Liquid Chromatography-electrospray Mass Spectrometry Research Articles

Streamlined Arsenolipid Identification via Direct Arsenic Detection Using RPLC-ESI-QTOF-MS with Collision-Induced Dissociation.

Arsenolipids are organoarsenicals with a long aliphatic chain that have been identified in a wide array of marine organisms. Precise analysis of arsenolipids is crucial for evaluating their toxicity, ensuring food safety, monitoring the environment, and gaining insights into the evolution of arsenic biogeochemistry. However, the discovery of new arsenolipids is often impeded by existing analytical challenges, notably the need for multiple instruments, such as the combination of liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) and inductively coupled plasma mass spectrometry (LC-ICP-MS). This study introduces a high-throughput untargeted analytical method on the basis of an unsophisticated instrumental configuration, LC-ESI-MS with collision-induced dissociation (CID) at 200 eV. This approach provides efficient dissociation of arsenic atoms from their precursor lipids and direct detection of the organic-bound arsenic as monatomic cations, As+. Application of this method has shown promise in rapidly characterizing arsenolipids in diverse samples, which has led to the discovery of a wide range of novel arsenolipids, including seven previously unidentified thioxoarsenolipids in ancient marine sediments.

Isolation, Antimicrobial Effect and Metabolite Analysis of Bacillus amyloliquefaciens ZJLMBA1908 against Citrus Canker Caused by Xanthomonas citri subsp. citri.

Citrus canker caused by Xanthomonas citri subsp. citri is a devastating bacterial disease with severe implications for the citrus industry. Microorganisms possessing biocontrol capabilities against X. citri subsp. citri offer a highly promising strategy for healthy citrus management. In the present study, a broad-spectrum antagonist strain ZJLMBA1908 with potent antibacterial activity against X. citri subsp. citri was isolated from symptomatic lemon leaves, and identified as Bacillus amyloliquefaciens. Cell-free supernatant (CFS) of strain ZJLMBA1908 also exhibited remarkable antimicrobial activity, especially suppressing the growth of X. citri subsp. citri and Nigrospora oryzae, with inhibition rates of 27.71% and 63.75%, respectively. The antibacterial crude extract (CE) derived from the CFS displayed effective activity against X. citri subsp. citri. A preventive treatment using the CE significantly reduced the severity and incidence of citrus canker in a highly susceptible citrus host. Additionally, the CE maintained activity in the presence of protease and under a wide range of temperature and pH treatments. Applying high-performance liquid chromatography (HPLC) to separate and purify the CE resulted in the discovery of one highly potent anti-X. citri subsp. citri subfraction, namely CE3, which could completely inhibit the growth of X. citri subsp. citri. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis revealed that CE3 mainly consisted of palmitic acid, surfactin C15, phytosphingosine and dihydrosphingosine. Taken together, the results contribute to the possible biocontrol mechanisms of B. amyloliquefaciens ZJLMBA1908, as well as providing a promising new candidate strain as a biological control agent for controlling citrus canker.

Proteome analysis of propolis deciphering the origin and function of its proteins

Propolis is a resinous honeybee product, rich in polyphenolic compounds and with high economic value. Although extensive studies regarding the chemical composition of different propolis extracts have been carried out, the propolis proteome remained unknown. The present study aimed at characterizing the proteome of two geographically-distinct propolis originating from Belgium and Iran. Propolis extracts were analyzed by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) followed by Matrix Assisted Laser Desorption Ionization – Time of Flight Mass Spectrometry (MALDI-TOF MS) and Liquid chromatography-electrospray ionization MS (LC-ESI-MS) analyses. Our comprehensive proteomic analysis led to the identification of honeybee venom and royal jelly proteins, and numerous plant-defense proteins in propolis, extending our understanding of the antimicrobial properties of this complex natural substance. The proteome of both propolis was mainly made up of proteins belonging to the poplar tree. Pathogenesis-related (PR) proteins, poplar PR-2 (β-1,3-glucanases) and PR-8 (acidic endochitinases) were found to be the major components of both propolis extracts, while the major bee venom allergen (Api m 1) and royal jelly proteins (MJRP 1, 2, 3 and 5) were identified in the propolis from Belgium. Overall, our proteome analyses revealed that propolis extracts from Belgium and Iran shared a core protein composition originating from the poplar proteome. The majority of identified proteins are involved in the plant defense against pathogens, most belonging to well-known pathogenesis-related families. Proteomics could thus represent a viable approach for origin authentication and allergen identification in poplar-type propolis.

Identification of nonvolatile organic compounds (NVOCs) in biopharmaceuticals through non-target analysis and quantification using complexation-precipitation extraction

Single-use systems in biopharmaceutical manufacturing can potentially release chemical constituents (leachables) into drug products. Prior to conducting toxicological risk assessments, it is crucial to establish the qualitative and quantitative methods for these leachables. In this study, we conducted a comprehensive screening and structure elucidation of 23 leachables (nonvolatile organic compounds, NVOCs) in two antibody drugs using multiple (self-built and public) databases and mass spectral simulation. We identified 7 compounds that have not been previously reported in medical or medicinal extractables and leachables. The confidence levels for identified compounds were classified based on analytical standards, literature references, and fragment assignments. Most of the identified leachables were found to be plasticizers, antioxidants, slip agents or polymer degradants. Polysorbate (namely Tween) is commonly used as an excipient for protein stabilization in biopharmaceutical formulations, but its ionization in liquid chromatography-electrospray ionization mass spectrometry can interfere with compound quantification. To address this, we employed a complexation-precipitation extraction method to reduce polysorbate content and quantify the analytes. The developed quantitative method for target NVOCs demonstrated high sensitivity (limit of quantification: 20 or 50 μg/L), accuracy (recoveries: 77.2 to 109.5 %) and precision (RSD ≤ 8.2 %). Overall, this established method will facilitate the evaluation of NVOC safety in drug products.

Redox-sensitive high-mobility group box-1 isoforms contribute to liver fibrosis progression and resolution in mice

High-mobility group box-1 (HMGB1) significantly increases and undergoes post-translational modifications (PTMs) in response to liver injury. Since oxidative stress plays a major role in liver fibrosis and induces PTMs in proteins, we hypothesized that redox-sensitive HMGB1 isoforms contribute to liver fibrosis progression and resolution. We used ESI-LC-MS (electrospray ionization-liquid chromatography-mass spectrometry) to study PTMs of HMGB1 during fibrosis progression and resolution. Conditional knockout mice were used for functional analyses. We identified that disulfide ([O]) and sulfonated ([SO3]) HMGB1 increase during carbon tetrachloride-induced liver fibrosis progression, however, while [O] HMGB1 declines, [SO3] HMGB1 drops but remains, during fibrosis resolution. Conditional knockout of Hmgb1 revealed that production of [O] and [SO3] HMGB1 occurs mostly in hepatocytes. Co-injection of [O] HMGB1 worsens carbon tetrachloride-induced liver fibrosis more than co-injection of [H] HMGB1. Conversely, ablation of [O] Hmgb1 in hepatocytes reduces liver fibrosis. Moreover, ablation of the receptor for advanced-glycation end-products (Rage) reveals that the profibrogenic effect of [O] HMGB1 is mediated by RAGE signaling in hepatic stellate cells (HSCs). Notably, injection of [SO3] HMGB1 accelerates fibrosis resolution due to RAGE-dependent stimulation of HSC apoptosis. Importantly, gene signatures activated by redox-sensitive HMGB1 isoforms in mice, classify patients with fibrosis according to fibrosis and inflammation scores. Dynamic changes in hepatocyte-derived [O] and [SO3] HMGB1 signal through RAGE-dependent mechanisms on HSCs to drive their profibrogenic phenotype and fate, contributing to progression and resolution of liver fibrosis. Since oxidative stress plays a major role in liver fibrosis and induces post-translational modifications of proteins, we hypothesized that redox-sensitive HMGB1 isoforms contribute to liver fibrosis progression and resolution. This study is significant because a rise in [H] HMGB1 could flag 'patient at risk', the presence of [O] HMGB1 could suggest 'disease in progress or active scarring', while the appearance of [SO3] HMGB1 could point at 'resolution under way'. The latter could be used as a readout for response to pharmacological intervention with anti-fibrotic agents.

Toxicological evaluation of ginsenoside-rich cell culture biomass of Panax japonicus produced in a large-scale bioreactor system

An environmentally friendly, resource-efficient substitute for the production of vegetative biomass and the bioactive components of medicinal plants is provided by novel biotechnology-based suspension cells cultured in bioreactors. Despite the cultured biomass is produced in the controlled conditions, it requires detailed chemical analysis and toxicological evaluation before being approved for human consumption. This study reports the results of biochemical, toxicological, and elemental composition analysis of the suspension cell culture of a medicinal species Panax japonicus (T. Nees) C.A. Mey. cultivated in a large-scale bioreactor system. For the scaling up the process of cultivation, the suspension culture was grown sequentially in a series of laboratory (20-L), pilot (75-L), and industrial (630-L) bioreactors. The biomass productivity of cell culture was comparable or higher than of wild plants or in vitro cultured hairy roots reported in the literature. The resulting cell biomass was analyzed for ginsenoside contents using ultra-high-performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC–ESI–MS). For the elemental composition of cells, inductively coupled argon plasma mass spectrometry (ICP–MS) was used. In addition, the biomass was evaluated for acute toxicity and skin sensibilization effects on in vivo animal models. The analyses confirmed that suspension cells cultured in bioreactors accumulate a broad spectrum of ginsenosides and their malonylated derivatives at a total concentration of 7.54 % (w/w) based on dry weight and contain essential macro- (K, Ca, Mg, Na) and micro- (Zn, Mn, Fe, B, Al, Cu) elements in dietary-safe concentrations. Acute toxicity tests using the administered doses of 2000 and 5000 mg dry biomass per kg animal weight resulted in no changes in animal organ weights and no or minor changes in hematological and biochemical parameters of blood. No skin irritation or sensitizing effects were observed. Therefore, bioreactor cultivation of P. japonicus cell suspension is highly productive and provides a ginsenoside-rich and non-toxic cell biomass which is a potentially valuable component of functional foods, food additives, and natural health products.

Condensed tannins from the leave of Dimocarpus longan Lour. exhibit anti-tyrosinase, anti-melanogenesis, anti-browning, anti-α-glucosidase, and anti-glycation activities

Dimocarpus longan Lour. is a valuable industrial crop for its broad applications in food, traditional Chinese medicine, and other fields. In this paper, condensed tannins were isolated from the leave of D. longan Lour., and their structure, anti-tyrosinase, anti-melanogenesis, anti-browning, anti-α-glucosidase, and anti-glycation properties were methodically clarified. The joint application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), electrospray ionization time-of-flight mass spectrometer (ESI+ TOF MS), and high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) confirmed that the longan leave condensed tannins (LLCTs) were polymers of procyanidins (majority) and prodelphinidins. The polymers powerfully inhibit tyrosinase (TY) with a competitive mechanism. These compounds altered the microenvironment of the binding site through hydrophobic interaction and hydrogen bond, and caused conformational modification of the enzyme by changing the polypeptide framework and secondary structure, leading to a decrease in TY activity. Cell assays further suggested that LLCTs exhibited a strong inhibition against intracellular TY activity and melanin production, and caused apoptosis in B16F10 cells. Anti-browning studies demonstrated that LLCTs suppress the browning of fresh-cut apples. Furthermore, the LLCTs exhibited an impressive inhibition on both α-glucosidase and nonenzymatic glycation. Therefore, this study confirms the potential of LLCTs as new inhibitors of tyrosinase, melanogenesis, browning, α-glucosidase, and nonenzymatic glycation, which provided a theoretical basis for the potential utilization of these polymers in the cosmetics, pharmaceutical, and food industries.

The male's scent triggered a neural response in females despite ambiguous behavioral response in Asian house rats.

Pheromones and olfactory communication play vital roles in sex recognition and mate choice in rodents. Asian house rats (Rattus tanezumi) (RT) often startle easily, making behavioral measurements difficult to carry out accurately in the laboratory. Here, the behavioral and olfactory preferences of the female RT between males and females were not observed using a conventional two-choice device; we then explored the neuro-immunohistochemical evidence in the brains of RT females. We found that male urine elicited significantly higher c-fos expression in the accessory olfactory system and sex-related brain regions in females than female urine did. On the other hand, the differences of volatile compounds and major urinary proteins (MUPs) in both voided urine and preputial glands (PGs) of the RT were detected using gas chromatography-mass spectrometer, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, and liquid chromatography-electrospray ionization mass spectrometry. We found that PG-derived 1-(4,5-dihydro-2-thiazolyl)-ethanone and total MUPs were more abundant in males versus females, suggesting these sexually divergent components might activate the female's accessory olfactory system. In conclusion, the neuro-immunohistochemical evidence indicated that potential sex pheromones might exist in RT; however, the strength of the chemical signal might be too weak to cause behavioral responses in females.

Preparation of low molecular weight chondroitin sulfate from different sources by H2O2/ascorbic acid degradation and its degradation mechanism

Low molecular weight chondroitin sulfate (LMCS) has attention for enhanced bioavailability and bioactivity compared to native CS. We optimized H2O2/ ascorbic acid (Vc) degradation conditions to prepare LMCS from chicken, bovine, and shark cartilages. Degradation kinetics models and chemical composition data of LMCS showed the GlcA residues of chondroitin-4-sulfate (CSA) may be preferentially attacked. Nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography-electrospray mass spectrometry (HPLC-MS) indicated that the CH of GlcA in CS was broken through a hydrogen abstraction reaction to break the β-(1 → 3) bond and form the hexendioic acid product. Standard density functional theory (DFT) calculations indicated that the energy required for the hydrogen abstraction from the C1-H bond in GlcA was lower than that of GalNAc. Molecular dynamics (MD) showed that CSA was more likely to interact with hydroxyl radicals (·OH) than non-sulfated chondroitin (CSO) and chondroitin-6-sulfate (CSC). These results provide guidance for producing LMCS.

Profiling of regioisomeric triacylglycerols in pistachio nuts by high-performance liquid chromatography-electrospray ionization mass spectrometry

In this contribution, a non-aqueous reversed-phase liquid chromatographic method coupled to electrospray ionization (ESI) tandem mass spectrometry (LC-MS) is used for the identification of triacylglycerol (TAG) down to the regioisomersíc level in pistachio lipids. Furthermore, for effective profiling of TAG, a two-dimensional (2D) map was employed to identify the entire homologous series of TAGs especially, odd-numbered fatty acid and long-chain derivatives. Notably, TAGs were observed as pairs of regioisomers with distinct retention times and tandem mass spectra. Furthermore, more than 100 TAGs containing 26 fatty acids with 51-63 carbon atoms and 0-3 double bonds were identified in different pistachio samples. The developed method is effective and facile for the fast quantification of complex TAGs in pistachio samples at high selectivity.

Characterization of Forced Degradants of Tegafur, Gimeracil, and Oteracil Potassium by Liquid Chromatographic-Electrospray Ionization-Mass Spectrometry and Simultaneous Estimation of Triple Combination in Drug Substance and Finished Pharmaceutical Dosage Form.

Tegafur, gimeracil, and oteracil potassium are widely used pharmaceuticals to treat lung cancers of the gastrointestinal tract, such as those of the oral cavity, esophagus, colon and rectum, and pancreas, as well as non-small cell lung cancers. The literature review revealed that no study has yet offered a completely stability-demonstrating, validated liquid chromatography-mass spectrometric approach for the concurrent estimation of tegafur, gimeracil, and oteracil potassium, along with all known degradation products. The simultaneous detection of tegafur, gimeracil, and oteracil potassium and their forced degradation product characterization necessitated the invention of a simpler, faster, and less expensive method. Therefore, this study aimed to follow the ICH method validation standards to develop and validate a fast, easy, and rugged liquid chromatography-mass spectrometry (LC-MS) technique for the concurrent estimation of tegafur, gimeracil, and oteracil potassium in the drug substance and the finished dosage form. Tegafur, gimeracil, and oteracil potassium were examined on the Waters HPLC Alliance system, coupled to the SCIEX QTRAP 5500 mass spectrometer, and endowed with an interface capable of carrying electrospray ionization. The tegafur, gimeracil, and oteracil peaks eluted at retention times of 2.338 min, 3.756 min, and 5.338 min, respectively. The limit of detection values of tegafur, gimeracil, and oteracil were detected to be 0.6, 0.174, and 0.474 μg/mL, respectively. The results for the quantification limit were calculated at 2.0, 0.58, and 1.58 µg/mL concentrations, respectively. Tegafur, gimeracil, and oteracil had linear ranges of 50-300 µg/ml, 14.5-87 µg/ml, and 39.5-237 µg/ml, with regression coefficients of 0.99956, 0.99986, and 0.999479, respectively. The accuracy values of tegafur, gimeracil, and oteracil in the ranges of 50%, 100%, and 150% were determined at 99.9%, 99.9%, and 99.4%, respectively. The RSD for the six replicates was less than 2% for precision. According to the ICH Q2 guidelines, this approach was effectively evaluated with LC-MS to validate the chemical structures of the freshly created tegafur, gimeracil, and oteracil degradation products. An accurate and sensitive LC-MS technique was developed and validated for the concurrent quantification of tegafur, gimeracil, and oteracil potassium in the drug material and the medicinal dosage form.

Immunoglobulin A Glycosylation Differs between Crohn's Disease and Ulcerative Colitis.

Inflammatory bowel diseases (IBD), such as Crohn's disease (CD) and ulcerative colitis (UC), are chronic and relapsing inflammations of the digestive tract with increasing prevalence, yet they have unknown origins or cure. CD and UC have similar symptoms but respond differently to surgery and medication. Current diagnostic tools often involve invasive procedures, while laboratory markers for patient stratification are lacking. Large glycomic studies of immunoglobulin G and total plasma glycosylation have shown biomarker potential in IBD and could help determine disease mechanisms and therapeutic treatment choice. Hitherto, the glycosylation signatures of plasma immunoglobulin A, an important immunoglobulin secreted into the intestinal mucin, have remained undetermined in the context of IBD. Our study investigated the associations of immunoglobulin A1 and A2 glycosylation with IBD in 442 IBD cases (188 CD and 254 UC) and 120 healthy controls by reversed-phase liquid chromatography electrospray-ionization mass spectrometry of tryptic glycopeptides. Differences of IgA O- and N-glycosylation (including galactosylation, bisection, sialylation, and antennarity) between patient groups were associated with the diseases, and these findings led to the construction of a statistical model to predict the disease group of the patients without the need of invasive procedures. This study expands the current knowledge about CD and UC and could help in the development of noninvasive biomarkers and better patient care.

Improving Quantitative Accuracy in Nontargeted Lipidomics by Evaluating Adduct Formation.

For large-scale lipidomic analyses, accurate and reproducible quantification of endogenous lipids is crucial for comparing results within and across studies. Many lipids present in liquid chromatography-electrospray ionization-mass spectrometry form various adducts with buffer components. The mechanisms and conditions that dictate adduct formation are still poorly understood. In a positive mode, neutral lipids like mono-, di-, and triacylglycerides and cholesteryl esters typically generate [M + NH4]+ adduct ions, although [M + Na]+, [M + K]+, and other (more complex) species can also be significantly abundant in MS1 precursor ion spectra. Variations in the ratios of these adducts (within and between matrices) can lead to dramatic inaccuracies during quantification. Here, we examine 48 unique diacylglycerol (DAG) species across 2366 mouse samples for eight matrix-specific data sets of plasma, liver, kidney, brain, heart muscle, gastrocnemius muscle, gonadal, and inguinal fat. Typically, no single adduct ion species accounted for more than 60% of the total observed abundance across each data set. Even within a single matrix, DAGs showed a high variability of adduct ratios. The ratio of [M + NH4]+ adduct ions was increased for longer-chain DAGs and for polyunsaturated DAGs, at the expense of reduced ratios of [M + Na]+ adducts. When using three deuterated internal DAG standards, we found that absolute concentrations were estimated with up to 70% error when only one adduct ion was used instead of all adducts combined. Importantly, when combining [M + NH4]+ and [M + Na]+ adduct ions, quantification results were within 5% accuracy compared to all adduct ions combined. Additional variance can be caused by other factors, such as instrument conditions or matrix effects.

Secondary Metabolomic Analysis and In Vitro Bioactivity Evaluation of Stems Provide a Comprehensive Comparison between Dendrobium chrysotoxum and Dendrobium thyrsiflorum.

The stems of Dendrobium chrysotoxum (DC) are commonly used as health-promoting foods due to their excellent biological activities. However, the stems of D. thyrsiflorum (DT) are often used to meet the scarcity of DC in production because of their highly similar morphology. However, the related metabolomic and bioactive information on the stems of DC and DT are largely deficient. Here, secondary metabolites of DC and DT stems were identified using an ultra-performance liquid chromatography-electrospray ionization-mass spectrometry, and their health-promoting functions were evaluated using several in vitro arrays. A total of 490 metabolites were identified in two stems, and 274 were significantly different. We screened out 10 key metabolites to discriminate the two species, and 36 metabolites were determined as health-promoting constituents. In summary, DT stems with higher extract yield, higher total phenolics and flavonoids, and stronger in vitro antioxidant activities demonstrated considerable potential in food and health fields.

In vitro anti-bactrical activity and its preliminary mechanism of action of the non-medicinal parts of Sanguisorba officinalis L. against Helicobacter pylori infection

Ethnopharmacological relevanceSanguisorba officinalis L. (S. officinalis L.), known as Di Yu (DY) in Traditional Chinese Medicine (TCM), are used to treat burns, vomiting of blood, asthma, intestinal infections, and dermatitis. It has been reported that the root of DY has a significant inhibitory effect on Helicobacter pylori (H. pylori). However, there is currently little research on the composition analysis and anti-H. pylori infection properties of the non-medicinal parts of DY, such as its stems, leaves, and flowers. Aim of studyThe commonly used eradication therapies for H. pylori infection are antibiotic-based therapies. With the increasing antibiotic resistance of H. pylori, it is urgent to find effective alternative therapies. To find alternative therapies and increase the utilization of DY, this study aims to investigate the phytochemistry profile, in vitro anti-H. pylori activity, and preliminary antibacterial mechanism of the non-medicinal parts of DY. Materials and methodsThe non-medicinal parts of DY extracts were obtained by using hot water reflux method. The chemical composition of these extracts was analyzed using colorimetric method, high-performance liquid chromatography (HPLC), and ultra-high-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS). The in vitro anti-H. pylori activity was investigated using broth microdilution method, checkerboard dilution method, time-kill curve, time-inhibition curve, scanning electron microscopy, and transmission electron microscopy. Transcriptional sequencing technology was used to study the effect of DY stems and flowers on the gene expression of H. pylori and explore possible antibacterial mechanisms. ResultsThe non-medicinal parts of DY contain abundant phytochemicals, such as total phenols and total flavonoids, and possess strong inhibitory and bactericidal activity against both standard and clinical strains of H. pylori in vitro. The MIC was 80–1280 μg/mL and the MBC was 80–2560 μg/mL, and the strength of the antibacterial effects was dependent on the concentration of phytochemicals (total polyphenols, gallic acid and ellagic acid). In addition, the combination of non-medicinal parts of DY with antibiotics, such as amoxicillin, metronidazole, levofloxacin, and clarithromycin, did not result in any antagonistic effects. All of them could disrupt the morphology, internal microscopic and cell wall structures of H. pylori thereby acting as an inhibitor. The mechanism of action was found to be the disruption of H. pylori morphology, internal microstructure, and cell wall. Transcriptomic analysis showed that the non-medicinal parts of DY significantly regulated the gene expression of H. pylori, especially the metabolic pathway. ConclusionsThis study analyzed the chemical composition of the non-medicinal parts of DY and confirmed its inhibitory and bactericidal activities against H. pylori, both standard and clinical strains. Additional, the mechanism of inhibition involves disrupting the structure of H. pylori cells, altering gene expression, and interfering with bacterial metabolic pathways. This study provides a reference for further resource utilization and the development of H. pylori drugs using the non-medicinal parts of DY.

Bacterial responses to plant antimicrobials: the case of alkannin and shikonin derivatives.

Alkannin, shikonin and their derivatives (A/S) are secondary metabolites produced in the roots of certain plants of the Boraginaceae family such as Lithospermum erythrorhizon Siebold & Zucc. and Alkanna tinctoria (L.) Tausch. These naphthoquinones express anti-cancer, wound healing, and antimicrobial activities. To study the interactions between endophytic bacteria isolated from A. tinctoria and the antimicrobials A/S, endophytic bacteria known to be resistant to the compounds were screened for their effect on A/S in liquid medium. Thereafter, the strain Pseudomonas sp. R-72008, was selected and tested for its ability to modify A/S in nutrient medium and minimal medium with A/S as sole carbon source. Bacterial growth was recorded, and high performance liquid chromatography-diode array and ultra-high performance liquid chromatography-electrospray ionization-mass spectrometry analyses were performed to detect and quantify metabolites. In nutrient medium inoculated with R-72008, a decrease in the amount of A/S monomers initially present was observed and correlated with an increase of A/S oligomers. Moreover, a significant decrease of initial A/S monomers in minimal medium was correlated with bacterial growth, showing for the first time that a bacterial strain, Pseudomonas sp. R-72008, was able to use the naphthoquinones A/S as sole carbon source. This study opens new perspectives on the interactions between bacteria and plant antimicrobials.

Production and characterization of a novel poly amino acid from a thermophilic bacterium, and preliminary testing of its coagulating potential for imminent wastewater treatment application

The increasing demand for biopolymers across diverse fields, such as food, medicine, cosmetics, and environmental applications, has prompted researchers to explore novel molecules with enhanced functionalities that meet these demands. In this study, a thermophilic strain of Bacillus licheniformis was employed to produce a unique polyamino acid. This thermophilic isolate exhibited rapid growth at 50 °C in a sucrose mineral salts medium, resulting in a biopolymer concentration of 7.4 g/L. Interestingly, the biopolymer produced at different temperatures exhibited varying glass-transition temperatures (ranging from 87.86 °C to 104.11 °C) and viscosities (7.5 cP to 16.3 cP), suggesting that the fermentation temperature significantly influenced the degree of polymerization. Furthermore, the biopolymer was characterized using various techniques, including Thin Layer Chromatography (TLC), Fourier Transform Infrared (FTIR) spectroscopy, Liquid Chromatography-Electrospray Ionization-Mass Spectroscopy (LC-ESI MS), Nuclear Magnetic Resonance (NMR), and Differential Scanning Calorimetry-Thermogravimetric Analysis (DSC-TGA). The results revealed that the obtained biopolymer was a poly amino acid, with poly-γ-glutamic acid as the major monomeric component in the polymer backbone with a few appendages of aspartic acid residues in its side chain. Finally, the biopolymer demonstrated significant coagulation potential for water treatment applications, as evidenced by coagulation studies conducted under varying pH conditions using kaolin-clay as a model precipitant.

Efficacy of Veronica incana for Treating Osteoarthritis Induced by Monosodium Iodoacetate in Rats.

The aim of this study is to investigate the efficacy and the underlying mechanism of Veronica incana in osteoarthritis (OA) induced by intraarticular injection of monosodium iodoacetate (MIA). The selected major four compounds (A-D) of V. incana were found from fractions 3 and 4. Its structure elucidation was determined by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) data analysis and nuclear magnetic resonance (NMR) data comparison with literature. MIA (50 μL with 80 mg/mL) for the animal experiment was injected into the right knee joint. The V. incana was administered orally every day to rats for 14 days from 7 days after MIA treatment. Finally, we confirmed the four compounds: (A) verproside; (B) catalposide; (C) 6-vanilloylcatapol; and (D) 6-isovanilloylcatapol. When we evaluated the effect of V. incana on the MIA injection-induced knee OA model, there were a noticeable initial decreased in hind paw weight-bearing distribution compared to the Normal group (P < .001), but V. incana supplementation resulted in a significant increase in the weight-bearing distribution to the treated knee (P < .001). Moreover, the V. incana treatment led to a decrease in the levels of liver function enzymes and tissue malondialdehyde (P < .05 and .01). The V. incana significantly suppressed the inflammatory factors through the nuclear factor-kappa B signaling pathway and downregulated the expression of matrix metalloproteinases, which are involved in the degradation of the extracellular matrix (P < .01 and .001). In addition, we confirmed the alleviation of cartilage degeneration through tissue stains. In conclusion, this study confirmed the major four compounds of V. incana and suggested that V. incana could serve as an anti-inflammatory candidate agent for patients with OA.

Estimation of dihydroartemisinin in human plasma using a highly sensitive LTQ Orbitrap mass spectrometer with Xcalibur software.

Background: Artemether (ARM), the O-methyl ether prodrug of dihydroartemisinin (DHA), is considered a first-line antimalarial agent. Artemether is extensively metabolized in vivo to its active metabolite DHA, and therefore its determination offers considerable difficulties. In the present study, DHA identification and estimation were accurately performed by the mass spectrometric analysis, using a high-resolution liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) LTQ Orbitrap hybrid mass spectrometer. Methods: The plasma samples were taken from healthy volunteers, and the spiked plasma was extracted by adding 1mL of a mixture of dichloromethane and tert.-methyl butyl ether (8:2v/v) to 0.5mL of plasma. The internal standard solution (artemisinin 500ng/mL) was added to the plasma samples. After vertexing and centrifugation, the organic layer was separated and transferred into another tube and dried under nitrogen. The residue was reconstituted in 100μL of acetonitrile and was injected onto the LC-MS system for analysis. Measurement of standards and samples was carried out isocratically on a Surveyor HPLC system combined with an LTQ Orbitrap mass spectrometer using an ACE 5 C18-PFP column. Mobile phase A consisted of 0.1% v/v formic acid in water, Mobile phase B consisted of acetonitrile only, and isocratic elution was carried out with A:B 20:80, v/v. The flow rate was 500μL/min. The ESI interface was operated in a positive ion mode with a spray voltage of 4.5kV. Results: Artemether is not a very biologically stable compound and is immediately metabolized to its active metabolite dihydroartemisinin, so no clear peak was observed for artemether. Both artemether and DHA after ionization undergo neutral losses of methanol and water, respectively, in the source of the mass spectrometer. The ions observed were (MH-H2O) m/z 267.15 for DHA and (MH-m/z 283.15 for internal standard artemisinin. The method was validated according to international guidelines. Discussion: The validated method was applied successfully for the determination and quantification of DHA in plasma samples. This method works well for the extraction of drugs, and the Orbitrap system with the help of Xcalibur software accurately and precisely determines the concentration of DHA in spiked as well as volunteer's plasma.

Polybutylene terephthalate-based stationary phase for ion-pair-free reversed-phase liquid chromatography of small interfering RNA. Part 2: Use for selective comprehensive two-dimensional liquid chromatography

With the increasing numbers of nucleic acid-based pharmaceuticals like antisense oligonucleotides (ASO), small interfering ribonucleic acid (siRNA) entering the market, research facilities, pharmaceutical industries and also regulatory authorities have been looking for efficient analytical methods for these synthetic oligonucleotides (ON). Besides of conventional one-dimensional (1D) reversed-phase liquid chromatography with or without ion-pairing (IP-RP-LC, RP-LC), hydrophilic liquid chromatography (HILIC) and mixed-mode chromatography (MMC), two-dimensional (2D) approaches combining two orthogonal chromatographic techniques also become more relevant due to the high structural complexity of oligonucleotides. Recently, we tested a polybutylene terephthalate(PBT)-based stationary phase under ion-pairing free RP mode for the liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) analysis of siRNA (Patisiran). In this study, retention profile and chromatographic orthogonality, respectively, were compared to other LC-modes like HILIC, IP-RPLC, another ion-pair free cholesterol-bonded RPLC and MMC considering their normalized retention times. Finally, because of higher orthogonality, the ion-pairing free PBT-bonded RPLC as first dimension (1D) was hyphenated with HILIC in the second dimension (2D) in a selective comprehensive 2D-LC setup leading to an enhanced resolution for peak purity evaluation of the main ON entities.