Liquid Chromatography Coupled With High-resolution Mass Spectrometry Research Articles

Implementation of a community-based LC-UV drug checking service: promising preliminary findings on feasibility and validity

BackgroundThe increasing diversity of psychoactive substances on the unregulated drug market poses significant health, psychological, and social risks to people who use drugs (PWUD). To address these risks, various harm reduction (HR) policies have been implemented, including drug checking services (DCS). Many analytical methods are used for DCS. While qualitative methods (e.g., thin layer chromatography, spectroscopy) are easier to implement, they are not as accurate as quantitative methods (e.g., LC-UV, LC-MS). Some HR programmes have implemented high-performance liquid chromatography coupled with UV detection (LC-UV). This article presents the cross-validation of this quantitative method with a reference liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) method.MethodsDrug samples were provided by PWUD to a DCS called DrugLab in Marseille, France. The samples were weighed and prepared through dissolution in methanol, followed by ultrasonic bathing. Samples were analysed onsite using LC-UV analysis. They were then subsequently analysed with the reference LC-HRMS method. The LC-UV instrument in DrugLab was calibrated after being purchased; analysis of standard solutions was routinely performed once a month and after maintenance operations. For the LC-HRMS instrument, calibration and quality control procedures followed European Medicines Agency (EMA) guidelines. Statistical analyses were conducted including Spearman correlation tests using IBM® SPSS® Statistics version 20.ResultsA total of 102 samples representing different product classes and cutting agents were cross-validated. Differences between both analyses methods for each molecule analysed were ≤ 20%, with significant correlations between both methods’ results for most substances. Notably, LC-HRMS provided lower concentration values for cocaine and acetaminophen, whereas it provided higher values for other substances. Correlations were significant for cocaine, ketamine, MDMA, heroin, amphetamine, caffeine, acetaminophen, and levamisole.ConclusionsThis study demonstrates that the results provided by DrugLab were accurate and reliable, making LC-UV an adaptable, stable, and suitable analytical method for simple matrices like drugs in a DCS context. However, this cross validation does not guarantee accuracy over time. A proficiency test project in HR laboratories across France is currently under development in order to address potential drifts in LC-UV accuracy.

Polyphenolic profiling of coffee beverages by Liquid Chromatography-High-Resolution mass Spectrometry for classification and characterization

The importance of monitoring the presence of bioactive compounds as food attributes for sample classification and characterization is increasing. In this study, targeted Liquid Chromatography coupled with High-Resolution Mass Spectrometry (LC-HRMS) was employed to analyze the chemical profile of polyphenolic compounds as the source of information for the characterization and classification of 306 commercial coffee samples. Coffee holds a distinguished position as one of the most widely popular beverages globally but also one of the most easily adulterated. Regrettably, in recent times, instances of coffee adulteration have been on the rise. Consequently, implementing rigorous quality control measures for coffee becomes imperative to guarantee its quality. The results obtained in this work confirm that the proposed chemical profiles serve as excellent descriptors for sample characterization and classification through the implementation of principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA), achieving classification rates higher than 83.3% in PLS-DA validation. Moreover, the proposed LC-HRMS polyphenolic approach was employed to identify and measure adulteration levels in coffee samples using partial least squares (PLS) regression with prediction errors below 7.8%.

Consolidating two-dimensional liquid chromatography–high-resolution tandem mass spectrometry (LC×LC-HRMS/MS) technique for the non-targeted analysis of poly- and perfluorinated substances: A trial on aqueous film-forming foams

To date, poly- and perfluoroalkyl substances (PFAS) represent a real threat for their environmental persistence, wide physicochemical variability, and their potential toxicity. Thus far a large portion of these chemicals remain structurally unknown. These chemicals, therefore, require the implementation of complex non-targeted analysis workflows using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) for their comprehensive detection and monitoring. This approach, even though comprehensive, does not always provide the much-needed analytical resolution for the analysis of complex PFAS mixtures such as fire-fighting aqueous film-forming foams (AFFFs). This study consolidates the advantages of the LC×LC technique hyphenated with high-resolution tandem mass spectrometry (HRMS/MS) for the identification of PFAS in AFFF mixtures. A total of 57 PFAS homolog series (HS) were identified in 3M and Orchidee AFFF mixtures thanks to the (i) high chromatographic peak capacity (n’2D,c ~ 300) and the (i) increased mass domain resolution provided by the “remainder of Kendrick Mass” (RKM) analysis on the HRMS data. Then, we attempted to annotate the PFAS of each HS by exploiting the available reference standards and the FluoroMatch workflow in combination with the RKM defect by different fluorine repeating units, such as CF2, CF2O, and C2F4O. This approach resulted in 12 identified PFAS HS, including compounds belonging to the HS of perfluoroalkyl carboxylic acids (PFACAs), perfluoroalkyl sulfonic acids (PFASAs), (N-pentafluoro(5)sulfide)-perfluoroalkane sulfonates (SF5-PFASAs), N-sulfopropyldimethylammoniopropyl perfluoroalkane sulfonamides (N-SPAmP-FASA), and N-carboxymethyldimethylammoniopropyl perfluoroalkane sulfonamide (N-CMAmP-FASA). The annotated categories of perfluoroalkyl aldehydes and chlorinated PFASAs represent the first record of PFAS HS in the investigated AFFF samples.

Nonclinical Pharmacokinetics Study of OLX702A-075-16, N-Acetylgalactosamine Conjugated Asymmetric Small Interfering RNA (GalNAc-asiRNA).

In this study, the nonclinical pharmacokinetics of OLX702A-075-16, an RNA interference therapeutic currently in development, were investigated. OLX702A-075-16 is a novel N-acetylgalactosamine conjugated asymmetric small-interfering RNA (GalNAc-asiRNA) used for the treatment of an undisclosed liver disease. Its unique 16/21-mer asymmetric structure reduces nonspecific off-target effects without compromising efficacy. We investigated the plasma concentration, tissue distribution, metabolism, and renal excretion of OLX702A-075-16 following a subcutaneous administration in mice and rats. For bioanalysis, high-performance liquid chromatography with fluorescence detection was used. The results showed rapid clearance from plasma (0.5 to 1.5 hours of half-life) and predominant distribution to the liver and/or kidney. Less than 1% of the liver concentration of OLX702A-075-16 was detected in the other tissues. Metabolite profiling using liquid chromatography coupled with high-resolution mass spectrometry revealed that the intact duplex OLX702A-075-16 was the major compound in plasma. The GalNAc moiety was predominantly metabolized from the sense strand in the liver, with the unconjugated sense strand of OLX702A-075-16 accounting for more than 95% of the total exposure in the rat liver. Meanwhile, the antisense strand was metabolized by the sequential loss of nucleotides from the 3'-terminus by exonuclease, with the rat liver samples yielding the most diverse truncated forms of metabolites. Urinary excretion over 96 hours was less than 1% of the administered dose in rats. High plasma protein binding of OLX702A-075-16 likely inhibited its clearance through renal filtration. SIGNIFICANCE STATEMENT: This study presents the first comprehensive characterization of the in vivo pharmacokinetics of GalNAc-asiRNA. The pharmacokinetic insights gained from this research will aid in understanding toxicology and efficacy, optimizing delivery platforms, and improving the predictive power of preclinical species data for human applications.

Prioritization, Identification, and Quantification of Emerging Contaminants in Recycled Textiles Using Non-Targeted and Suspect Screening Workflows by LC-ESI-HRMS.

Recycled textiles are becoming widely available to consumers as manufacturers adopt circular economy principles to reduce the negative impact of garment production. Still, the quality of the source material directly impacts the final product, where the presence of harmful chemicals is of utmost concern. Here, we develop a risk-based suspect and non-targeted screening workflow for the detection, identification, and prioritization of the chemicals present in consumer-based recycled textile products after manufacture and transport. We apply the workflow to characterize 13 recycled textile products from major retail outlets in Sweden. Samples were extracted and analyzed by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). In positive and negative ionization mode, 20,119 LC-HRMS features were detected and screened against persistent, mobile, and toxic (PMT) as well as other textile-related chemicals. Six substances were matched with PMT substances that are regulated in the European Union (EU) with a Level 2/3 confidence. Forty-three substances were confidently matched with textile-related chemicals reported for use in Sweden. For estimating the relative priority score, aquatic toxicity and concentrations were predicted for 7416 features with tandem mass spectra (MS2) and used to rank the non-targeted features. The top 10 substances were evaluated due to elevated environmental risk linked to the recycling process and potential release at end-of-life.

Staphylococcal Enterotoxins: Description and Importance in Food.

Staphylococcus aureus stands out as one of the most virulent pathogens in the genus Staphylococcus. This characteristic is due to its ability to produce a wide variety of staphylococcal enterotoxins (SEs) and exotoxins, which in turn can cause staphylococcal food poisoning (SFP), clinical syndromes such as skin infections, inflammation, pneumonia, and sepsis, in addition to being associated with the development of inflammation in the mammary glands of dairy cattle, which results in chronic mastitis and cell necrosis. SEs are small globular proteins that combine superantigenic and emetic activities; they are resistant to heat, low temperatures, and proteolytic enzymes and are tolerant to a wide pH range. More than 24 SE genes have been well described (SEA-SEE, SEG, SEH, SEI, SEJ, SElK, SElL, SElM, SElN, SElO, SElP, SElQ, SElR, SElS, SElT, SElU, SElV, SElW, SElX, SElY, and SElZ), being a part of different SFP outbreaks, clinical cases, and isolated animal strains. In recent years, new genes (sel26, sel27, sel28, sel31, sel32, and sel33) from SEs have been described, as well as two variants (seh-2p and ses-3p) resulting in a total of thirty-three genes from Ses, including the nine variants that are still in the process of genetic and molecular structure evaluation. SEs are encoded by genes that are located in mobile genetic elements, such as plasmids, prophages, pathogenicity islands, and the enterotoxin gene cluster (egc), and housed in the genomic island of S. aureus. Both classical SEs and SE-like toxins (SEls) share phylogenetic relationships, structure, function, and sequence homology, which are characteristics for the production of new SEs through recombination processes. Due to the epidemiological importance of SEs, their rapid assessment and detection have been crucial for food security and public health; for this reason, different methods of identification of SEs have been developed, such as liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS), molecular methods, and whole-genome sequencing; providing the diagnosis of SEs and a better understanding of the occurrence, spread, and eradication of SEs. This review provides scientific information on the enterotoxins produced by S. aureus, such as structural characteristics, genetic organization, regulatory mechanisms, superantigen activity, mechanisms of action used by SEs at the time of interaction with the immune system, methods of detection of SEs, and recent biocontrol techniques used in food.

Investigating the etiology of Haff disease: Optimization and validation of a sensitive LC-MS/MS method for palytoxins analysis in directly associated freshwater and marine food samples from Brazil

Haff disease typically develops after eating contaminated marine or freshwater species, especially fish. Despite still having an unknown etiology, recent reports have suggested its possible correlation with palytoxins. Therefore, the present work aimed to optimize and perform a validation of a sensitive method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the analysis of palytoxin and some of its analogs, with the main purpose of investigating their presence in marine and freshwater food samples associated with Haff disease in Brazil. The method optimization was performed using a central composite rotatable design and fish samples fortified with the palytoxin standard. Then, the optimized method was validated for different food matrices, including freshwater and marine fish, mollusks, and crustaceans. The sample preparation involved a solid–liquid extraction using methanol and water, solid-phase extraction using Strata-X cartridges, and on-column palytoxin oxidation. The detection of the main oxidized fragments (amino and amide aldehydes) was achieved by LC-MS/MS with electrospray ionization in positive mode, using a C18 column, as well as acetonitrile and water as mobile phases, both acidified with 0.1 % of formic acid. After optimization and validation, the etiological investigation involved the analysis of 16 Brazilian Haff disease-related food samples (in natura and leftover meals) from 2022. The method was demonstrated to be appropriate for quantitative analysis of freshwater and marine species. So far, it has proven to be one of the most sensitive methods related to palytoxin detection (LOD 10 μg/kg), being able to work in a range that includes the provisional ingestion limit (30 μg/kg). Regarding the Haff disease-related samples analysis, there is a strong indication of palytoxin contamination since the amino aldehyde (common fragment for all palytoxins) was detected in 15 of the 16 samples. Selected results were confirmed using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS).

The Potential of Multi-Screening Methods and Omics Technologies to Detect Both Regulated and Emerging Mycotoxins in Different Matrices.

Mycotoxins are well-known secondary metabolites produced by several fungi that grow and occur in different crops during both pre-harvest and post-harvest conditions. The contamination and occurrence of mycotoxins currently represent some of the major issues in the entire agri-food system. The quantification of mycotoxins in different feeds and foodstuffs is extremely difficult because of the low concentration ranges; therefore, both sample collection and preparation are essential to providing accurate detection and reliable quantification. Currently, several analytical methods are available for the detection of mycotoxins in both feed and food products, and liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) represents the most reliable instrumental approach. In particular, the fast development of high-throughput methods has made it possible to screen and analyze, in the same analytical run and with high accuracy, multiple mycotoxins, such as those regulated, masked, or modified, and emerging ones. Therefore, the aim of this review is to provide an overview of the state of the art of mycotoxins occurrence, health-related concerns, and analyses, discussing the need to perform multi-screening approaches combined with omics technologies to simultaneously analyze several mycotoxins in different feed and food matrices. This approach is expected to provide more comprehensive information about the profile and distribution of emerging mycotoxins, thus enhancing the understanding of their co-occurrence and impact on the entire production chain.

Antiviral Activity of Flavonoids from Bauhinia holophylla Leaves against Zika virus

Zika virus (ZIKV) is involved in the etiology of serious nervous system pathologies. Currently, there are no specific and effective vaccines or antiviral drugs to prevent the diseases caused by ZIKV. This study aimed to assess the activity of flavonoids present in crude hydroethanolic extract (CHE) and fractions obtained from B. holophylla leaves against ZIKV. O-glycosylated flavonoids were characterized by high-performance liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS/MS). The cytotoxic concentration and the effective concentration for 50% of the cells (CC50 and EC50, respectively) were determined, and the selectivity index (SI) was calculated. Molecular networks were constructed based on the chemical composition of the samples and global antiviral activity data using the Global Natural Products Social Molecular Networking (GNPS) platform. Protein–ligand docking was performed in the NS2B-NS3 protease, NS3 helicase, and NS5 methyltransferase of the ZIKV. CHE showed greater antiviral activity at a multiplicity of infection (MOI) of 1.0, with an EC50 of 11.93 µg/mL, SI = 13.38, and reduced cytopathic effects. Molecular networks indicated that O-glycosylated flavonoids are responsible for the activity against ZIKV, being quercetin-O-deoxyhexoside more selective and effective. Molecular docking confirmed the inhibitory activity of quercetin-O-deoxyhexoside, which showed an affinity for the tested targets, especially for NS2B-NS3 protease. The results showed that B. holophylla has flavonoids with potential for future therapeutic applications against ZIKV.

The Discovery of a Multidomain Mannanase Containing Dual-Catalytic Domain of the Same Activity: Biochemical Properties and Synergistic Effect.

In recent years, the wide application of mannan has driven the demand for the exploration of mannanase. As one of the main components of hemicellulose, mannan is an important polysaccharide that ruminants need to degrade and utilize, making rumen a rich source of mannanases. In this study, gene mining of mannanases was performed using bioinformatics, and potential dual-catalytic domain mannanases were heterologously expressed to analyze their properties. The hydrolysis pattern and enzymatic products were identified by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). A dual-catalytic domain mannanase Man26/5 with the same function as the substrate was successfully mined from the genome of cattle rumen microbiota. Compared to the single-catalytic domain, its higher thermal stability (≤50 °C) and catalytic efficiency confirm the synergistic effect between the two catalytic domains. It exhibited a unique "crab-like" structure where the CBM located in the middle is responsible for binding, and the catalytic domains at both ends are responsible for cutting. The exploration of its multidomain structure and synergistic patterns could provide a reference for the artificial construction and molecular modification of enzymes.

Metabolomic changes in adults with status epilepticus: Ahuman case-control study.

Status epilepticus (SE) is a life-threatening prolonged epileptic seizure that affects ~40 per 100 000 people yearly worldwide. The persistence of seizures may lead to excitotoxic processes, neuronal loss, and neuroinflammation, resulting in long-term neurocognitive and functional disabilities. A better understanding of the pathophysiological mechanisms underlying SE consequences is crucial for improving SE management and preventing secondary neuronal injury. We conducted a comprehensive untargeted metabolomic analysis, using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS), on plasma and cerebrospinal fluid (CSF) samples from 78 adult patients with SE and 107 control patients without SE, including 29 with CSF for both groups. The metabolomic fingerprints were compared between patients with SE and controls. Metabolites with differences in relative abundances that could not be attributed to treatment or nutrition provided in the intensive care unit were isolated. Enrichment analysis was performed on these metabolites to identify the most affected pathways. We identified 76 metabolites in the plasma and 37 in the CSF that exhibited differential expression in patients with SE compared to controls. The enrichment analysis revealed that metabolic dysregulations in patients with SE affected primarily amino acid metabolism (including glutamate, alanine, tryptophan, glycine, and serine metabolism), pyrimidine metabolism, and lipid homeostasis. Specifically, patients with SE had elevated levels of pyruvate, quinolinic acid, and keto butyric acid levels, along with lower levels of arginine, N-acetylaspartylglutamate (NAAG), tryptophan, uracil, and uridine. The tryptophan kynurenine pathway was identified as the most significantly altered in SE, resulting in the overproduction of quinolinic acid, an N-methyl-d-aspartate (NMDA) receptor agonist with pro-inflammatory properties. This study has identified several pathways that may play pivotal roles in SE consequences, such as the tryptophan kynurenine pathway. These findings offer novel perspectives for the development of neuroprotective therapeutics.

Development of a generic sample preparation method using dispersive liquid-liquid microextraction for the monitoring of leachable compounds in hospital pharmacy-prepared prefilled drug products.

Performant sample preparation is mandatory in any leachable study to clean and preconcentrate analytes within the sample to offer the best possible extraction recovery as well the best precision for any given substance. The aim consists in developing a sample preparation method for hospital pharmacy-prepared drug products such as long-term storage prefilled syringes, vials and IV bags for the screening of leachable compounds. The Quality Control Laboratory of the Pharmacy of the Lausanne University Hospital (Switzerland) has developed a time- and cost-effective, highly sensitive, robust, and fast method using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) for the analysis of 205 plastic additives. An innovative setup, based on postcolumn infusion (PCI) using 2% ammonium hydroxide in methanol was used to boost the signal intensity of the analytes in MS detection. A database for extractable and leachable trace assessment (DELTA) was built to assist in the screening process of 205 plastic packaging-related compounds. The development of the sample preparation was based on 33 plastic additive candidates in different hospital pharmacy compounding solutions, and their extraction recovery rates as well as their relative standard deviation were taken into consideration. In conclusion, the developed DLLME was assigned with ultrasound assistance and triple extraction, which brought about extraction recovery rates between 67% and 92%, a good RSD <10%, and a preconcentration factor of 50×. Therefore, DLLME could be considered suitable for the semiquantitative screening of leachable additives in simple hospital pharmacy-prepared prefilled drug products.

Metabolomics of 3D cell co-culture reveals alterations in energy metabolism at the cross-talk of colorectal cancer-adipocytes.

Colorectal cancer (CRC) is the third most incident and the second most lethal malignant tumor. Despite the recognized association between obesity and CRC, further clarification is necessary regarding the lipids that are overexpressed during the development of CRC. In this scenario, the combination of metabolomics and a three-dimensional (3D) co-culture model involving CRC tumor cells and lipids can enhance the knowledge of energy metabolism modifications at the cross-talk between colorectal cancer and adipocytes. This study aimed to screen potential metabolites in the three dimensional (3D) co-culture of CRC and adipocytes by investigating the metabolome composition of this co-culture released into the extracellular space, which is known as the secretome. Pre-adipocyte cells (3T3-L1), human colon carcinoma (HT-29), and the 3D co-culture (3T3-L1 + HT-29) were cultured for the secretome obtention. Then, ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) was employed to analyze the metabolomics of each secretome. Overall, 3.731 molecules were detected independent of the cell culture. When comparing the three cultures, 105 molecules presented a statistically significant difference in abundance between groups. Among these molecules, 16 were identified, with a particular emphasis on six lipids (PG 20:0, octadecenal, 3-Hydroxytetracosanoyl-CoA, 9,10-dihydroxy-octadecenoic acid, palmitoleic acid, and PA 18:4) and one amino acid derivative (acetylglutamic acid), which presented significant scores during the partial least-squares discriminant analysis (PLS-DA). Although it is too early to determine the possible impact of such molecules in a CRC microenvironment, these results open new avenues for further studies on the energy metabolism at the cross-talk of colorectal cancer adipocytes.

Distinct metabolic profiles in ocular hypertensive individuals responding

Aims/Purpose: To determine the effect of hypoxia on the plasma metabolic signature of patients with normal‐tension glaucoma (NTG) and ocular hypertension (OHT), compared to healthy controls.Methods: Metabolomic profiles of plasma from patients with NTG (n = 10) and OHT (n = 10) were compared to age‐matched controls (n = 10) to investigate the signature of hypoxia. Participants underwent two hours of exposure to normobaric hypoxia, followed by a 30‐min recovery period in normobaric normoxia. Blood samples were collected at baseline, during hypoxia, and recovery. Plasma samples were analysed using a non‐targeted metabolomics approach based on liquid chromatography coupled with high‐resolution mass spectrometry (LC‐HRMS). Multivariate analyses were conducted using principal component analysis (PCA) and orthogonal partial least squares‐discriminant analysis (OPLS‐DA), as well as univariate analysis with Wilcoxon signed‐rank test and false discovery rate (FDR) correction.Results: Overall, controls, OHT, and NTG patients had similar regulatory patterns. Only few metabolites involved in fatty acid biosynthesis, ketone body metabolism and kynurenine pathway were affected in OHT patients exposed to hypoxia, whereas metabolites related to energy production were not modified in NTG and OHT patients. No differences were found when comparing them pairwise at baseline, or during hypoxia and recovery.Conclusions: OHT patients have altered regulation of ketogenesis and kynurenine pathway, that may explain why they do not suffer from degeneration of the optic nerve. The metabolomes of NTG and OHT patients are regulated differently from controls, and omit to regulate metabolites that are important for energy production. This can potentially contribute to neurodegeneration and ultimately cell death in retinal ganglion cells.

Distinct Metabolic Profiles of Ocular Hypertensives in Response to Hypoxia.

Glaucoma is a neurodegenerative disease that affects the retinal ganglion cells (RGCs). The main risk factor is elevated intraocular pressure (IOP), but the actual cause of the disease remains unknown. Emerging evidence indicates that metabolic dysfunction plays a central role. The aim of the current study was to determine and compare the effect of universal hypoxia on the metabolomic signature in plasma samples from healthy controls (n = 10), patients with normal-tension glaucoma (NTG, n = 10), and ocular hypertension (OHT, n = 10). By subjecting humans to universal hypoxia, we aim to mimic a state in which the mitochondria in the body are universally stressed. Participants were exposed to normobaric hypoxia for two hours, followed by a 30 min recovery period in normobaric normoxia. Blood samples were collected at baseline, during hypoxia, and in recovery. Plasma samples were analyzed using a non-targeted metabolomics approach based on liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). Multivariate analyses were conducted using principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA), and univariate analysis using the Wilcoxon signed-rank test and false discovery rate (FDR) correction. Unique metabolites involved in fatty acid biosynthesis and ketone body metabolism were upregulated, while metabolites of the kynurenine pathway were downregulated in OHT patients exposed to universal hypoxia. Differential affection of metabolic pathways may explain why patients with OHT initially do not suffer or are more resilient from optic nerve degeneration. The metabolomes of NTG and OHT patients are regulated differently from control subjects and show dysregulation of metabolites important for energy production. These dysregulated processes may potentially contribute to the elevation of IOP and, ultimately, cell death of the RGCs.

Targeting antimalarial metabolites from the actinomycetes associated with the Red Sea sponge Callyspongia siphonella using a metabolomic method

Malaria is a persistent illness that is still a public health issue. On the other hand, marine organisms are considered a rich source of anti‑infective drugs and other medically significant compounds. Herein, we reported the isolation of the actinomycete associated with the Red Sea sponge Callyspongia siphonella. Using "one strain many compounds" (OSMAC) approach, a suitable strain was identified and then sub-cultured in three different media (M1, ISP2 and OLIGO). The extracts were evaluated for their in-vitro antimalarial activity against Plasmodium falciparum strain and subsequently analyzed by Liquid chromatography coupled with high-resolution mass spectrometry (LC-HR-MS). In addition, MetaboAnalyst 5.0 was used to statistically analyze the LC-MS data. Finally, Molecular docking was carried out for the dereplicated metabolites against lysyl-tRNA synthetase (PfKRS1). The phylogenetic study of the 16S rRNA sequence of the actinomycete isolate revealed its affiliation to Streptomyces genus. Antimalarial screening revealed that ISP2 media is the most active against Plasmodium falciparum strain. Based on LC-HR-MS based metabolomics and multivariate analyses, the static cultures of the media, ISP2 (ISP2-S) and M1 (M1-S), are the optimal media for metabolites production. OPLS-DA suggested that quinone derivatives are abundant in the extracts with the highest antimalarial activity. Fifteen compounds were identified where eight of these metabolites were correlated to the observed antimalarial activity of the active extracts. According to molecular docking experiments, saframycin Y3 and juglomycin E showed the greatest binding energy scores (-6.2 and -5.13) to lysyl-tRNA synthetase (PfKRS1), respectively. Using metabolomics and molecular docking investigation, the quinones, saframycin Y3 (5) and juglomycin E (1) were identified as promising antimalarial therapeutic candidates. Our approach can be used as a first evaluation stage in natural product drug development, facilitating the separation of chosen metabolites, particularly biologically active ones.

Hunting the missing fluorine in aqueous film-forming foams containing per- and polyfluoroalkyl substances

Since aqueous film-forming foams (AFFFs) are major sources of per- and polyfluoroalkyl substances (PFAS), understanding the quantity and type of PFAS present in AFFFs is crucial for assessing environmental risk and remediation. We characterized 25 foams from Canada and Europe, including two non-AFFFs and two fluorine-free AFFFs. We used liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) to identify novel PFAS, as well as total oxidizable precursor assays (TOP) and total organofluorine (TOF) measurements for comparison. LC-HRMS showed that the two non-AFFF foams and two PFAS-free AFFFs contained little or no PFAS, confirmed by TOF measurement using combustion ion chromatography (CIC). The PFAS-containing AFFFs, however, spanned a wide concentration range of TOF (2200–45,000 mg F/L) and contained 22 new classes of polyfluoroalkyl substances not previously reported. As a result of identifying new compounds, LC-HRMS was fully able to capture the oxidizable precursors determined by TOP assay in all tested fluorotelomer (FT) AFFFs, while unknown compounds still constituted a significant fraction (19–53 mol%) in most electrochemical fluorination (ECF) AFFFs. A fluorine mass balance was achieved by comparing the amounts of compounds identified by LC-HRMS with those detected by CIC, although LC-HRMS overestimated TOF with a recovery of 127 ± 36%.

LC-HRMS analysis of 13 classes of pharmaceutical substances in food supplements

ABSTRACT Food supplements should not contain substances considered unsafe or pose a health risk to consumers. In recent years illegal adulterants have been found in various functional foods without notification of their presence or amount in the labelling. In this study, a validated method was developed and applied as a screening method to detect 124 forbidden substances belonging to 13 classes of compounds in food supplements. Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) and a simple and rapid extraction protocol was applied to 110 food supplements collected from the internet market or during official controls in Italy. The percentage of non-compliant samples was 4.5%, relatively high compared with the official control results for these substances usually obtained on other food matrices. The results suggested the need to strengthen controls in this field to detect food supplement adulteration, which represents a potential health risk for the consumer.

Development of a candidate reference measurement procedure by ID-LC-MS/MS for total tau protein measurement in cerebrospinal fluid (CSF).

In clinical pratice, tau protein measurement generally relies on immunoassays (IAs), whose major drawback is the lack of results comparability due to differences in selectivity and/or calibration. This underlines the importance of establishing a traceability chain for total tau (t-tau) measurements. The objective of this work is to develop a higher order candidate reference measurement procedure (RMP) for the absolute quantification of t-tau in cerebrospinal fluid (CSF). To calibrate the candidate RMP and establish metrological traceability to the SI units, a primary calibrator consisting in a highly purified recombinant protein was sourced. Its purity was evaluated by liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) and the protein mass fraction in solution was certified by amino acid analysis (AAA). An isotopically-labelled homologue was obtained to develop a candidate RMP by isotope dilution mass spectrometry (IDMS) for t-tau absolute quantification in CSF. Calibration blends and quality control (QC) materials were gravimetrically prepared and subjected to the same preparation workflow as CSF samples, followed by LC-HRMS analysis in Parallel Reaction Monitoring (PRM) mode. A primary calibrator has been developed and an IDMS candidate RMP has been validated for CSF t-tau. The candidate RMP was used to certify t-tau concentration in three pools of CSF (low, medium, high). The candidate RMP will pave the road towards global standardization of CSF t-tau measurements. Together with commutable Certified Reference Materials (CRMs), it will allow evaluating and improving the accuracy and comparability of results provided by IAs.

The Fate of Sulfur Radical Cation of N-Acetyl-Methionine: Deprotonation vs. Decarboxylation

In the present study, we investigated the photooxidation of the biomimetic model of C-terminal methionine, N-Acetyl-Methionine (N-Ac-Met), sensitized by a 3-Carboxybenzophenone (3CB) excited triplet in neutral and basic aqueous solutions. The short-lived transient species that formed in the reaction were identified and quantified by laser flash photolysis and the final stable products were analyzed using liquid chromatography coupled with high-resolution mass spectrometry (LC-MS) and tandem mass spectrometry (MSMS). Based on these complementary methods, it was possible to calculate the quantum yields of both competing reactions, and the deprotonation was found to be favored over decarboxylation (for neutral pH: ϕ-H = 0.23 vs. ϕ-CO2 = 0.09, for basic pH: ϕ-H = 0.23 vs. ϕ-CO2 = 0.05). Findings on such a model system, which can possibly mimic the complex protein environment, are important in understanding complicated biological systems, for example, the studied compound, N-Ac-Met, can, to some extent, mimic the methionine in the C-terminal domain of β-amyloid, which is thought to be connected with the pathogenesis of Alzheimer’s disease.