High-resolution Liquid Chromatography-tandem Mass Spectrometry Research Articles

UHPLC-ESI-QqTOF Analysis and In Vitro Rumen Fermentation for Exploiting Fagus sylvatica Leaf in Ruminant Diet.

In recent years, animal husbandry has aimed at improving the conditions of livestock animals useful for humans to solve environmental and health problems. The formulation of animal feeds or supplements based on antioxidant plant compounds is considered a valuable approach and an alternative for livestock productivity. Forest biomass materials are an underestimated source of polyphenolic compounds whose sustainable recovery could provide direct benefits to animals and, indirectly, human nutrition. In this context, an alcohol extract from leaves of Fagus sylvatica L. was first investigated through an untargeted ultra-high-performance liquid chromatography–high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) approach. Then, it was fractionated into a fatty acid-rich and a polyphenolic fraction, as evidenced by total lipid, phenol, and flavonoid content assays, with antiradical and reducing activity positively correlated to the latter. When tested in vitro with rumen liquor to evaluate changes in the fermentative parameters, a significant detrimental effect was exerted by the lipid-rich fraction, whereas the flavonoid-rich one positively modulated the production of volatile fatty acids (i.e., acetate, butyrate, propionate, etc.).

Identification and characterization of Fe3O4/peroxodisulfate advanced oxidation products from sulfameter

Sulfonamides (SAs) are one of the most widely used antibiotics and their residuals in the environment could cause some negative environmental issues. Advanced oxidation such as Fenton-like reaction has been widely applied in the treatment of SAs polluted water. Degradation rates of 95%-99.7% were achieved in this work for the tested 8 SAs, including sulfisomidine, sulfameter (SME), phthalylsulfathiazole, sulfamethoxypyridazine, sulfamonomethoxine, sulfisoxazole, sulfachloropyridazine, and sulfadimethoxine, in the Fe3O4/peroxodisulfate (PDS) oxidation system after the optimization of PDS concentration and pH. Meanwhile, it was found that a lot of unknown oxidation products were formed, which brought up the uncertainty of health risks to the environment, and the identification of these unknown products was critical. Therefore, SME was selected as the model compound, from which the oxidation products were never elucidated, to identify these intermediates/products. With liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS), 10 new products were identified, in which 2-amino-5-methoxypyrimidine (AMP) was confirmed by its standard. The investigation of the oxidation process of SME indicated that most of the products were not stable and the degradation pathways were very complicated as multiple reactions, such as oxidation of the amino group, SO2 extrusion, and potential cross-reaction occurred simultaneously. Though most of the products were not verified due to the lack of standards, our results could be helpful in the evaluation of the treatment performance of SAs containing wastewater.

Phytochemical and multi-biological characterization of two Cynara scolymus L. varieties: A glance into their potential large scale cultivation and valorization as bio-functional ingredients

Artichoke leaf (Cynarae folium) extracts are used as traditional herbal medicinal products to treat a wide range of human ailments, being widely commercialized as nutraceutical or pharmaceutical preparations. In the current study, the hydromethanolic dried leaf extracts of Cynara scolymus L. var. major Brotero and C. scolymus L. var. redonensis N.H.F. Desp. were phytochemically and biologically investigated. The liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) metabolite profiling showed a complex composition, with phenolic acids (mostly mono- and di-caffeoylquinic acids), flavonoids and sesquiterpene lactones as the most representative classes. The strong antioxidant activity of the two C. scolymus varieties was evidenced in DPPH [64.84–65.21 mg trolox equivalents (TE)/g] and ABTS (86.39–95.55 mg TE/g) radical scavenging, cupric (160.49–171.07 mg TE/g) and ferric (71.47–78.95 mg TE/g) reducing capacity, metal chelating and phosphomolybdenum assays. In addition, the two extracts also displayed anti-enzymatic effects, as assessed in cholinesterase, tyrosinase, glucosidase and amylase tests. Lastly, the artichoke samples (at the concentration of 20 μg/mL) proved a very potent inhibition of the production of several pro-inflammatory cytokines, namely interleukin (IL)-1β [7.55–15.75% of lipopolysaccharide (LPS) + cells], IL-8 (11.72–13.46% of LPS + cells) and tumor necrosis factor (TNF)-α (4.07–10.35% LPS + cells), in LPS-stimulated human neutrophils. Overall, the results of our study indicate that the two C. scolymus varieties could be regarded as a rich source of biologically active compounds, opening thus the perspectives for their future large scale cultivation and valorization as bio-functional ingredients with putative antioxidant, anti-enzymatic and anti-inflammatory effects.

Determination of glyphosate exposure in the Iberian hare: A potential focal species associated to agrosystems

Glyphosate is the most used herbicide worldwide. It is a small and highly polar pesticide whose physicochemical properties makes its analytical determination difficult. Here, a procedure based on liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) was developed for glyphosate determination in samples of gastric content from wildlife. Iberian hare (Lepus granatensis), a herbivorous mammal species, strongly associated to agrosystems was selected as model species. The procedure involves direct analysis of sample without derivatization or instead of neither further cleaning steps. The procedure was validated by inter-day accuracy and precision studies with gastric content of hare spiked with glyphosate at ecologically relevant concentrations for the species (0.1–6 μg/g), and with 1 μg/g of isotopically labelled internal standard (glyphosate-2-13C,15N). Finally, glyphosate residues in hunted animals from pesticide-treated and pesticide-free areas (n = 75 and 28, respectively), as well as from hares found dead in the field (n = 11) were analysed. The linearity of both standards in extraction solutions and procedural calibration curves with spiked samples was similar, both with determination coefficients (r2) higher than 0.99. Satisfactory recoveries in spiked samples were achieved within the range of 95% to 118% (CV ≤ 20%). The limit of detection of glyphosate in hare gastric content was 0.03 μg/g. Prevalence of glyphosate in hunted animals from pesticide-treated areas ranged between 9 and 22%, increasing to 45% in animals found dead. The glyphosate concentrations detected in the gastric content of hares ranged from 0.11 to 16 μg/g. No residues were detected in animals from pesticide-free areas. In practice, the developed methodology may be particularly useful in the context of research and other work on the exposure in wildlife of one of the most used pesticides nowadays.

Stereocalpin B, a New Cyclic Depsipeptide from the Antarctic Lichen Ramalina terebrata.

Stereocalpin B, a new cyclic depsipeptide (1), and a new dibenzofuran derivative (3), were isolated from the Antarctic lichen, Ramalina terebrata (Ramalinaceae), along with a known cyclic depsipeptide (2). The structures of new compounds were characterized by comprehensive spectrometric analyses; high-resolution fast atom bombardment mass spectrometry (HR-FABMS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Stereocalpin B (1) existed in a rotameric equilibrium, which was confirmed using nuclear Overhauser effect spectroscopy (NOESY)/exchange spectroscopy (EXSY) spectrum. Absolute configurations of the amino acid units in 1 were assigned using the advanced Marfey’s method and subsequent NOESY analysis of the 5-hydroxy-2,4-dimethyl-3-oxo-decanoic acid residue confirmed the complete stereochemistry of 1. Compounds 1-3 exhibited moderate antimicrobial activities against E. coli, with the IC50 values ranging from 18–30 μg/mL. Compound 2 exhibited cell growth inhibition against HCT116 cell lines, with the IC50 value of 20 ± 1.20 μM, and compounds 1 and 2 also showed potent anti-inflammatory activities against lipopolysaccharide (LPS)-induced RAW264.7 macrophages with the IC50 values ranging from 5–7 μM.

In silico deconjugation of glucuronide conjugates enhances tandem mass spectra library annotation of human samples

Mass spectral library annotation of liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) data is a reliable approach for fast identification of organic contaminants and toxicants in complex environmental and biological matrices. While determining the exposure of humans or mammals, it is indispensable to include phase I and phase II metabolites (conjugates) along with the parent compounds, but often, tandem mass spectra for these are unavailable. In this study, we present and evaluate a strategy for annotating glucuronide conjugates in LC-HRMS/MS scans by applying a neutral loss search for detection, then truncating the spectra which we refer to as in silico deconjugation, and finally searching these against mass spectral libraries of the aglycones. The workflow was tested on a dataset of in vitro–generated glucuronides of reference standard mixtures and a dataset of 51 authentic urine samples collected from patients with known medication status, acquired on different instrumentations. A total number of 75 different glucuronidated molecular structures were identified by in silico deconjugation and spectral library annotation. We also identified specific molecular structures (sulfonamides, ether bonds, di-glucuronides), which resulted in slightly different fragmentation patterns between the glucuronide and the unconjugated compound. This led to a decreased spectral matching score and in some cases to a false-negative identification. Still, by applying this method, we revealed a reliable annotation of most common glucuronides, leading to a new strategy reducing the need for deconjugation steps or for recording many reference glucuronide spectra for screening approaches.Graphical

Comparison of Methods for Quantitative Analysis of a Monoclonal Antibody and its Related Fab Fragment in Human Plasma Using Various Bioanalytical Techniques, Including Microfluidic Immunoassay, Triple Quadrupole, and High-Resolution Liquid Chromatography-Tandem Mass Spectrometry Approaches

Comparison of Methods for Quantitative Analysis of a Monoclonal Antibody and its Related Fab Fragment in Human Plasma Using Various Bioanalytical Techniques, Including Microfluidic Immunoassay, Triple Quadrupole, and High-Resolution Liquid Chromatography-Tandem Mass Spectrometry Approaches

Optimization of the Everolimus Intermediate Preparation Process: Impurity Identification Followed by One-Pot Transformation to the Desired Product

An investigation of the everolimus intermediate PG-EVR preparation process resulted in the identification of three impurities PG-D,E,F constantly formed as byproducts in large amounts. Their structures were reliably established based on two-dimensional (2D) NMR and liquid chromatography–high-resolution tandem mass spectrometry (LC–HRMS/MS) data as dialkylated rapamycin derivatives. Finding that one of these impurities PG-E is hydrolyzable to the target everolimus intermediate resulted in optimized technology based on changing the selectivity of byproduct formation to PG-E as the major one followed by its one-pot quantitative transformation to PG-EVR. The yield of the everolimus intermediate increased by 10% under mild conditions, while lower amounts of reagents and solvents were required. The active pharmaceutical ingredient (API) that meets the requirements of the European Pharmacopoeia was prepared from thus obtained intermediate with no need for high-performance liquid chromatography (HPLC) purification, indicating a significant increase in the efficiency of large-scale production using new technology.

Metabolomic Profile and Biological Properties of Sea Lavender (Limonium algarvense Erben) Plants Cultivated with Aquaculture Wastewaters: Implications for Its Use in Herbal Formulations and Food Additives.

Water extracts from sea lavender (Limonium algarvense Erben) plants cultivated in greenhouse conditions and irrigated with freshwater and saline aquaculture effluents were evaluated for metabolomics by liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS/MS), and functional properties by in vitro and ex vivo methods. In vitro antioxidant methods included radical scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), ferric-reducing antioxidant power (FRAP), and copper and iron chelating assets. Flowers’ extracts had the highest compounds’ diversity (flavonoids and its derivatives) and strongest in vitro antioxidant activity. These extracts were further tested for ex vivo antioxidant properties by oxidative haemolysis inhibition (OxHLIA), lipid peroxidation inhibition by thiobarbituric acid reactive substances (TBARS) formation, and anti-melanogenic, anti-tyrosinase, anti-inflammation, and cytotoxicity. Extract from plants irrigated with 300 mM NaCl was the most active towards TBARS (IC50 = 81 µg/mL) and tyrosinase (IC50 = 873 µg/mL). In OxHLIA, the activity was similar for fresh- and saltwater-irrigated plants (300 mM NaCl; IC50 = 136 and 140 µg/mL, respectively). Samples had no anti-inflammatory and anti-melanogenic abilities and were not toxic. Our results suggest that sea lavender cultivated under saline conditions could provide a flavonoid-rich water extract with antioxidant and anti-tyrosinase properties with potential use as a food preservative or as a functional ingredient in herbal supplements.

A Meta-Omics Analysis Unveils the Shift in Microbial Community Structures and Metabolomics Profiles in Mangrove Sediments Treated with a Selective Actinobacterial Isolation Procedure

Mangrove sediment ecosystems in the coastal areas of the Yucatan peninsula are unique environments, influenced by their karstic origin and connection with the world’s largest underground river. The microbial communities residing in these sediments are influenced by the presence of mangrove roots and the trading chemistry for communication between sediment bacteria and plant roots can be targeted for secondary metabolite research. To explore the secondary metabolite production potential of microbial community members in mangrove sediments at the “El Palmar” natural reserve in Sisal, Yucatan, a combined meta-omics approach was applied. The effects of a cultivation medium reported to select for actinomycetes within mangrove sediments’ microbial communities was also analyzed. The metabolome of the microbial communities was analyzed by high-resolution liquid chromatography-tandem mass spectrometry, and molecular networking analysis was used to investigate if known natural products and their variants were present. Metagenomic results suggest that the sediments from “El Palmar” harbor a stable bacterial community independently of their distance from mangrove tree roots. An unexpected decrease in the observed abundance of actinomycetes present in the communities occurred when an antibiotic-amended medium considered to be actinomycete-selective was applied for a 30-day period. However, the use of this antibiotic-amended medium also enhanced production of secondary metabolites within the microbial community present relative to the water control, suggesting the treatment selected for antibiotic-resistant bacteria capable of producing a higher number of secondary metabolites. Secondary metabolite mining of “El Palmar” microbial community metagenomes identified polyketide synthase and non-ribosomal peptide synthetases’ biosynthetic genes in all analyzed metagenomes. The presence of these genes correlated with the annotation of several secondary metabolites from the Global Natural Product Social Molecular Networking database. These results highlight the biotechnological potential of the microbial communities from “El Palmar”, and show the impact selective media had on the composition of communities of actinobacteria.

Vertical Inheritance Facilitates Interspecies Diversification in Biosynthetic Gene Clusters and Specialized Metabolites.

ABSTRACTWhile specialized metabolites are thought to mediate ecological interactions, the evolutionary processes driving chemical diversification, particularly among closely related lineages, remain poorly understood. Here, we examine the evolutionary dynamics governing the distribution of natural product biosynthetic gene clusters (BGCs) among 118 strains representing all nine currently named species of the marine actinobacterial genus Salinispora. While much attention has been given to the role of horizontal gene transfer (HGT) in structuring BGC distributions, we find that vertical descent facilitates interspecies BGC diversification over evolutionary timescales. Moreover, we identified a distinct phylogenetic signal among Salinispora species at both the BGC and metabolite level, indicating that specialized metabolism represents a conserved phylogenetic trait. Using a combination of genomic analyses and liquid chromatography–high-resolution tandem mass spectrometry (LC-MS/MS) targeting nine experimentally characterized BGCs and their small molecule products, we identified gene gain/loss events, constrained interspecies recombination, and other evolutionary processes associated with vertical inheritance as major contributors to BGC diversification. These evolutionary dynamics had direct consequences for the compounds produced, as exemplified by species-level differences in salinosporamide production. Together, our results support the concept that specialized metabolites, and their cognate BGCs, can represent phylogenetically conserved functional traits with chemical diversification proceeding in species-specific patterns over evolutionary time frames.

Improved structural annotation of triterpene metabolites of traditional Chinese medicine in vivo based on quantitative structure-retention relationships combined with characteristic ions: Alismatis Rhizoma as an example

As a fast, sensitive and selective method, liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS) has been used for studying the in vivo metabolism of traditional Chinese medicine (TCM). However, the rapid discovery and characterization of metabolites, especially isomers, remain challenging due to their complexity and low concentration in vivo. This study proposed a strategy to improve the structural annotation of prototypes and metabolites through characteristic ions and a quantitative structure-retention relationship (QSRR) model, and Alismatis Rhizoma (AR) triterpenes were used as an example. This strategy consists of four steps. First, based on an in-house database reported previously, prototypes and metabolites in biosamples were preliminarily identified. Second, the candidate structures of prototype compounds and metabolites were determined by characteristic ions, databases or potential metabolic pathways. Then, a QSRR model was established to predict the retention times of the proposed structure. Finally, the structures of unknown prototypes and metabolites were determined by matching experimental retention times with the predicted values. The QSRR model built by the genetic algorithm-multiple linear regression (GA-MLR) has excellent regression correlation (R2 = 0.9966). Based on this strategy, a total of 118 compounds were identified, including 47 prototypes and 71 metabolites, among which 61 unknown compounds were reasonably characterized. The typical compound identified by this strategy was successfully validated using a triterpene standard. This strategy can improve the annotation confidence of in vivo metabolites of TCM and facilitate further pharmacological research.

LC-HRMS/MS study of the prodrug ciclesonide and its active metabolite desisobutyryl-ciclesonide in plasma after an inhalative administration to horses for doping control purposes.

Ciclesonide (CIC) is the first inhaled highly potent corticosteroid that does not cause any cortisol suppression. It has been developed for the treatment of asthma in human and more recently in equine. CIC is the active compound of Aservo® EquiHaler® (Boehringer Ingelheim Vetmedica GmbH), the pre-filled inhaler generating a medicated mist based on Soft Mist™ technology. This prodrug is rapidly converted to desisobutyryl-ciclesonide (des-CIC), the main pharmacologically active compound. Due to its anti-inflammatory properties, CIC is prohibited for use in horse competitions. To set up an appropriate control, the determination of detection times and screening limits are required. Therefore, a highly sensitive analytical method based on supported liquid extraction (SLE) combined with liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) was developed to detect CIC and its active metabolite des-CIC in plasma. The lower limit of detection of CIC and des-CIC was approximately 1 pg/ml in plasma. After a pilot study conducted on a single horse at the recommended dose (eight actuations twice daily corresponding to 5.5mg/day for the first 5days, followed by 12 actuations once daily corresponding to 4.1mg/day in the last 5days), the same protocol was applied in the main study using six horses. In all horses, CIC and des-CIC levels were less than 5 and 10pg/ml, respectively, at 36 h after the end of the administration. The outcome of this risk assessment study should be useful to draw any recommendations for horse competitions.

Combining OSMAC Approach and Untargeted Metabolomics for the Identification of New Glycolipids with Potent Antiviral Activity Produced by a Marine Rhodococcus.

Natural products of microbial origin have inspired most of the commercial pharmaceuticals, especially those from Actinobacteria. However, the redundancy of molecules in the discovery process represents a serious issue. The untargeted approach, One Strain Many Compounds (OSMAC), is one of the most promising strategies to induce the expression of silent genes, especially when combined with genome mining and advanced metabolomics analysis. In this work, the whole genome of the marine isolate Rhodococcus sp. I2R was sequenced and analyzed by antiSMASH for the identification of biosynthetic gene clusters. The strain was cultivated in 22 different growth media and the generated extracts were subjected to metabolomic analysis and functional screening. Notably, only a single growth condition induced the production of unique compounds, which were partially purified and structurally characterized by liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). This strategy led to identifying a bioactive fraction containing >30 new glycolipids holding unusual functional groups. The active fraction showed a potent antiviral effect against enveloped viruses, such as herpes simplex virus and human coronaviruses, and high antiproliferative activity in PC3 prostate cancer cell line. The identified compounds belong to the biosurfactants class, amphiphilic molecules, which play a crucial role in the biotech and biomedical industry.

In silico prediction, LC-HRMS/MS analysis, and targeted/untargeted data-mining workflow for the profiling of phenylfentanyl in vitro metabolites

Illicit fentanyl and analogues have been involved in many fatalities and cases of intoxication across the United States over the last decade, and are becoming a health concern in Europe. New potent analogues emerge onto the drug market every year to circumvent analytical detection and legislation, and little pharmacological/toxicological data are available when the substances first appear. However, pharmacokinetic data are crucial to determine specific biomarkers of consumption in clinical and forensic settings, considering the low active doses and the rapid metabolism of fentanyl analogues. Phenylfentanyl is a novel analogue that was first detected in seized material in 2017, and little is currently known about this substance and its metabolism.We studied phenylfentanyl metabolic fate using in silico predictions with GLORYx freeware, human hepatocyte incubations, and liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS). We applied a specific targeted/untargeted workflow using data-mining software to allow the rapid and partially automated screening of LC-HRMS/MS raw data. Approximately 90,000 substances were initially individuated after 3-h incubation with hepatocytes, and 115 substances were automatically selected for a manual check by the operators. Finally, 13 metabolites, mostly produced by N-dealkylation, amide hydrolysis, oxidation, and combinations thereof, were identified. We suggest phenylnorfentanyl as the main biological marker of phenylfentanyl use, and we proposed the inclusion of its fragmentation pattern in mzCloud and HighResNPS online libraries. Other major metabolites include N-Phenyl-1-(2-phenylethyl)-4-piperidinamine (4-ANPP), 1-(2-phenylethyl)-4-piperidinol, and other non-specific metabolites. Phase II transformations were infrequent, and the hydrolysis of the biological samples is not required to increase the detection capability of non-conjugated metabolites. The overall workflow is easily adaptable for the metabolite profiling of other novel psychoactive substances.

Novel Insight in Idiopathic Normal Pressure Hydrocephalus (iNPH) Biomarker Discovery in CSF

Idiopathic normal pressure hydrocephalus (iNPH) is a potentially reversible neurological disease, causing motor and cognitive dysfunction and dementia. iNPH and Alzheimer’s disease (AD) share similar molecular characteristics, including amyloid deposition, t-tau and p-tau dysregulation; however, the disease is under-diagnosed and under-treated. The aim was to identify a panel of sphingolipids and proteins in CSF to diagnose iNPH at onset compared to aged subjects with cognitive integrity (C) and AD patients by adopting multiple reaction monitoring mass spectrometry (MRM-MS) for sphingolipid quantitative assessment and advanced high-resolution liquid chromatography–tandem mass spectrometry (LC–MS/MS) for proteomic analysis. The results indicated that iNPH are characterized by an increase in very long chains Cer C22:0, Cer C24:0 and Cer C24:1 and of acute-phase proteins, immunoglobulins and complement component fragments. Proteins involved in synaptic signaling, axogenesis, including BACE1, APP, SEZ6L and SEZ6L2; secretory proteins (CHGA, SCG3 and VGF); glycosylation proteins (POMGNT1 and DAG1); and proteins involved in lipid metabolism (APOH and LCAT) were statistically lower in iNPH. In conclusion, at the disease onset, several factors contribute to maintaining cell homeostasis, and the protective role of very long chains sphingolipids counteract overexpression of amyloidogenic and neurotoxic proteins. Monitoring specific very long chain Cers will improve the early diagnosis and can promote patient follow-up.

Effect of Sevelamer and Nicotinamide on Albumin Carbamylation in Patients with End-Stage Kidney Disease.

Background and ObjectiveIn end-stage kidney disease, high urea levels promote the carbamylation of lysine side chains on a variety of proteins, including albumin. Albumin carbamylation has been identified as a risk factor for mortality and sevelamer led to a decrease in urea levels in dialysis patients. In the present secondary analysis of the NICOREN trial, we investigated the putative impacts of sevelamer and nicotinamide on albumin carbamylation, and the potential correlation between carbamylation and vascular calcifications.MethodsAll possible carbamylation of circulating albumin were screened for with high-resolution liquid chromatography-tandem mass spectrometry. Levels of three carbamylated peptides were then measured as a guide to the extent of albumin carbamylation. Carbamylation was measured at baseline in 55 patients included in the NICOREN trial and 29 patients at 24 weeks of treatment. Calcifications on plain radiographs were quantified as the Kauppila score and the Adragao score.ResultsBaseline albumin carbamylation was present at three different sites in subjects with end-stage kidney disease. At baseline, we observed only a correlation between urea and the KQTA carbamylation site in these patients. Albumin carbamylation levels did not decrease after 24 weeks of treatment with either sevelamer or nicotinamide. Furthermore, the proportion of carbamylated serum albumin was not correlated with vascular calcification scores in this population.ConclusionsOur results confirmed the presence of carbamylated albumin in patients with end-stage kidney disease and demonstrated the presence of carbamylation beyond the LRVP residues. The results also demonstrated the lack of impact of sevelamer or nicotinamide on albumin carbamylation levels. Therapeutic strategies to lower carbamylation load should probably be focused on direct anti-carbamylation processes and/or potentially anti-inflammatory therapies.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40268-021-00350-7.

Ontogenetic Variation in the Mineral, Phytochemical and Yield Attributes of Brassicaceous Microgreens

Microgreens constitute novel gastronomic ingredients that combine visual, kinesthetic and bioactive qualities. The definition of the optimal developmental stage for harvesting microgreens remains fluid. Their superior phytochemical content against mature leaves underpins the current hypothesis of significant changes in compositional profile during the brief interval of ontogeny from the appearance of the first (S1) to the second true leaf (S2). Microgreens of four brassicaceous genotypes (Komatsuna, Mibuna, Mizuna and Pak Choi) grown under controlled conditions and harvested at S1 and S2 were appraised for fresh and dry yield traits. They were further analyzed for macro- and micromineral content using inductively coupled plasma optical emission spectrometry (ICP-OES), carotenoid content using high-performance liquid chromatography with a diode-array detector (HPLC-DAD), volatile organic compounds using solid-phase microextraction followed by gas chromatography-mass spectrometry (SPME-GC/MS), anthocyanins and polyphenols using liquid chromatography-high resolution-tandem mass spectrometry (LC-MS/MS) with Orbitrap technology and for chlorophyll and ascorbate concentrations, well as antioxidant capacity by spectrophotometry. Analysis of compositional profiles revealed genotype as the principal source of variation for all constituents. The response of mineral and phytochemical composition and of antioxidant capacity to the growth stage was limited and largely genotype-dependent. It is, therefore, questionable whether delaying harvest from S1 to S2 would significantly improve the bioactive value of microgreens while the cost-benefit analysis for this decision must be genotype-specific. Finally, the lower-yielding genotypes (Mizuna and Pak Choi) registered higher relative increase in fresh yield between S1 and S2, compared to the faster-growing and higher-yielding genotypes. Although the optimal harvest stage for specific genotypes must be determined considering the increase in yield against reduction in crop turnover, harvesting at S2 seems advisable for the lower-yielding genotypes.

Sponge-associated sp. RM66 metabolome induction with N-acetylglucosamine: Antibacterial, antifungal and anti-trypanosomal activities.

The marine sponge Amphimedon sp., collected from Hurghada (Egypt) was investigated for its sponge-derived actinomycetes diversity. Nineteen actinomycetes were cultivated and phylogenetically identified using 16S rDNA gene sequencing were carried out. The strains belong to genera Kocuria, Dietzia, Micrococcus, Microbacterium and Streptomyces. Many silent biosynthetic genes clusters were investigated using genome sequencing of actinomycete strains and has revealed in particular the genus Streptomyces that has indicated their exceptional capacity for the secondary metabolites production that not observed under classical cultivation conditions. In this study, the effect of N-acetylglucosamine on the metabolome of Streptomyces sp. RM66 was investigated using three actinomycetes media (ISP2, M1 and MA). In total, twelve extracts were produced using solid and liquid fermentation approaches. Liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) data were analysed using metabolomics tools to compare natural product production across all crude extracts. Our study highlighted the elicitation effect of N-acetylglucosamine on the secondary metabolite profiles of Streptomyces sp. RM66. These results highlight the of N-acetylglucosamine application as an elicitor to induce the cryptic metabolites and for increasing the chemical diversity. All the twelve extracts were tested for their antibacterial activity was tested against Staphylococcus aureus NCTC 8325, antifungal activity against Candida albicans 5314 (ATCC 90028) and anti-trypanosomal activity against Trypanosoma brucei brucei. Extract St1 showed the most potent one with activities 2.3, 3.2 and 4.7 ug/ml as antibacterial, antifungal and anti-trypanosomal, respectively.

High-Coverage Screening of Sulfonamide Metabolites in Goat Milk by Magnetic Doped S Graphene Combined with Ultrahigh-Performance Liquid Chromatography-High-Resolution Mass Spectrometry.

Currently, there are more than 1000 varieties of synthetic sulfonamides universally used as antibiotics causing severe results of potential carcinogenicity and drug resistance for human health due to excessive residue of animal-derived food. A facile and novel approach for untargeted screening of sulfonamides (SAs) and metabolites was proposed based on magnetic solid-phase extraction-ultrahigh-performance liquid chromatography-tandem high-resolution mass spectrometry (MSPE-UHPLC-HRMS). Compared with QuEChERS without the clean-up procedure and SPE in terms of matrix effect and absolute recovery, magnetic doped S graphene (S-doping level: 2.82%) synthesized via a solid-state microwave approach and the aggregation wrap mechanism was used as a novel adsorbent for nonspecific extraction of desired analytes by the noncovalent interaction between electron-deficient thiophene sulfur and electron donors such as amino or amide as well as π-π stacking interactions. Combined with variable data-independent acquisition, characteristic fragment-ion filtering (m/z 156.01138 or m/z 108.04439) and assignment of extracted-ion chromatograms of marked fragment ions were successfully utilized to screen the desired analytes and subsequently confirmed with the availability of reference standards. The optimized and validated approach for spiked 26 SAs and 9 metabolites in control goat milk demonstrated satisfactory accuracy (80.1-112.6%) and precision (RSDs < 6.4%) for matrix-matched standard addition. After applying suspect goat milk samples, untargeted SA analytes including sulfanilamide or an N4-acetylsulfamethazine metabolite with concentration ranging from 66.3 to 398.5 ng L-1 were determined in 5 of 45 goat milk samples.