PNS in a depression model induced by chronic unpredictable mild stress (CUMS). Over a period of three weeks, rats were administered ginsenoside Rg1 at a dose of 30 mg/kg and PNS at dosages ranging from 100 to 200 mg/kg body weight per day. To assess how ginsenoside Rg1 and PNS influence depression-like behaviours in rats, various assessments were conducted, including coat state evaluation, forced swim test, and elevated plus maze test. The levels of cortisol and testosterone in serum samples were analysed using the liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) method. LC-ESI-MS/MS method provides precise and accurate results. The lower limit of quantification values for cortisol and testosterone were determined as 100 and 2 pg/mL, respectively. Our data demonstrated that both ginsenoside Rg1 and PNS significantly reversed depression-like behaviour in rats by improving coat condition, reducing immobility time in the forced swim test, and increasing time spent in the open arms of the elevated plus maze test. Furthermore, ginsenoside Rg1 and PNS exhibited a regulatory effect on cortisol and testosterone levels in plasma. These findings suggest that ginsenoside Rg1 and PNS may be potential antidepressants in clinical treatment.

Tandem mass spectrometry imaging to assess illudin S distributions in Omphalotus illudens mushrooms

Poisoning metabolites within mushrooms typically exert varying physiological and biological effects; however, their distributions within mushrooms are seldom investigated. This study focused on imaging illudin S, a representative poisoning metabolite within Omphalotus illudens mushroom, using desorption electrospray ionization–tandem mass spectrometry (DESI–MS/MS) coupled with liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS). Initially, LC–ESI–MS/MS conditions were established for the selective detection of illudin S (m/z 247→159, positive mode) from extracted mushroom. Based on this MS/MS condition, each mushroom was divided into nine sections, and each section was subjected to specific DESI–MS/MS mode imaging (pixel size: 200 × 200 μm). The results revealed high concentrations of illudin S in the stalks of the mushrooms growing on trees. Further, re-evaluations using the LC–MS/MS assay revealed a seven-fold difference in the illudin S between the stalk and other regions. This study marks the first attempt at assessing toxic metabolite distributions within poisonous mushrooms, thus offering valuable insights including the efficient utilization of illudin analogs in biological applications.

Quantitative analysis of erythromycin, its major metabolite and clarithromycin in chicken tissues and eggs via QuEChERS extraction coupled with ultrahigh-performance liquid chromatography-tandem mass spectrometry

A simple, rapid and novel method involving ultrahigh-performance liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry (UHPLC-ESI–MS/MS) was developed to simultaneously detect erythromycin, its major metabolite and clarithromycin in chicken tissues (muscle, liver and kidney) and eggs (whole egg, albumen and yolk). Samples were extracted using acetonitrile–water (80:20, v/v), and a Cleanert MAS-Q cartridge was used to perform quick, easy, cheap, effective, rugged, and safe (QuEChERS) purification. The average recoveries were 87.78–104.22 %, and the corresponding intraday and interday relative standard deviations were less than 7.10 %. The decision limits and detection capabilities of the chicken tissues and eggs were 2.15–105.21 μg/kg and 2.26–110.42 μg/kg, respectively. For chicken tissues and eggs, the limits of detection and limits of quantification were 0.5 μg/kg and 2.0 μg/kg, respectively. The proposed method was successfully employed to analyse real samples, demonstrating its applicability.

A Reliable Quantification of Cholesterol and 25-Hydroxycholesterol in Liposomal Adjuvant Formulation by Liquid Chromatography High-Resolution Tandem Mass Spectrometry.

Cholesterol, as one of the major components of liposomes, plays a critical role in modulating membrane bilayer permeability, fluidity, and structural stability. Controlling these quality attributes is essential to maintaining the efficacy and fitness of the liposomes in various applications. However, during the manufacture and storage of liposomes, cholesterol has a propensity to undergo oxidative degradation. Hence, an analytical tool that is capable of determining not only the identity and quantity of cholesterol but also its associated degradants is a prerequisite to effective process control and product quality and safety assessments. In this view, a new liquid chromatography electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method with parallel reaction monitoring (PRM) was developed and qualified to accurately quantify cholesterol and monitor the formation of 25-hydroxycholesterol degradant in liposomal drug formulations without the use of an isotopic internal standard (IS). The method was qualified according to the FDA Quality Guidance for Industry: Q2(R1). Study results showed that the method presents good specificity for cholesterol and 25-hydroxycholesterol detection in the liposomal matrix, good sensitivity characterized by LOD/LOQ in the nanomolar range, and accuracy within the range of 80 to 120%. The described method enables accurate evaluation of in-process and product release samples of Army Liposome Formulation with QS21 (ALFQ).

Untargeted Metabolomic Profiling of Fructus Chebulae and Fructus Terminaliae Billericae

This study aims to identify the differences in metabolites between Fructus Chebulae (FC) and Fructus Terminaliae Billericae (FTB). Untargeted metabolomics was used to analyze differentially expressed metabolites (DEMs) with ultra-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC-ESI-MS/MS). A grand total of 558 metabolites were detected, with 155 in positive ion mode and 403 in negative ion mode. Further differential analysis yielded 110 and 87 significantly different metabolites, which were mainly polyphenols, flavonoids, terpenoids, and alkaloids. Analysis of KEGG data showed that differentially expressed metabolites (DEMs) in both positive and negative ion modes were found to be enriched in 5 and 18 metabolic pathways, respectively, with metabolic pathways being the most enriched among them. In sum, this study reveals the differential metabolic profiles of FC and FTB and provides support for their further applications in traditional Chinese medicine.

Molecular identification and management of mycotoxigenic fungi in stored corn Grains

Mycotoxin-producing molds which considered as common maize grains contaminants are the genera Fusarium, Aspergillus and Penicillium. There are natural and safe ways to protect grains from mold contamination as the use of essential oils and chemical treatments. A total number of 25 samples were used to study the natural frequency in five governorates in Egypt, Molecular identification indicated that the most frequent fungi were Fusarium verticillioides, Aspergillus niger, Talaromyces verruculosus, Aspergillus flavus and Aspergillus terreus. The in vitro studies have been done to determine mycelial growth and spore germination inhibition of the two A. flavus; isolated and reference isolates. Thyme and acetic acid were tested in direct contact assay to study their effects on mycelial growth. Treatments showed significant impact on mycelial growth and spore germination inhibition of both A. flavus isolates. In the postharvest application treatments: as vapour and carrier contact assay, Thyme and Acetic acid were tested to determine their influence on growth and aflatoxin production in A. flavus isolates by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS). Results indicated that both treatments were effective in inhibition of aflatoxin production in both vapour and carrier assays as they succeeded in reducing AFB1 while they inhibited completely the production of AFB2. The extent of the inhibition of aflatoxin production was dependent on the concentration and storage duration of treatments applied.

Anti-Cholinergic Effects of the Phenolic Extract from the Astragalus crenatus Plant: A Computational and Network Pharmacology Study.

Investigations into cholinesterase inhibition have received attention from researchers in recent years for the treatment of Alzheimer's disease. Cholinesterase enzymes, namely, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), hold pivotal significance in Alzheimer's disease (AD) treatment. In this study, we utilized the ethanolic extract of Astragalus crenatus followed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to separate and identify at least 21 compounds in the extract. Rosmarinic acid exhibited the highest concentration (96.675 ± 1.3 mg/g extract), succeeded by hesperidin (79.613 ± 1.2 mg/g extract), hesperetin (75.102 ± 1.4 mg/g extract), rutin (68.156 ± 1.6 mg/g extract), chlorogenic acid (67.645 ± 1.5 mg/g extract), fisetin (66.647 ± 2.3 mg/g extract), and hyperoside (63.173 ± 1.5 mg/g extract). A. crenatus extract efficiently inhibited both AChE and BChE activities in a dosage-dependent manner. Molecular docking was employed to scrutinize the anticholinesterase mechanisms of the identified phytocompounds. Notably, a network pharmacology analysis was executed for the most efficacious compound. Based on binding energies, hesperidin emerged as the most potent inhibitor against both AChE and BChE, exhibiting scores of -10.5 Kcal/mol and -9.8 Kcal/mol, respectively. Due to its dual inhibition of AChE and BChE activities, hesperidin from Astragalus crenatus holds promise for the development of novel therapeutics aimed at neurological disorders, particularly AD.

Optimized Conditions for the Extraction of Phenolic Compounds from Aeginetia indica L. and Its Potential Biological Applications.

Aeginetia indica L., a parasitic root in the Orobanchaceae family, is used as a food colorant in traditional Thai desserts. However, scant information is available on its food applications as well as medicinal properties, while overharvesting by the local people has severely depleted wild plant populations. This research, thus, aimed to extract optimized total phenolic content (TPC) in varying extraction conditions using response surface methodology (RSM) and the Box-Behnken design (BBD). Results indicated that an extraction temperature of 90 °C, 80% (v/v) aqueous ethanol, and 0.5% (w/v) solid-to-liquid ratio yielded the highest TPC at 129.39 mg gallic acid equivalent (GAE)/g dry weight (DW). Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) identified the predominant phenolics as apigenin (109.06 mg/100 g extract) and luteolin (35.32 mg/100 g extract) with trace amounts of naringenin and rutin. Under the optimal extraction condition, the plant extract exhibited antioxidant activities of 5620.58 and 641.52 µmol Trolox equivalent (TE)/g DW determined by oxygen radical absorbance capacity (ORAC) and ferric ion reducing antioxidant power (FRAP) assay, while the scavenging capacity of total radicals at 50% (SC50) was determined to be 135.50 µg/mL using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. The plant extract also exhibited inhibitory activities against the key enzymes relevant to type II diabetes, obesity, and Alzheimer's disease, suggesting the potential for medicinal applications.

Signal interference between drugs and metabolites in LC-ESI-MS quantitative analysis and its evaluation strategy

Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS) is a widely utilized technique for in vivo pharmaceutical analysis. Ionization interference within electrospray ion source, occurring between drugs and metabolites, can lead to signal variations, potentially compromising quantitative accuracy. Currently, method validation often overlooks this type of signal interference, which may result in systematic errors in quantitative results without matrix-matched calibration. In this study, we conducted an investigation using ten different groups of drugs and their corresponding metabolites across three LC-ESI-MS systems to assess the prevalence of signal interference. Such interferences can potentially cause or enhance nonlinearity in the calibration curves of drugs and metabolites, thereby altering the relationship between analyte response and concentration for quantification. Finally, we established an evaluation scheme through a step-by-step dilution assay and employed three resolution methods: chromatographic separation, dilution, and stable labeled isotope internal standards correction. The above strategies were integrated into the method establishment process to improve quantitative accuracy.