Liquid Chromatography-electrospray Ionization-tandem Mass Spectrometry Research Articles

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.

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.

Overweight/bowel dysmotility crosslinking and analogous laxative actions of two edible wild fruits in obese/constipated rats.

The prompt development of obesity/constipation is the most serious problem for both children and adults. Limited studies suggested an association between them but lacked preclinical studies. This study allows to evaluate their crosslink and to compare the aqueous extracts laxative actions of two edible wild fruits of Arbutus unedo (AUAE) and Crataegus monogyna (CMAE) in constipated high-fat diet (HFD) rats. Wistar rats were divided into experimental groups for 13 weeks: standard (SD) and HFD groups. SD-rats were randomly redivided into 2 groups: SD and SD + Loperamide (LOP, 3 mg/kg, b.w.). HFD-rats were randomly reseparated into HFD-group, (HFD + LOP)-group, [HFD + Yohimbine (YOH, 2 mg/kg, b.w.)]-group, [HFD+ LOP]-groups+ various doses of AUAE or CMAE (75, 150, and 300 mg/kg, b.w.). Diversified indicators were investigated to achieve the expected objectives, including; fecal parameters, gastrointestinal transit (GIT), gastric emptying (GE), oxidative stress-(OxS), blood biochemical analysis, and accompanied small/large bowel histological modification. The liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS) analysis of AUAE and CMAE allowed the identification of 11 and 6 phenolic compounds, respectively. In HFD-rats, the subsequent dysregulation of GI motility was markedly aggravated. More importantly, with the same way (CMAE and AUAE)-treated groups showed alleviated outcomes for the following: most stool parameters, GIT, and GE were remarkably recovered; a similar recovery pattern was observed in the histopathological structure, OxS, and blood biochemical indicators. Our results experimentally confirmed the crosslink between overweight and constipation and both fruits have potential as functional foods to reduce metabolic risk of the obesity associated with bowel dysmotility.

Metabolism of quercitrin in the colon and its beneficial regulatory effects on gut microbiota.

Quercitrin is a dietary flavonoid widely found in plants with various physiological activities. However, whether quercitrin alters gut microbiota in vivo is not well understood. The aim of this study was to investigate metabolism of quercitrin in the colon and its regulation on gut microbiota in mice. Herein, 22 flavonoids related to quercitrin metabolism were identified based on ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS). Gas chromatography and 16S rDNA gene sequencing were used to investigate short-chain fatty acid (SCFA) content and diversity of composition of gut microbiota, respectively. The results showed that quercitrin significantly alters the beta-diversity of the gut microbiota, probiotics such as Akkermansia and Lactococcus were significantly increased, and the production of propanoate, isovalerate and hexanoate of the quercitrin group were enhanced significantly. The Spearman's association analysis provided evidence that Gardnerella and Akkermansia have obvious correlations with most of quercitrin metabolites and SCFAs. Quercitrin and its metabolites in the colon altered the structure of the mice gut microbiota and increased the content of SCFAs. Our experiments provide valuable insights into quercitrin research and application. © 2024 Society of Chemical Industry.

Production and biological activity of β-1,3-xylo-oligosaccharides using xylanase from Caulerpa lentillifera

In this study, β-1,3-xylanase (Xyl3088) was designed and prepared by constructing the expression vector plasmid and expressing and purifying the fusion protein. β-1,3-xylo-oligosaccharides were obtained through the specific enzymatic degradation of β-1, 3-xylan from Caulerpa lentillifera. The enzymolysis conditions were established and optimized as follows: Tris-HCl solution 0.05 mol/L, temperature of 37 °C, enzyme amount of 250 μL, and enzymolysis time of 24 h. The oligosaccharides' compositions and structural characterization were identified by thin-layer chromatography (TLC), ion chromatography (IC) and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS). The IC50 values for scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azino-bis-3-ethyl-benzothiazoline-p-sulfonic acid (ABTS+), and superoxide anion radical (•O2−) were 13.108, 1.258, and 65.926 mg/mL for β-1,3-xylo-oligosaccharides, respectively, and 27.588, 373.048, and 269.12 mg/mL for β-1,4-xylo-oligosaccharides, respectively. Compared with β-1,4-xylo-oligosaccharides, β-1,3-xylo-oligosaccharides had substantial antioxidant activity and their antioxidant effects were concentration dependent. β-1,3-xylo-oligosaccharides also possessed a stronger anti-inflammatory effect on RAW 264.7 cells stimulated by lipopolysaccharide (LPS) than β-1,4-xylo-oligosaccharides. At a working concentration of 100 μg/mL, β-1,3-xylo-oligosaccharides inhibited the release of NO and affected the expression of IL-1β, TNF-α, and other proteins secreted by cells, effectively promoting the release of pro-inflammatory mediators by immune cells in response to external stimuli and achieving anti-inflammatory effects. Therefore, β-1,3-xylo-oligosaccharides are valuable products in food and pharmaceutical industries.

Widely Targeted Metabolomic Analysis Reveals the Improvement in Panax notoginseng Triterpenoids Triggered by Arbuscular Mycorrhizal Fungi via UPLC-ESI-MS/MS.

Panax notoginseng is a highly valued perennial medicinal herb in China and is widely used in clinical treatments. The main purpose of this study was to elucidate the changes in the composition of P. notoginseng saponins (PNSs), which are the main bioactive substances, triggered by arbuscular mycorrhizal fungi (AMF) via ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS). A total of 202 putative terpenoid metabolites were detected, of which 150 triterpene glycosides were identified, accounting for 74.26% of the total. Correlation analysis, principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) of the metabolites revealed that the samples treated with AMF (group Ce) could be clearly separated from the CK samples. In total, 49 differential terpene metabolites were identified between the Ce and CK groups, of which 38 and 11 metabolites were upregulated and downregulated, respectively, and most of the upregulated differentially abundant metabolites were mainly triterpene glycosides. The relative abundances of the two major notoginsenosides (MNs), ginsenosides Rd and Re, and 13 rare notoginsenosides (RNs), significantly increased. The differential saponins, especially RNs, were more easily clustered into one branch and had a high positive correlation. It could be concluded that the biosynthesis and accumulation of some RNs share the same pathways as those triggered by AMF. This study provides a new way to obtain more notoginsenoside resources, particularly RNs, and sheds new light on the scientization and rationalization of the use of AMF agents in the ecological planting of medicinal plants.

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.

Simultaneous determination of semi-essential nutrients taurine, l-carnitine and choline in infant formulas and adult/pediatric nutritional formulas by hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry.

The nutritional intake of formula-fed newborns is often limited to a single source, so it must be supplemented with essential nutrients for the growth and proper development of infants. Taurine, l-carnitine, and choline are considered conditionally essential nutrients especially in newborns and infants. In this work, a simple routine hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry (HILIC-ESI-MS/MS) method was developed and validated for the simultaneous determination of these semi-essential nutrients in infant and adult/pediatric milk formulas. The extraction recoveries were between 90% and 114%. Precision of the method offered relative standard deviation below 5% and 7% for intra-day and inter-day precision, respectively. The proposed method was successfully applied to quantification of taurine, l-carnitine, and choline in milk formula. The contents found were in good agreement with those provided on the product label for almost all samples. In view of these results, it can be concluded that the developed method can be a useful approach for the simultaneous determination of taurine, l-carnitine and choline in powdered milk samples, so it can be useful in the routine quality control of this kind of samples. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Improvement of Alginate Extraction from Brown Seaweed (Laminaria digitata L.) and Valorization of Its Remaining Ethanolic Fraction.

This study aimed to improve the conventional procedure of alginate isolation from the brown seaweed (Laminaria digitata L.) biomass and investigate the possibility of further valorization of the ethanolic fraction representing the byproduct after the degreasing and depigmentation of biomass. The acid treatment of biomass supported by ultrasound was modeled and optimized regarding the alginate yield using a response surface methodology based on the Box-Behnken design. A treatment time of 30 min, a liquid-to-solid ratio of 30 mL/g, and a treatment temperature of 47 °C were proposed as optimal conditions under which the alginate yield related to the mass of dry biomass was 30.9%. The use of ultrasonic radiation significantly reduced the time required for the acid treatment of biomass by about 4 to 24 times compared to other available conventional procedures. The isolated alginate had an M/G ratio of 1.08, which indicates a greater presence of M-blocks in its structure and the possibility of forming a soft and elastic hydrogel with its use. The chemical composition of the ethanolic fraction including total antioxidant content (293 mg gallic acid equivalent/g dry weight), total flavonoid content (14.9 mg rutin equivalent/g dry weight), contents of macroelements (the highest content of sodium, 106.59 mg/g dry weight), and microelement content (the highest content of boron, 198.84 mg/g dry weight) was determined, and the identification of bioactive compounds was carried out. The results of ultra high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis confirmed the presence of 48 compounds, of which 41 compounds were identified as sugar alcohol, phenolic compounds, and lipids. According to the 2,2-diphenyl-1-picrylhydrazyl assay, the radical scavenging activity of the ethanolic fraction (the half-maximal inhibitory concentration of 42.84 ± 0.81 μg/mL) indicated its strong activity, which was almost the same as in the case of the positive control, synthetic antioxidant butylhydroxytoluene (the half-maximal inhibitory concentration of 36.61 ± 0.79 μg/mL). Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis, and Bacillus cereus) were more sensitive to the ethanolic fraction compared to Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Shigella sonnei). The obtained results indicated the possibility of the further use of the ethanolic fraction as a fertilizer for plant growth in different species and antifouling agents, applicable in aquaculture.

Impact of radish seeds (Semen Raphani) on the absorption and transportation of ginsenosides in the Caco-2 cell model: a UPLC-ESI-MS analysis

This study examined the impact of Semen raphani on the absorption of ginsenosides from Panax ginseng C.A. Meyer (ginseng) using a Caco-2 cell model and Ultra-High-Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (UPLC-ESI-MS). Six primary ginsenosides (Rg1, Re, Rb1, Rb2, Rc, Rd) were quantified. Results showed that Semen Raphani increased the efflux rate of ginsenosides, particularly at higher concentrations, suggesting it inhibits their absorption. The research elucidates the intestinal absorption process of ginsenosides and the antagonistic mechanism of Semen Raphani against ginseng.

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.

Characterization of spironolactone and metabolites derivatized using Girard's reagent P using mass spectrometry and ion mobility spectrometry.

Spironolactone is a steroidal drug prescribed for a variety of medical conditions and is extensively metabolized quickly after administration. Measurement of spironolactone and its metabolites remains challenging using mass spectrometry (MS) due to in-source fragmentation and relatively poor ionization using electrospray ionization. Therefore, improved methods of measurements are needed, particularly in the case of small sample volumes. Girard's reagent P (GP) derivatization of spironolactone was employed to improve response and provide an MS-based solution to the measurement of spironolactone and its metabolites. We performed ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) and ion mobility spectrometry (IMS)-high-resolution mass spectrometry (HRMS) to fully characterize the GP derivatization products. Analytes were studied in positive ionization mode, and MS/MS was performed using nonresonance and resonance excitation collision-induced dissociation. We observed the successful GP derivatization of spironolactone and its metabolites using authentic chemical standards. A signal enhancement of 1-2 orders of magnitude was observed for GP-derivatized versions of spironolactone and its metabolites. Further, GP derivatization eliminated in-source fragmentation. Finally, we performed GP derivatization and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) in a small volume of murine serum (20 μL) from spironolactone-treated and control animals and observed multiple spironolactone metabolites only in the spironolactone-treated group. GP derivatization was proven to have advantageous mass spectral performance (e.g., limiting in-source fragmentation, enhancing signals, and eliminating isobaric analytes) for spironolactone and its metabolites. This work and the detailed characterization using ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) and IMS serve as the foundation for future developments in reaction optimization and/or quantitative assay development.

Association between caffeine metabolites in urine and muscle strength in young and older adults: A cross-sectional study from NHANES 2011–2012

Elevated levels of reactive oxygen species may contribute to the gradual decline in muscle strength over time. Although caffeine and its metabolites have antioxidant properties that can mitigate oxidative stress, the association of caffeine and its metabolites with muscle strength remains unknown. To investigate whether caffeine metabolites in urine are associated with muscle strength in young and older adults. A cross-sectional study was conducted with 1145 individuals aged over 20 years (n=801<60 years and n=344≥60 years) from the National Health and Nutrition Examination Survey (NHANES) 2011-2012. Muscle strength was assessed using a handgrip dynamometer, and combined grip strength was determined by summing the highest value from each hand. Caffeine and its metabolites in urine were quantified using ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (1-methyluric acid, 3-methyluric acid, 7-methyluric acid, 1,3-dimethyluric acid, 1,7-dimethyluric acid, 3,7-dimethyluric acid, 1,3,7-trimethyluric acid, 1-methylxanthine, 3-methylxanthine, 7-methylxanthine, 1,3-dimethylxanthine, 1,7-dimethylxanthine, 3,7-dimethylxanthine, 1,3,7-trimethylxanthine, 5-acetylamino-6-amino-3-methyluracil). Linear regression analyses were performed to determine the association of caffeine and its metabolites with muscle strength in young and older adults, adjusting for confounders. Positive associations between muscle strength and levels of 7-methyluric acid (β=0.029; p=0.021), 1,3-dimethyluric acid (β=0.008; p=0.004), 3,7-dimethyluric acid (β=0.645; p=0.012), 3-methylxanthine (β=0.020; p=0.002), 7-methylxanthine (β=0.020; p=0.006), 1,3-dimethylxanthine (theophylline) (β=0.030; p=0.004) and 3,7-dimethylxanthine (theobromine) (β=0.035; p=0.029) were observed in older adults. In contrast, no such associations were noted in young adults. Our study indicates a positive association between certain caffeine metabolites in urine and muscle strength in older adults, but not in younger individuals. These findings indicate that specific caffeine metabolites may contribute to an antioxidant role especially in older adults.

Unveiling Pharmacokinetics and Drug Interaction of Linagliptin and Pioglitazone HCl in Rat Plasma Using LC-MS/MS.

The linagliptin (LIN) and pioglitazone HCl (PIO) combination, currently undergoing phase III clinical trials for diabetes mellitus treatment, demonstrated significant improvements in glycemic control. However, the absence of an analytical method for simultaneous determination in biological fluids highlights a crucial gap. This underscores the pressing need for sensitive bioanalytical methods, emphasizing the paramount importance of developing such tools to advance diabetes management strategies and enhance patient care. Herein, a sensitive reverse-phase high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed for simultaneous determination of LIN and PIO in rat plasma using alogliptin as an internal standard. Chromatographic separation was performed on an Agilent Eclipse Plus C18 (4.6 × 100 mm, 3.5 μm) using an isocratic mobile phase system consisting of ammonium formate (pH 4.5) and methanol using an acetonitrile-induced protein precipitation technique for sample preparation. Multiple reaction monitoring in positive ion mode was used for quantitation of the precursor to production at m/z 473.2 → 419.9 for LIN, 357.1 → 134.2 for PIO, and 340.3 → 116.1 for ALO. The linearity range was 0.5 to 100 and 1 to 2000 ng/mL for LIN and PIO, respectively. The developed method was validated as per US-FDA guidelines and successfully applied to clinical pharmacokinetic and drug-drug interaction studies with a single oral administration of LIN and PIO in rat plasma. Pharmacokinetic parameters of LIN were significantly influenced by the concomitant administration of PIO and vice versa. Molecular modeling revealed the significant interaction of LIN and PIO with P-glycoprotein. Therefore, the drug-drug interaction between LIN and PIO deserves further study to improve drug therapy and prevent dangerous adverse effects.

In vitro study on the competitive reactions between arsenite and selenite with glutathione.

Exposure to arsenic can cause various biological effects by increasing the production of reactive oxygen species (ROS). Selenium acts as a beneficial element by regulating ROS and limiting heavy metal uptake and translocation. There are studies on the interactive effects of As and Se in plants, but the antagonistic and synergistic effects of these elements based on their binding to glutathione (GSH) molecules have not been studied yet. In this study, we aimed to investigate the antagonistic or synergistic effects of As and Se on the binding mechanism of Se and As with GSH at pH3.0, 5.0, or 6.5. The interaction of As and Se in Se(SG)2+ As(III) or As(SG)3+ Se(IV) binary systems and As(III) + Se(IV) + GSH ternary system were examined depending on their ratios via liquid chromatography diode array detector/electrospray mass spectrometry (LC-DAD/MS) and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). The results showed that the formation of As(GS)3 was not detected in the As(III) + Se(SG)2 binary system, indicating that As(III) did not affect the stability of Se(SG)2 complex antagonistically. However, in the Se(IV) + As(SG)3 binary system, the addition of Se(IV) to As(SG)3 affected the stability of As(SG)3 antagonistically. Se(IV) reacted with GSH, disrupting the As(SG)3 complex, and consequently, Se(SG)2 formation was measured using LC-MS/DAD. In the Se(IV) + GSH + As(III) ternary system, Se(SG)2 formation was detected upon mixing As(III), Se(IV), and GSH. The increase in the concentration of As(III) did not influence the stability of the Se(SG)2 complex. Additionally, Se(IV) has a higher affinity than As(III) to the GSH, regardless of the pH of the solution. In both binary and ternary systems, the formation of the by-product glutathione trisulfide (GSSSG) was detected using LC-ESI-MS/MS.

Coupling of a streamlined solid-liquid extraction protocol with LC-ESI-MS/MS for the regular oversight of illegal bromate additive use: An accurate and sensitive determination method in preliminary and bakery products

A reliable solid-liquid extraction protocol coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry in the negative-ion mode was developed and validated for illegal bromate determination in preliminary and bakery products. Crude and dried-treated samples were directly extracted with acetonitrile-water (4:1, v/v). Bromate was determined using a Phenomenex Synergi™ Polar reversed-phase column and MS/MS under multiple reaction monitoring. The chosen solvent efficiently extracted bromate with all applied extraction-assisting techniques (p > 0.05). Although this assay avoids cleanup procedures, matrix effect of <−11% was achieved. Rapid bromate separation in only 8 min was attained by a reversed-phase column. In both commodities, linearity range, R2, recovery%, repeatability, intermediate precision, LOD and LOQ results were 0.05–100 ng mL−1, >0.9999, 88.6–103%, 2.93–9.80% and 9.64–10.10%, 0.015 μg kg−1 and 0.05 μg kg−1, respectively. Out of 288 tested real samples, 13.9% of violations were observed. This high-sensitivity protocol offers effective oversight and consumer protection.

Widely targeted metabolomics reveals differences in metabolites of Paeonia lactiflora cultivars.

Paeonia lactiflora contains diverse active constituents and exhibits various pharmacological activities. However, only partial identification of biologically active substances from P. lactiflora has been achieved using low-throughput techniques. Here, the roots of P. lactiflora, namely, Fenyunu (CK), Dafugui (DFG), and Red Charm (HSML), were studied. The primary and secondary metabolites were investigated using ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESIMS/MS). The chemical compounds and categories were detected using broadly targeted UPLC-MS/MS. Principal component analysis (PCA), orthogonal partial least-squares discriminant analysis (OPLS-DA), and hierarchical clustering analysis (HCA) were carried out for metabolites of different varieties of P. lactiflora. A total of 1237 compounds were detected and classified into 11 categories. HCA, PCA, and OPLS-DA of these metabolites indicated that each variety of P. lactiflora was clearly separated from the other groups. Differential accumulated metabolite analysis revealed that the three P. lactiflora varieties contained 116 differentially activated metabolites (DAMs) involved in flavonoid, flavone, and flavonol metabolism. KEGG pathway analysis revealed that, in 65 pathways, 336 differentially abundant metabolites (DMs) were enriched in the CK and DFG groups; moreover, the type and content of terpenoids were greater in the CK group than in the DFG group. The CK and HSML groups contained 457 DMs enriched in 61 pathways; the type and amount of flavonoids, terpenoids, and tannins were greater in the CK group than in the HSML group. The DFG and HSML groups contained 497 DMs enriched in 65 pathways; terpenoids and alkaloids were more abundant in the HSML variety than in the DFG variety. A total of 1237 compounds were detected, and the results revealed significant differences among the three P. lactiflora varieties. Among the three P. lactiflora varieties, phenolic acids and flavonoids composed the largest and most diverse category of metabolites, and their contents varied greatly. Therefore, CK is suitable for medicinal plant varieties, and DFG and HSML are suitable for ornamental plant varieties. Twelve proanthocyanidin metabolites likely determined the differences in color among the three varieties.

Development of a high-throughput liquid chromatography-tandem mass spectrometry platform for the determination of intact natriuretic peptides in human plasma

We present an innovative, reliable, and antibody-free analytical method to determine multiple intact natriuretic peptides in human plasma. These biomolecules are routinely used to confirm the diagnosis and monitor the evolution of heart failure, so that their determination is essential to improve diagnosis and monitor the efficacy of treatment. However, common immunoassay kits suffer from main limitations due to high cross-reactivity with structurally similar species. In our method, we pre-treated the sample by combining salting-out with ammonium sulfate with microextraction by packed sorbent technique. Analyses were then carried out by ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry. The use of 3-nitrobenzyl alcohol as a supercharger reagent enhanced the ESI ionization and improved the signal-to-noise ratio. The analytical protocol showed good linearity over one order of magnitude, recovery in the range of 94–105 %, and good intra- and inter-day reproducibility (RSD<20 %), and the presence of a matrix effect. Limits of detection were in the range of pg/mL for all peptides (0.2–20 pg/mL). Stability study in plasma samples demonstrated that proper protease inhibitors need to be included in blood collection tubes to avoid peptide degradation. Preliminary analyses on plasma samples from heart failure patients allow the quantification of ANP 1–28 as the most abundant species and the detection of ANP 5–28, BNP 1–32, and BNP 5–32. The method could be used to investigate how cross-reactivity issues among structurally similar species impact determinations by ELISA kits.

Dynamic changes of key metabolites in Longjing green tea during processing revealed by widely targeted metabolomic profiling and sensory experiments

In this study, widely targeted metabolomics and chemometrics were utilized to comprehensively analyse the formation of taste compounds in Longjing green tea. A total of 580 non-volatile metabolites were identified by using ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, and alterations in three metabolic pathways were investigated. Notably, the fixation process reduced phosphatidic acid levels, resulting in the formation of lyso-phosphatidylcholine and lyso-phosphatidylethanolamine, as well as the release of esterified polyunsaturated fatty acids. Baiye No.1 had high levels of L-glutamic acid and l-glutamine, while Longjing 43 showed elevated levels of flavones. Correlation analysis and sensory verification indicated that the specific concentration of L-leucine could decrease the umami of the tea. These findings advance our understanding of Longjing green tea quality improvement and cultivar development.

Liangxue-Qushi-Zhiyang Decoction Ameliorates DNCB-Induced Atopic Dermatitis in Mice through the MAPK Signaling Pathway Based on Network Pharmacology.

The traditional prescription of Liangxue-Qushi-Zhiyang decoction (LQZ) has been demonstrated to be efficacious in treating atopic dermatitis (AD), a chronic inflammatory skin disorder marked by intense itching, redness, rashes, and skin thickening. Nevertheless, there has been an inadequate systematic exploration of the potential targets, biological processes, and pathways for AD treatment through LQZ. The study objective was to evaluate the efficacy and possible mechanism of LQZ in AD mice. In our study, we identified the primary compounds of LQZ, analyzed hub targets, and constructed a network. Subsequently, the predicted mechanisms of LQZ in AD were experimentally studied and validated in vivo, as determined by network pharmacological analysis. A total of 80 serum components of LQZ were identified through ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS), among which 49 compounds were absorbed into the bloodstream. Our results indicated that LQZ targets six putative key factors in the MAPK signaling pathway, which play essential roles in AD, namely, EGFR, p-MAPK1/3, p-MAPK14, IL-1β, IL-6, and TNF-α. We observed spleen coefficient, dermatitis scores, and ear thickness were all downregulated in 2,4-dinitrochlorobenzene (DNCB)-induced mice after LQZ treatment. Histological analysis of the dorsal and ear skin further revealed that LQZ significantly decreased skin inflammation, epidermal thickness, and mast cell numbers compared to the DNCB group. Our study demonstrated the effectiveness of LQZ in reducing epidermal and dermal damage in a mouse model of AD. Furthermore, our findings suggest that downregulating the MAPK signaling pathway could be a potential therapeutic strategy for the treatment of AD.