Glycoproteomics of a Single Protein: Revealing Tens of Thousands of Myozyme Glycoforms by Hybrid HPLC-MS Approaches.

Characterization of highly glycosylated biopharma-ceuticals by mass spectrometry is challenging because of the huge chemical space of coexistent glycoforms present. Here, we report the use of an array of HPLC-mass spectrometry-based approaches at different structural levels of released glycan, glycopeptide, and hitherto unexplored intact glycoforms to scrutinize the biopharmaceutical Myozyme, containing the highly complex lysosomal enzyme recombinant acid α-glucosidase. The intrinsic heterogeneity of recombinant acid α-glucosidase glycoforms was unraveled using a novel strong anion exchange HPLC-mass spectrometry approach involving a pH-gradient of volatile buffers to facilitate chromatographic separation of glycoforms based on their degree of sialylation, followed by the acquisition of native mass spectra in an Orbitrap mass spectrometer. Upon considering the structures of 60 different glycans attached to seven glycosylation sites in the intact protein, the large set of interdependent data acquired at different structural levels was integrated using a set of bioinformatic tools and allowed the annotation of intact glycoforms unraveling more than 1,000,000 putative intact glycoforms. Detectable isoforms also included several mannose-6-phosphate variants, which are essential for directing the drug toward its target, the lysosomes. Finally, for the first time, we sought to validate the intact glycoform annotations by integrating experimental data on the enzymatically dissected proteoforms, which reduced the number of glycoforms supported by experimental evidence to 42,104. The latter verification clearly revealed the strengths but also intrinsic limitations of this approach for fully characterizing such highly complex glycoproteins by mass spectrometry.

Technical challenges in defining RNA modifications.

It is well established that DNA base modifications play a key role in gene regulation during development and in response to environmental stress. This type of epigenetic control of development and environmental responses has been intensively studied over the past few decades. Similar to DNA, various RNA species also undergo modifications that play important roles in, for example, RNA splicing, protein translation, and the avoidance of immune surveillance by host. More than 160 different types of RNA modifications have been identified. In addition to base modifications, RNA modification also involves splicing of pre-mRNAs, leading to as many as tens of transcript isoforms from a single pre-RNA, especially in higher organisms. However, the function, prevalence and distribution of RNA modifications are poorly understood. The lack of a suitable method for the reliable identification of RNA modifications constitutes a significant challenge to studying their functions. This review focuses on the technologies that enable de novo identification of RNA base modifications and the alternatively spliced mRNA transcripts.

A validated HPLC-MS/MS method for the simultaneous determination of ecdysteroid hormones in subminimal amounts of biological material.

Ecdysteroids represent a large class of polyhydroxylated steroids which, due to their anabolic properties, are marketed as dietary supplements. Some ecdysteroids also act as important hormones in arthropods, where they regulate molting, development, and reproduction and many of these insects are miniature organisms that contain submicroliter levels of circulating biofluids. Analysis of ecdysteroids is further complicated by their very low abundance, large fluctuations during development, and difficult access to a pooled sample, which is important for quantitative measurements. In this work, we propose a new method that overcomes the described difficulties and allows validated quantification of four ecdysteroids in minimal amounts of biological material. After methanolic extraction, detectability of the ecdysteroids is increased 16- to 20-fold by conversion to their 14,15-anhydrooximes. These are further purified by pipette tip solid-phase extraction on a three-layer sorbent and subjected to HPLC-MS/MS analysis. Full validation was achieved using hemolymph from larvae of the firebug Pyrrhocoris apterus as a blank matrix and by the determination of ecdysteroids in a single Drosophila larva. The lower limit of quantifications for the four target ecdysteroids (20-hydroxyecdysone, ecdysone, makisterone A, and 2-deoxyecdysone) were 0.01; 0.1; 0.05; and 0.025 pg·ml-1 (20; 200; 100; 50 fmol ml-1), respectively, with very good accuracy, precision (expressed as relative standard deviation <15%) and recoveries (96%-119.9%). The application potential of the new method was demonstrated by quantification of ecdysteroids in various biological materials including human serum.

Simultaneous determination of plasma protein binding of five C-glycosylflavones from TFDS by rapid equilibrium dialysis.

The total flavonoids of Desmodium styracifolium (TFDS) are flavonoid-rich extracts obtained from Desmodii Styracifolii Herba, which is approved for the treatment of urolithiasis in China. C-glycosylflavones including schaftoside, vicenin-1, vicenin-2, vicenin-3, and isovitexin are the main active constituents. In this study, the plasma protein binding of these compounds was determined for the first time in rat and human plasma by rapid equilibrium dialysis combined with HPLC-MS/MS method. The developed method was validated in terms of specificity, linearity, accuracy, precision, extraction effect, matrix effect, and stability. Schaftoside, vicenin-1, vicenin-2, and vicenin-3 exhibited moderate plasma protein binding, ranging from 56.6% to 61.5% in rat plasma and 55.0%-62.9% in human plasma. In comparison, isovitexin demonstrated a higher plasma protein binding in the range of 92.3-93.1% and 95.1-96.2% in rat and human plasma, respectively. Furthermore, the potential interactions mediated via plasma protein binding between isovitexin and nonsteroidal anti-inflammatory drugs (NSAIDs) were investigated by rapid equilibrium dialysis. No significant changes were observed, indicating a lower likelihood of interaction between TFDS and NSAIDs due to plasma protein binding in the treatment of urinary system disorders.

Miniaturized matrix solid-phase dispersion assisted by deep eutectic solvent for acrylamide determination in bread samples.

Acrylamide determination is important to state its quantity in baked food preventing any potential carcinogenic effects. Matrix solid-phase dispersion (MSPD) extraction is an extraction procedure based on a homogenization phase between a solid sample and a solid dispersing material to break sample increasing analyte extraction yield, often used for acrylamide determination. The addition of a green deep eutectic solvent (DES) during the MSPD homogenization phase improves the analyte extraction, giving the possibility to reduce the amount of organic solvent used. In this work, a miniaturized MSPD extraction assisted by a DES was developed to determine acrylamide in bread, using high-performance liquid chromatography coupled with mass spectrometry detection. The optimized procedure provides 1:1 (w/w) matrix-to-dispersing material ratio, 2 mL of methanol as extraction solvent, and 50 µL of choline chloride-glycerol DES added during the homogenization phase. Method validation ensured good results with minimum recoveries of 90%, high precision with a maximum intra-day error of 4%, and inter-day error of 6%. Limit of detection and limit of quantification resulted to be 16 µg/kg and 35 µg/kg, respectively. This miniaturized extraction procedure represents a good alternative to those reported in the literature, guaranteeing great performance and respecting green chemistry principles.

On the utility of ultrafast MS1-only proteomics in drug target discovery studies based on thermal proteome profiling method.

Advances in high-throughput high-resolution mass spectrometry and the development of thermal proteome profiling approach (TPP) have made it possible to accelerate a drug target search. Since its introduction in 2014, TPP quickly became a method of choice in chemical proteomics for identifying drug-to-protein interactions on a proteome-wide scale and mapping the pathways of these interactions, thus further elucidating the unknown mechanisms of action of a drug under study. However, the current TPP implementations based on tandem mass spectrometry (MS/MS), associated with employing lengthy peptide separation protocols and expensive labeling techniques for sample multiplexing, limit the scaling of this approach for the ever growing variety of drug-to-proteomes. A variety of ultrafast proteomics methods have been developed in the last couple of years. Among them, DirectMS1 provides MS/MS-free quantitative proteome-wide analysis in 5-min time scale, thus opening the way for sample-hungry applications, such as TPP. In this work, we demonstrate the first implementation of the TPP approach using the ultrafast proteome-wide analysis based on DirectMS1. Using a drug topotecan, which is a known topoisomerase I (TOP1) inhibitor, the feasibility of the method for identifying drug targets at the whole proteome level was demonstrated for an ovarian cancer cell line.

Analytical methods based on liquid chromatography for the analysis of albumin adducts involved in retrospective biomonitoring of exposure to mustard agents.

The objective of the present review is to list, describe, compare, and critically analyze the main procedures developed in the last 20 years for the analysis of digested alkylated peptides, resulting from the adduction of albumin by different mustard agents, and that can be used as biomarkers of exposure to these chemical agents. While many biomarkers of sulfur mustard, its analogues, and nitrogen mustards can easily be collected in urine such as their hydrolysis products, albumin adducts require blood or plasma collection to be analyzed. Nonetheless, albumin adducts offer a wider period of detectability in human exposed patients than urine found biomarkers with detection up to 25 days after exposure to the chemical agent. The detection of these digested alkylated peptides of adducted albumin constitutes unambiguous proof of exposure. However, their determination, especially when they are present at very low concentration levels, can be very difficult due to the complexity of the biological matrices. Therefore, numerous sample preparation procedures to extract albumin and to recover alkylated peptides after a digestion step using enzymes have been proposed prior to the analysis of the targeted peptides by liquid chromatography coupled to mass spectrometry method with or without derivatization step. This review describes and compares the numerous procedures including a number of different steps for the extraction and purification of adducted albumin and its digested peptides described in the literature to achieve detection limits for biological samples exposed to sulfur mustard, its analogues, and nitrogen mustards in the ng/mL range.

A liquid chromatography-mass spectrometry method for the enantioselective multiresidue determination of nine chiral agrochemicals in urine using an enrichment procedure based on graphitized carbon black.

Many agrochemicals are chiral molecules, and most of them are marketed as racemates or diastereomeric mixtures. Stereoisomers that are not the active enantiomer have little or no pesticidal activity and can exert serious toxic effects towards non-target organisms. Thus, investigating the possible exposure to different isomers of chiral pesticides is an urgent need. The present work was aimed at developing a new enantioselective high-performance liquid chromatography-mass spectrometry method for the simultaneous determination of nine chiral pesticides in urine. Two solid-phase extraction (SPE) procedures, based on different carbon-based sorbents (graphitized carbon black (GCB) and buckypaper (BP)), were developed and compared. By using GCB, all analytes were recovered with yields ranging from 60 to 97%, while BP allowed recoveries greater than 54% for all pesticides except those with acid characteristics. Baseline separation was achieved for the enantiomers of all target agrochemicals on a Lux Cellulose-2 column within 24 min under reversed-phase mode. The developed method was then validated according to the FDA guidelines for bioanalytical methods. Besides recovery, the other evaluated parameters were precision (7-15%), limits of detection (0.26-2.21 µg/L), lower limits of quantitation (0.43-3.68 µg/L), linear dynamic range, and sensitivity. Finally, the validated method was applied to verify the occurrence of the pesticide enantiomers in urine samples from occupationally exposed workers.

HPLC-MS/MS-based quantification of human monoclonal antibodies targeting SARS-CoV-2 in the presence of endogenous SARS-CoV-2 antibodies in human serum.

Antibodies for treatment and prophylaxis against SARS-CoV-2 are needed particularly for immunocompromised individuals, who cannot adequately benefit from vaccination. To address this need, Aerium Therapeutics is developing antibodies targeting the SARS-CoV-2 spike protein. A bioanalytical method to quantify fully human monoclonal antibodies in a population with widely varying anti-spike antibody titers is required to investigate the pharmacokinetics of these antibodies in clinical trials. To eliminate interference from endogenous anti-spike protein antibodies, an HPLC-MS/MS assay was developed to quantify the investigational monoclonal antibodies (AER001 and AER002) by targeting signature peptides spanning the monoclonal antibodies' CDR regions. By optimizing and comparing affinity capture and ammonium sulphate precipitation, it was demonstrated that both procedures allowed accurate and precise quantification of AER001 and AER002 in human serum with comparable sensitivity. Ammonium sulphate precipitation outperformed immunocapture due to its simplicity and speed at lower cost and a full bioanalytical method validation was performed in human serum. The assay was also validated for human nasal lining fluid extract with a 50-fold lower limit of quantification and was shown to deliver similar sensitivity to previously published affinity capture HPLC-MS/MS assays. Finally, the CDR-derived signature peptides were also generated by tryptic digestion of blank serum in some individuals, an important caveat for HPLC-MS/MS strategies targeting human monoclonal antibodies. In summary, the presented results show that ammonium sulphate precipitation and HPLC-MS/MS allow accurate and precise quantification of monoclonals in clinical studies. The developed methods demonstrate that HPLC-MS/MS can reliably quantify human monoclonal antibodies even when endogenous antibodies with overlapping specificities are present and are crucial for the clinical testing of two investigational COVID-19 monoclonals.

Development, validation, and implementation of an ultratrace analysis method for the determination of moenomycin A, in aquatic animal products.

Moenomycin A, an antimicrobial growth promoter widely used as an additive in aquaculture feedstuffs, has been restricted for use in the European Union and China due to its potential risk of promoting resistant strains of pathogenic bacteria and causing residues in aquatic animal products. Although methods for analyzing moenomycin A in feedstuffs have been developed, no established method exists for aquatic matrices. In this study, we present, for the first time, a sensitive and validated high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the determination of moenomycin A in aquatic animal products. Samples were extracted using methanol and purified with the QuEChERS method employing C18 sorbent. The aliquot was dried under a nitrogen stream, reconstituted with methanol-water solvent, and analyzed by HPLC-MS/MS. The developed method exhibited good linearity (r2 > 0.995) over a wide concentration range (1-100 µg/L) and a low limit of detection (1 µg/kg). Average recoveries ranged between 70 and 110% at spiked concentrations of 1, 50, and 100 µg/kg, with associated intra- and inter-day relative standard deviations of 1.25 to 7.32% (n = 6) and 2.91 to 10.08% (n = 3), for different representative aquatic animal production, respectively. To the best of our knowledge, this is the first reported HPLC-MS/MS method for the quantification of moenomycin A in aquatic animal products. The new approach was effectively employed in the analysis of moenomycin A across various aquatic samples.

Structural Elucidation of Glycosaminoglycans in the Tissue of Flounder and Isolation of Chondroitin Sulfate C.

Glycosaminoglycans (GAGs) are valuable bioactive polysaccharides with promising biomedical and pharmaceutical applications. In this study, we analyzed GAGs using HPLC-MS/MS from the bone (B), muscle (M), skin (S), and viscera (V) of Scophthalmus maximus (SM), Paralichthysi (P), Limanda ferruginea (LF), Cleisthenes herzensteini (G), Platichthys bicoloratus (PB), Pleuronichthys cornutus (PC), and Cleisthenes herzensteini (CH). Unsaturated disaccharide products were obtained by enzymatic hydrolysis of the GAGs and subjected to compositional analysis of chondroitin sulfate (CS), heparin sulfate (HS), and hyaluronic acid (HA), including the sulfation degree of CS and HS, as well as the content of each GAG. The contents of GAGs in the tissues and the sulfation degree differed significantly among the fish. The bone of S. maximus contained more than 12 µg of CS per mg of dry tissue. Although the fish typically contained high levels of CSA (CS-4S), some fish bone tissue exhibited elevated levels of CSC (CS-6S). The HS content was found to range from 10-150 ug/g, primarily distributed in viscera, with a predominant non-sulfated structure (HS-0S). The structure of HA is well-defined without sulfation modification. These analytical results are independent of biological classification. We provide a high-throughput rapid detection method for tissue samples using HPLC-MS/MS to rapidly screen ideal sources of GAG. On this basis, four kinds of CS were prepared and purified from flounder bone, and their molecular weight was determined to be 23-28 kDa by HPGPC-MALLS, and the disaccharide component unit was dominated by CS-6S, which is a potential substitute for CSC derived from shark cartilage.

Cellular metabolomics study of the antitumor mechanism of Sijunzi decoction combined with mitomycin C.

Mitomycin C (MMC) has an antitumor effect and is considered as a broad-spectrum antibiotic. Sijunzi Decoction (SJZD), a well-known ancient Chinese prescription, is widely used in the treatment of cancer when combined with chemotherapy drugs. Studies have shown that SJZD can be combined with other drugs to enhance the therapeutic effect against cancer and inhibit the toxicity of chemotherapy drugs, but the specific mechanism is not clear. Thus, we hope to further explore the antitumor mechanism of combined SJZD and MMC. 3-(4,5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide assay, flow cytometry, western blot, 1H NMR and HPLC-MS were used to study the mechanism at the cellular level. The results show that the combined administration can have a more significant effect on inhibiting the proliferation of cancer cells, promoting their apoptosis. Based on metabolomics, 38 biomarkers were found in the MMC group and 43 biomarkers were found in the combined administration group. Among them, 18 unique biomarkers were discovered in the combined administration group. Studies have shown that the antitumor mechanism of combined administration is related to amino acid metabolism, energy metabolism, lipid metabolism and nucleotide metabolism, among which amino acid metabolism is the most important. In addition, SJZD achieves the effect of toxin reduction and efficiency enhancement by improving the body's immunity and improving the oxidative stress environment.

Comparative analysis of six aconitine-type alkaloids in decocting extracts between the different processed Fuzi (Aconiti Lateralis Radix praeparata) and its pharmacokinetic behavior in rats by HPLC-MS/MS.

Aconiti Lateralis Radix praeparata (also known as Fuzi in Chinese) has been extensively used in clinic. However, the toxicity issues limit the therapeutic range of Fuzi. Thus, a rapid and sensitive HPLC-MS/MS method was developed to simultaneously quantify and compare six active/toxic constituents in decocting extracts from four different processed Fuzi products and in rat plasma after oral administration of its decocting extracts. The selectivity, linearity, sensitivity, precisions, accuracy, matrix effects, extraction recoveries, and stability were validated. The comparative analysis of six alkaloids in decocting extracts between the four kinds of Fuzi products were conducted by the validated HPLC-MS/MS. Principal component analysis (PCA) and orthogonal partial least-squares discriminate analysis (OPLS-DA) were adopted to compare the differences of decocting extracts from the different processed Fuzi products. Besides, selecting Heishunpian (HSP) as the representative of the processed Fuzi products, we investigated the pharmacokinetic behaviors of these major alkaloids after oral administration. The developed HPLC-MS/MS method to simultaneously analyze these aconitine-type alkaloids in decocting extracts, and its pharmacokinetic behavior after oral administration may pave a way for quality control of Fuzi and monitoring the safety and rationality of clinical prescriptions.

Simultaneous quantification of icaritin and its novel 3-methylcarbamate prodrug in rat plasma using HPLC-MS/MS and its application to pharmacokinetic study.

A sensitive, rapid, and simple HPLC-MS/MS method was first developed and fully validated to determine the icaritin (ICT) and its novel 3-methylcarbamate prodrug (3N) simultaneously in rat plasma. Analytes were extracted from rat plasma using a liquid-liquid extraction (LLE) method. Chromatographic separation was performed on ACE Excel 2 C18-Amide column. Quantitation of analytes was conducted on an LCMS-8060 triple-quadrupole tandem mass spectrometer. The quantitation mode was the multiple reaction monitoring via positive electrospray ionization. The calibration curve was linear over the concentration range of 1 to 200 ng/ml for ICT with a correlation coefficient of r = 0.9950 and 1 to 400 ng/ml for 3N with a correlation coefficient of r = 0.9956. The intra-precision RSDs were ≤12% for ICT and 3N. The inter-day precision RSDs were ≤10% for ICT and 3N. The accuracy RE was between -2.6% and 7.8% for ICT and 3N. The average ICT, 3N and IS recoveries were 87.9%, 83.6%, and 84.3%. The plasma matrix of ICT and 3N complied with the guidelines. ICT and 3N were stable in rat plasma under various tested conditions. This work has been successfully applied to studying the pharmacokinetics of ICT and 3N.

Assessment of drug-drug interaction of dapagliflozin with LCZ696 based on an LC-MS/MS method.

The co-administration of dapagliflozin (DPF) and sacubitril/valsartan (LCZ696) has emerged as a promising therapeutic approach for managing heart failure. Given that DPF and LCZ696 are substrates for P-glycoprotein, there is a plausible potential for drug-drug interactions when administered concomitantly. To investigate the pharmacokinetic changes when these drugs are co-administered, we have established and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of simultaneously detecting DPF, LBQ657 (the active metabolite of sacubitril) and valsartan in rat plasma. This method has demonstrated selectivity, sensitivity, and accuracy. Drug-drug interactions were examined by the LC-MS/MS method. The mechanisms were investigated using everted intestinal sac models and Caco-2 cells. The results showed that DPF significantly increased the area under the curve (AUC(0-t)) (3,563.3 ± 651.7 vs. 7,146.5 ± 1,714.9 h µg/L) of LBQ657 (the active metabolite of sacubitril) and the AUC(0-t) (24,022.4 ± 6,774.3 vs. 55,728.3 ± 32,446.3 h µg/L) of valsartan after oral co-administration. Dapagliflozin significantly increased the amount of LBQ657 and valsartan in intestinal sacs by 1- and 1.25-fold at 2.25 h. Caco-2 cell uptake studies confirmed that P-glycoprotein is the transporter involved in this interaction. This finding enhances the understanding of drug-drug interactions in the treatment of heart failure and provides a guidence for clinical therapy.

Pharmacokinetic study of multicomponent in Hong-Hua-Xiao-Yao tablet.

Hong-Hua-Xiao-Yao tablet (HHXYT) is attracting attention increasingly because of its use in treatment of mammary gland hyperplasia (MGH) and menopausal syndrome. However, its pharmacokinetics remains unclear. This study developed a sensitive and rapid method for simultaneous determination of 10 compounds of HHXYT in rat plasma by liquid chromatography-tandem mass spectrometry and to compare the pharmacokinetics of these compounds in MGH rats and sham operated rats. The linearity, accuracy, precision, stability and matrix effect were within acceptable ranges. This established method was successfully applied to a pharmacokinetics study of 10 compounds in sham operated and MGH rats. According to the results, the bioavailability of glycyrrhetinic acid was highest in MGH rats and sham operated rats. The mean residence times of glycyrrhetinic acid and glycyrrhetinic acid 3-O-glucuronide were higher than those of the other compounds while the mean residence time and half-life of liquiritin, isoliquiritin and paeoniflorin were lower. Some pharmacokinetic parameters of ormononetin, liquiritigenin, isoliquiritigenin, liquiritin, isoliquiritin, paeoniflorin, protocatechuic acid and senkyunolide I were significantly different between MGH rats and sham operated rats. This study elucidated the dynamic changes of multiple components in rats after oral administration of HHXYT systematically and comprehensively, which provided guidance for clinical application.

Assessment of baby disposable diapers application for urine collection and determination of phthalate metabolites.

The baby disposable diapers were investigated as a sampling material for urine collection and validated for the evaluation of the exposure of children to xenobiotics. Phthalate metabolites detected in urine samples were chosen as proof-of-concept analytes. For the determination of phthalate metabolites in children's urine samples, high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) was used. Two sampling approaches were compared, namely sterile containers and baby disposable diapers. Thirty urine samples from infants and toddlers were analyzed by both methods in parallel and the results were compared. It was found that for diaper sampling, lower concentrations of the metabolites were observed, however, the general distribution for particular metabolites remains the same for both methods. For most of the metabolites high determination coefficients were obtained, namely 0.9929 for MEHHP, 0.9836 for MMP, 0.9796 for MECPP, and 0.9784 for 2-cx-MMHP. For MEOHP the determination correlation coefficient was 0.9154, while for MBP was - 0.7771 and MEHP was - 0.5228. In general, for diaper sampling an underestimation for 2-cx-MMHP and MEOHP was observed, while for MMP diaper-based approach provides overestimation. However, the proposed procedure confirms the possibility of using baby disposable diapers as a material for the collection of urine samples for biomonitoring purposes and fast screening of phthalates exposure.

Dissipation behaviours, residues, and health risk of six herbicides in sugar beets under field conditions.

This study established a residue detection method based on the QuEChERS pre-treatment method and combined it with high-performance liquid chromatography-tandem mass spectrometry to test six herbicides (metamitron, clopyralid, desmedipham, phenmedipham, ethofumesate, and haloxyfop-p-methyl) in sugar beet plants, soil, and roots. The degradation dynamics and terminal residues of each herbicide in sugar beets were analysed. Finally, the dietary risks of various herbicides in sugar beets were evaluated based on the dietary structure of Chinese people, and the risk quotient values were below 100%. Using this detection method, all reagents exhibited good linearity (0.9724 ≤ R2 ≤ 0.9998), The limit of quantification (LOQ) ranged from 0.01 to 0.05 mg/L, the matrix effect ranged from -1.2% to -50%, the addition recovery rate ranged from 77.00% to 103.48%, and the relative standard deviation ranged from 1.61% to 16.17%; therefore, all indicators of this method met the residue detection standards. Under field conditions, the half-lives (t1/2) ranged about 0.65 ∼ 2.96 d and 0.38 ∼ 27.59 d in sugar beet plants and soil, respectively. All herbicides were easily degraded in sugar beet plants and soil (t1/2 < 30 d). The terminal residue amounts in the beet plants, soil, and roots ranged from < LOQ to 0.243 mg/kg. The dietary risk assessment of each pesticide was conducted based on the residual median of the terminal residues and the highest residual values on the edible part of the beetroot. The chronic exposure risk quotient (RQc) and acute exposure risk quotient (RQa) values were < 100%, indicating that the residue of each pesticide in beetroot posed low risks to consumers in China at the recommended dosage.

Symbiotic Antidiabetic Effect of Lactobacillus casei and the Bioactive Extract of Cleome droserifolia (Forssk.) Del. on Mice with Type 2 Diabetes Induced by Alloxan.

The consumption of probiotics protects pancreatic ß-cells from oxidative damage, delaying the onset of type 2 diabetes mellitus (T2DM) and preventing microvascular and macrovascular complications. This study aimed to evaluate the antidiabetic activity of CDE fermented by Lactobacillus casei (ATCC 39539) (LC) in alloxan-induced diabetic rats. The oxidative stress identified by catalase (CAT), serum AST, ALT, ALP, creatinine, urea, and uric acid were measured. The chemical profiles of the plant extract and the fermented extract were studied using HPLC/MS. The potential of the compounds towards the binding pockets of aldose reductase and PPAR was discovered by molecular docking. A significant reduction in fasting blood glucose in alloxan-treated rats. The CAT showed a significant decrease in diabetic rats. Also, serum AST, ALT, ALP, creatinine, urea, and uric acid were significantly decreased in the mixture group. Mild histological changes of pancreatic and kidney tissues suggested that the mixture of probiotics and cleome possesses a marked anti-diabetic effect. Overall, the study suggests that the combination of Cleome droserifolia fermented by Lactobacillus casei exhibits significant antidiabetic activity (p-value=0.05), reduces oxidative stress, improves lipid profiles, and shows potential for the treatment of diabetes.

Core-shell magnetic covalent organic polymer nanocomposites as an adsorbent for the extraction of flavonoids.

A novel 3D magnetic nanocomposite material based on covalent organic polymers was successfully synthesized and utilized as an efficient sorbent for magnetic solid-phase extraction. It exhibited a regular core-shell structure, large specific surface area, superior stability, and paramagnetism. To evaluate its extraction efficiency, six flavonoids were tested, demonstrating maximum adsorption capacities ranging from 90 to 218 mg/g. Additionally, the material exhibited remarkable reusability and mechanical stability, maintaining its original state over eight cycles with consistent recovery. An analytical strategy combining magnetic solid-phase extraction with high performance liquid chromatography and tandem mass spectrometry was developed for the determination of flavonoids in orange, honey, soybean, and Dioscorea bulbifera L. samples. The low limits of detection (0.01-0.1 ng/mL) and limits of quantification (0.05-0.5 ng/mL), as well as satisfactory recovery (80.4-114.8%), were obtained. The linear range started from the limits of quantification to 500 ng/mL with R2 ≥ 0.9929. These results suggest that the prepared adsorbent possesses excellent adsorption capabilities for flavonoids, highlighting its significant potential for detecting these compounds in complex sample matrices.