High-resolution Mass Spectrometry Research Articles

Synthesis of an Insulated Oligo(phenylene ethynylene) Dimer Through Cyclodextrin-Based [c2]Daisy Chain Rotaxane

Oligo(phenylene ethynylene)s (OPEs) are π-conjugated systems with promising optical, bioactive, and electrical properties, making them valuable candidates for molecular electronics and biosensors. Controlling the arrangement and orientation of π-conjugated systems is crucial in developing molecular devices. Recently, we developed insulated diarylacetylene dimers using a [c2]daisy chain rotaxane strategy, which brings two cores into close proximity without covalent bonding and shields them with permethylated α-cyclodextrins. Here, we synthesized an insulated OPE dimer using a similar rotaxane strategy to investigate its optical properties. The rotaxane structure and optical properties were evaluated using nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization high-resolution mass spectrometry (ESI-HRMS), and absorption and fluorescence spectroscopy. This study is expected to contribute to the development of optical and electronic materials utilizing OPEs.

Hydroxyl radical oxidation of chemical contaminants on indoor surfaces and dust

Humans are widely exposed to semivolatile organic contaminants in indoor environments. Many contaminants have long lifetimes following partitioning to the large surface reservoirs present indoors, which leads to long exposure times to gas-phase oxidants and multiphase chemistry. Studies have shown selective multiphase oxidation of organics on indoor surfaces, but the presence of hydroxyl radicals with nonselective reactivity and evidence of multiphase OH radical reactivity toward common indoor contaminants indicates that there may be additional unknown transformation chemistry indoors. We screened genuine indoor samples for 60 OH radical oxidation products of the common plasticizer and endocrine-disrupting contaminant bis(2-ethylhexyl) phthalate (DEHP) identified in laboratory experiments using nontargeted high-resolution mass spectrometry. At least 30 and 10 of these products are observed in indoor dust and DEHP films exposed to ambient indoor conditions, respectively, indicating that multiphase OH reactions occur indoors. Using the PROTEX model and a multimedia indoor chemical fate model, we demonstrate that these products have long indoor lifetimes and cause a higher potential for human exposure than DEHP. Some of these products are more active endocrine disruptors than DEHP itself, but most have unknown toxicities. Coexposure to all oxidation products will likely have an additive effect, leading to higher human health risks from indoor organic contaminants than previously thought. Due to the nonselective reactivity of OH radicals, it is likely that most indoor contaminants follow similar chemistry, and further study is needed to understand the prevalence and human health implications of such multiphase chemistry.

Malformin C preferentially kills glioblastoma stem-like cells via concerted induction of proteotoxic stress and autophagic flux blockade.

Glioblastoma is a highly aggressive brain tumor for which there is no cure. The dire prognosis of this disease is largely attributable to a high level of heterogeneity, including the presence of a subpopulation of tumor-initiating glioblastoma stem-like cells (GSCs), which are refractory to chemo- and radiotherapy. Here, in an unbiased marine-derived fungal extract screen, together with bioguided dereplication based on high-resolution mass spectrometry, we identified malformin C to preferentially induce cell death in patient-derived GSCs and explore the potential of this cyclic peptide as a therapeutic agent for glioblastoma. Malformin C significantly reduced tumor growth in an in vivo xenograft model of glioblastoma. Using transcriptomics and chemoproteomics, we found that malformin C binds to many proteins, leading to their aggregation, and rapidly induces the unfolded protein response, including autophagy, in GSCs. Crucially, chemical inhibition of translation using cycloheximide rescued malformin C-induced cell death in GSCs, demonstrating that the proteotoxic effect of the compound is necessary for its cytotoxicity. At the same time, malformin C appears to accumulate in lysosomes, disrupting autophagic flux, and driving cells to death. Supporting this, malformin C synergizes with chloroquine, an inhibitor of autophagy. Strikingly, we observed that autophagic flux is differentially regulated in GSCs compared with normal astrocytes. The sensitivity of GSCs to malformin C highlights the relevance of proteostasis and autophagy as a therapeutic vulnerability in glioblastoma.

Characterization of Trivalently Crosslinked C-Terminal Telopeptide of Type I Collagen (CTX) Species in Human Plasma and Serum Using High-Resolution Mass Spectrometry.

With an aging population, the increased interest in the monitoring of skeletal diseases such as osteoporosis led to significant progress in the discovery and measurement of bone turnover biomarkers since the 2000s. Multiple markers derived from type I collagen, such as CTX, NTX, PINP, and ICTP, have been developed. Extensive efforts have been devoted to characterizing these molecules; however, their complex crosslinked structures have posed significant analytical challenges, and to date, these biomarkers remain poorly characterized. Previous attempts at characterization involved gel-based separation methods and MALDI-TOF analysis on collagen peptides directly extracted from bone. However, using bone powder, which is rich in collagen, does not represent the true structure of the peptides in the biofluids as it was cleaved. In this study, our goal was to characterize plasma and serum CTX for subsequent LC-MS/MS method development. We extracted and characterized type I collagen peptides directly from human plasma and serum using a proteomics workflow that integrates preparative LC, affinity chromatography, and HR-MS. Subsequently, we successfully identified numerous CTX species, providing valuable insights into the characterization of these crucial biomarkers.

Elucidation of endogenous and exogenous chemicals in maternal serum using high-resolution mass spectrometry

The increasing exposure to environmental chemicals calls for comprehensive non-targeted analysis to detect unrecognized substances in human samples. We examined human serum samples to classify compounds as endogenous or exogenous using public databases and to explore the relationships between exposure markers and metabolic patterns. Serum samples from 84 pregnant women at 32 weeks gestation were analyzed using LC-QToFMS. Using the PubChemLite for Exposomics database, we annotated and classified 106 compounds (51 endogenous, 55 exogenous). The compound patterns were analyzed using three dimensional reduction methods: Principal Component Analysis (PCA), regularized Generalized Canonical Correlation Analysis (rGCCA), and Uniform Manifold Approximation and Projection (UMAP). OPTICS clustering applied to these methods revealed two distinct clusters, with 89 % of significant compounds overlapping between clusters. The detected exogenous compounds included dietary substances, phthalates, nitrogenous compounds, and parabens. Pathway enrichment analysis showed that chemical exposure was linked to changes in amino acid metabolism, protein and mineral transport, and energy metabolism. While we found associations between exposure and metabolite changes, we could not establish causality. Our approach of analyzing both exogenous and endogenous chemicals from the same dataset using PubChemLite database presents a new method for exposome research, despite limitations in sample size and peak annotation accuracy. These findings contribute to understanding multiple chemical exposures and their metabolic effects in human biomonitoring.

Semi-Preparation and X-ray Single-Crystal Structures of Sophocarpine-Based Isoxazoline Derivatives and Their Pesticidal Effects and Toxicology Study.

Recently, research and development of novel pesticides from natural plant products have received much attention. To accelerate the application of sophocarpine as the agrochemical candidate, a series of novel sophocarpine-based isoxazoline derivatives were prepared by the 1,3-dipolar [2 + 3] cycloaddition reaction of sophocarpine with different chloroximes. Their structures were well characterized by high-resolution mass spectra, infrared spectra, and proton/carbon-13 nuclear magnetic resonance spectra. Eight steric configurations of compounds 5a, 5e', 5f, 5g, 5h, 5i, 5r, and 5u' were further determined by X-ray single-crystallography. Against Aphis citricola Van der Goot, compounds 5n (LD50: 0.032 μg/nymph) and 5o (LD50: 0.024 μg/nymph) exhibited greater than 3.7- and 4.9-fold potent aphicidal activity compared to sophocarpine (LD50: 0.118 μg/nymph). Against Tetranychus cinnabarinus Boisduval, derivative 5g displayed the most promising acaricidal activity with the LC50 value of 0.247 mg/mL, which was 14.2-fold that of sophocarpine. Compounds 5d and 5g also exhibited good control efficacy against T. cinnabarinus. Scanning electron microscopy images indicated that compound 5g can destroy the mite cuticle layer. These results will provide the foundation for the structural modification and use of sophocarpine derivatives as agrochemicals in the future.

Anti-pathogenic and probiotic-friendly effect of benzyloxy pyridine derivative impregnated into bacterial cellulose matrix

Bacterial cellulose (BC) has emerged as a promising biomaterial for biomedical applications due to its unique properties. In this study, firstly, the potential of BC as a carrier material for the originally synthesized benzyloxy pyridine derivative (BeOxP), and also an antimicrobial and probiotic bacteria-friendly (on Lactiplantibacillus plantarum 299v (LP299V®) (DSM 9843)) effect of BC- BeOxP was investigated. The BC- BeOxP composite material was developed using a simple and efficient method, ensuring the uniform dispersion of BeOxP within the BC matrix. Characterization of BeOxP has been carried out with High-Resolution Mass Spectroscopy (HRMS) and Nuclear Magnetic Resonance (NMR). Additionally, BC- BeOxP was characterized by Thermogravimetric analysis (TGA), Differential thermal analysis (DTA), Scanning Electron Microscopy (SEM), Texture analysis, and Fourier-transform infrared spectroscopy (FTIR), confirming the successful integration of BeOxP without compromising the structural integrity of BC. The mechanical properties of the BC- BeOxP composites were also evaluated to ensure their suitability for drug-carrying or food-packaging applications. Antimicrobial activity of the BC- BeOxP was analyzed against Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 while the probiotic-friendly effect was tested on Lactiplantibacillus plantarum 299v (LP299V®) (DSM 9843). The findings open avenues for further research of BC-based drug carriers or packaging materials for advanced biomedical applications.

Non-target screening analysis of hazardous noxious substances using gas chromatography-quadrupole time-of-flight mass spectrometry

The forensic investigation of hazardous noxious substances (HNS) is paramount for an effective response to chemical spill emergencies and other accidents. Analyzing unknown emergency samples poses a challenge due to the limited availability of background information, making the selection of appropriate sample preparation and analytical methodologies difficult. The utilization of high-resolution mass spectrometers (HRMS) in screening both target and non-target substances proves instrumental in revealing hazardous substances that may be overlooked alongside the intended analytes. In this study, a gas chromatography-quadruple time-of-flight mass spectrometer (GC-QTOF-MS) was employed to identify numerous organic compounds in an indoor dust sample. The compounds detected encompassed normal alkanes, fatty acids (saturated and unsaturated), alcohols, phenols, sterols, drugs, polycyclic aromatic hydrocarbons (PAHs), pesticides, flame retardants (such as polybrominated biphenyl ethers, PBDEs), plasticizers (such as phthalates and phosphates), among others. Notably, concentrations of n-alkanes, fatty acids, and phthalates were relatively high, while PAHs and pesticides were present at trace levels. The application of GC-QTOF-MS provides a swift and confirmative approach for analyzing target, suspect, and non-target compounds in both routine and emergency scenarios. This methodology proves invaluable in enhancing our capability to comprehensively assess and address chemical incidents, ensuring a more thorough and accurate response.

Development and application of a dual isotopic labeling method for enhanced detection and quantification of stimulants in urine samples using high-resolution mass spectrometry.

Given the critical nature of anti-doping efforts, the detection of stimulant substances is shifting from accurate qualitative analysis to precise quantitative analysis. Additionally, the use of liquid chromatography-high-resolution mass spectrometry (LC-HRMS) in detecting stimulants is becoming more widespread. However, the lack of isotope-labeled internal standards is causing increasing issues with quantitative accuracy. Furthermore, challenges such as the mass spectrometric response of small molecules and the separation of isomers present additional difficulties. We have developed a quantitative method for stimulant substances containing amine or phenol hydroxyl groups, using a dual-label derivatization system. This method offers a new perspective for analyzing and detecting low molecular weight substances, isomers, or those with poor LC-MS response, and proposes a solution to the problem of missing isotope-labeled internal standards. Methodological validation has shown that this approach has promising application potential.

Synthesis of novel N-substituted tetrabromophthalic as corrosion inhibitor and its inhibition of microbial influenced corrosion in cooling water system

This study investigates the efficacy of newly synthesized inhibitor with a dual function of corrosion inhibition and biocide for control of microbial influenced corrosion (MIC) in carbon steel API 5LX in the cooling tower water (CTW) environment. Four types of N-substituted tetrabromophthalic inhibitor (N-TBI) were synthesized, and the structural characterization was performed via proton nuclear magnetic resonance spectroscopy, thermogravimetric analysis and high-resolution mass spectrometry. These studies revealed the distinctive optical, thermal, and dielectric properties of the synthesized inhibitors. The corrosion inhibition efficiency has been evaluated by the weight loss (WL) analysis and electrochemical measurements (ECM) and biofilm assay. Biofilm assays and WL showed that inhibitor II exhibited the highest inhibition efficiency 74% and 79% respectively than others. Further ECM showed that the higher charge transfer resistance and the lower corrosion current, suggesting a protective film formed on the metal surface which was due to the adsorption of the N-TBI. Fourier transform infrared spectroscopy confirmed the adsorption of the N-TBI as C–O stretching and C–H bending with the Fe complex. X-ray diffractometer revealed that the presence of inhibitors in the corrosion product (Fe3O4, Fe2O3, FeH2O2, FeS) were highly reduced than the control system. Overall, this study highlighted the potential application of N-TBI with dual function of corrosion inhibition and biocide to control the MIC for carbon steel API 5LX used in the CTW environment.

Nontargeted urine metabolomic analysis of acute intermittent porphyria reveals novel interactions between bile acids and heme metabolism: New promising biomarkers for the long-term management of patients.

Acute intermittent porphyria is an inherited error of heme synthesis. The underlying pathophysiology, involving mainly hepatic heme synthesis, is poorly understood despite its occurrence, and the severity of acute porphyria attack is still difficult to control. A better understanding of the interactions between heme synthesis and global metabolism would improve the management of AIP patients. An untargeted metabolomic analysis was performed on the urine of 114 patients with overt AIP and asymptomatic carriers using liquid chromatography coupled to high-resolution mass spectrometry. The collected data were analyzed by combining univariate and multivariate analyses. A total of 239 metabolites were annotated in urine samples by matching chromatographic and mass spectral characteristics with those from our chemical library. Twenty-six metabolites, including porphyrin precursors, intermediates of tryptophan or glycine metabolism and, unexpectedly, bile acids, showed significant concentration differences between the phenotypic groups. Dysregulation of bile acid metabolism was confirmed by targeted quantitative analysis, which revealed an imbalance in favor of hydrophobic bile acids associated with changes in conjugation, which was more pronounced in the severe phenotype. Using a random forest model, the cholic acid/chenodeoxycholic acid ratio enables the differential classification of severe patients from other patients with a diagnostic accuracy of 84%. The analysis of urine samples revealed significant modifications in the metabolome of AIP patients. Alteration in bile acids provides new insights into the pathophysiology of chronic complications, such as primary liver cancer, while also providing new biomarker candidates for predicting the most severe phenotypes.

Improved quality control of allergen products: Assessing the molecular allergen composition by mass spectrometry.

Natural allergen sources contain a variety of allergens, against which allergic subjects have developed individual sensitization profiles. Ideal allergen products for skin prick testing (SPT) and allergen immunotherapy (AIT) should contain the complete set of allergens of the respective allergen sources to cover all sensitization profiles. However, commercially available allergen products were shown to vary regarding their allergen composition. The qualitative allergen composition of different SPT and AIT products produced from pollen of grasses, birch, mugwort and from house dust mites was assessed by a consistent high-resolution liquid chromatography-coupled tandem mass spectrometry method (LC-MS/MS). All major, mid-tier and most minor allergens were detected in each of the investigated three batches of SPT and AIT products, demonstrating the completeness of the allergen composition and a high degree of batch-to-batch consistency. This is the first study using a single consistent high-resolution LC-MS/MS method to provide solid data on the qualitative allergen composition of SPT and AIT products manufactured from various common allergen sources. The applied method showed high reliability in qualitative batch-to-batch consistency testing and can be performed fast and with high throughput. High-resolution LC-MS/MS is applicable for process development and quality control to ensure market availability of allergen products corresponding to the composition of the respective natural allergen sources.

Discovery and Heterologous Expression of Trilenodin, an Antimicrobial Lasso Peptide with a Unique Tri-Isoleucine Motif.

Lasso peptides are an increasingly relevant class of peptide natural products with diverse biological activities, intriguing physical properties, and unique chemical structures. Most characterized lasso peptides have been from Actinobacteria and Proteobacteria, despite bioinformatic analyses suggesting that other bacterial taxa, particularly those from Firmicutes, are rich in biosynthetic gene clusters (BGCs) encoding lasso peptides. Herein, we report the bioinformatic identification of a lasso peptide BGC from Paenibacillus taiwanensis DSM18679 which we termed pats. We used a bioinformatics-guided isolation approach and high-resolution tandem mass spectrometry (HRMS/MS) to isolate and subsequently characterize a new lasso peptide produced from the pats BGC, which we named trilenodin, after the tri-isoleucine motif present in its primary sequence. This tri-isoleucine motif is unique among currently characterized lasso peptides. We confirmed the connection between the pats BGC and trilenodin production by establishing the first Bacillus subtilis 168-based heterologous expression system for expressing Firmicutes lasso peptides. We finally determined that trilenodin exhibits potent antimicrobial activity against B. subtilis and Klebsiella pneumoniae, making trilenodin the first characterized biologically active lasso peptide from Firmicutes. Collectively, we demonstrate that bacteria from Firmicutes can serve as high-potential sources of chemically and biologically diverse lasso peptides.

Profiling of urinary extracellular vesicle protein signatures from patients with cribriform and intraductal prostate carcinoma in a cross-sectional study

Prognostic tests and treatment approaches for optimized clinical care of prostatic neoplasms are an unmet need. Prostate cancer (PCa) and derived extracellular vesicles (EVs) proteome changes occur during initiation and progression of the disease. PCa tissue proteome has been previously characterized, but screening of tissue samples constitutes an invasive procedure. Consequently, we focused this study on liquid biopsies, such as urine samples. More specifically, urinary small extracellular vesicle and particles proteome profiles of 100 subjects were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC–MS/MS). We identified 171 proteins that were differentially expressed between intraductal prostate cancer/cribriform (IDC/Crib) and non-IDC/non-Crib after correction for multiple testing. However, the strong correlation between IDC/Crib and Gleason Grade complicates the disentanglement of the underlying factors driving this association. Nevertheless, even after accounting for multiple testing and adjusting for ISUP (International Society of Urological Pathology) grading, two proteins continued to exhibit significant differential expression between IDC/Crib and non-IDC/non-Crib. Functional enrichment analysis based on cancer hallmark proteins disclosed a clear pattern of androgen response down-regulation in urinary EVs from IDC/Crib compared to non-IDC/non-Crib. Interestingly, proteome differences between IDC and cribriform were more subtle, suggesting high proteome heterogeneity. Overall, the urinary EV proteome reflected partly the prostate pathology.

Characterization of Endophytic Streptomyces rhizosphaericola Ahn75 and Its Potential for Biocontrol against Rice Blast.

Plant endophyte Streptomyces are excellent candidates as biocontrol agents against the rice blast fungus, Magnaporthe oryzae. In this study, a novel strain Ahn75 with antifungal activity was isolated from healthy rice stem and identified as Streptomyces rhizosphaericola by phenotypic characterization and phylogenetic analysis based on 16S rRNA, multilocus and genome sequences. Inhibition test using culture filtrate showed that Ahn75 could effectively suppress M. oryzae, with mycelia growth inhibition rate of 80.88% and spore germination inhibition rate of 78.26%. Genome sequence analysis of strain Ahn75 showed 40 gene clusters of secondary metabolites and several genes related to plant growth promotion were predicted in the genome of Ahn75. Several antimicrobial compounds including valinomycin, tetrabutylammonium, and benzalkonium chloride, were also detected in the antifungal fraction from Ahn75 culture filtrate by liquid chromatography and high resolution mass spectrometry. Meanwhile, strain Ahn75 demonstrates UV tolerance under UV irradiation for 60 min, pH tolerance between pH6 and pH9, and a high halotolerance in 7% (w/v) of NaCl. Greenhouse experiments indicated that Ahn75 is able to colonize rice stems, roots, and leaves, which help rice to reduce the rice leaf blast incidence by 59.76%. All these findings suggest that strain Ahn75 could be a potential biocontrol agent for rice blast.

Introducing Molecular Hypernetworks for Discovery in Multidimensional Metabolomics Data.

Orthogonal separations of data from high-resolution mass spectrometry can provide insight into sample composition and address challenges of complete annotation of molecules in untargeted metabolomics. "Molecular networks" (MNs), as used in the Global Natural Products Social Molecular Networking platform, are a prominent strategy for exploring and visualizing molecular relationships and improving annotation. MNs are mathematical graphs showing the relationships between measured multidimensional data features. MNs also show promise for using network science algorithms to automatically identify targets for annotation candidates and to dereplicate features associated with a single molecular identity. This paper introduces "molecular hypernetworks" (MHNs) as more complex MN models able to natively represent multiway relationships among observations. Compared to MNs, MHNs can more parsimoniously represent the inherent complexity present among groups of observations, initially supporting improved exploratory data analysis and visualization. MHNs also promise to increase confidence in annotation propagation, for both human and analytical processing. We first illustrate MHNs with simple examples, and build them from liquid chromatography- and ion mobility spectrometry-separated MS data. We then describe a method to construct MHNs directly from existing MNs as their "clique reconstructions", demonstrating their utility by comparing examples of previously published graph-based MNs to their respective MHNs.

Simultaneous analysis of depurinated nucleic acid stem-loop and free adenine for ricin toxicity assay by hydrophilic interaction liquid chromatography-high-resolution mass spectrometry (HILIC-HRMS).

A simple, accurate method for measuring ricin activity was developed by detecting depurinated nucleic acid stem-loops and adenine using a commercially available hydrophilic interaction liquid chromatography (HILIC) column and a quadrupole-Orbitrap tandem mass spectrometer. Ricin in beverages was isolated using magnetic beads conjugated with ricin B-chain antibodies, and then incubated with a 14 mer RNA or a 12 mer RNA/DNA chimera, in which adenosine at the depurination site of RNA was replaced by deoxyadenosine. The adenine and depurinated nucleic acids were separated by HILIC and both analytes were detected by high-resolution mass spectrometry. The depurinated RNA was detectable at concentrations as low as 100 pM (6.5 μg mL-1) in orange juice and coffee, and 10 pM (0.65 μg mL-1) in milk and sake after incubation with the RNA substrate for 4 h. Free adenine was detectable at 10 pM in all matrices, although free adenine was also detected in all blanks and could not be distinguished from the coffee and orange juice blanks at 10 pM. When using the chimera as the substrate, the depurinated chimera and adenine were detected up to concentrations of 10 pM as larger peaks. However, since the depurinated chimera and adenine were also detected in blanks, careful judgment was needed to determine whether they were active. Following the assay, the captured ricin could be analyzed by enzymatic digestion and nano liquid chromatography-high-resolution mass spectrometry. The ricin A chain-specific T7A peptide was detectable at 10 pM for sake and at 100 pM for milk, orange juice, and coffee. Using the present method, a toxicity assay and qualitative analysis of ricin were feasible with a 0.2 mL beverage sample.

Emerging pollutants: Risks in salmon fish migration. A review

The release of pollutants into the environment is a serious ecological problem due to the significant risks they pose both to ecosystems and to human health. The review considers the issues of detection and distribution of new pollutants, as well as sources of their entry into the environment and potential impact on ecosystems. The problem of wastewater pollution is especially urgent in the context of conservation of salmonid fish species. Pharmaceutical and veterinary drugs, components of personal hygiene products, microplastics, per- and polyfluorinated substances, brominated flame retardants, oil and a number of other toxicants enter freshwater bodies, which are especially important for the early stages of the salmon development and its following migration. The presence of pollutants in the aquatic environment depends on a source, their ability to bioaccumulate, as well as to degrade in the processes of biological and abiotic purification. High-resolution mass spectrometry in combination with gas or liquid chromatography can be recommended for detection of new pollutants. Due to high mass measurement accuracy and high specificity, this method is able to effectively identify pollutants even in the complex fish matrix. When conducting non-targeted and targeted screening with highly sensitive full scanning, it is advisable to use high-resolution mass analyzers (TOF, Q-TOF, Q-Orbitrap), which allow determining a number of new compounds. A promising direction in the complex assessment of the impact of pollutants on salmon fish species and the identification of indicators characterizing this impact is metabolomics. It is based on the study of low-molecular organic compounds — metabolites, both intermediate and final metabolic products in body fluids and tissues. Metabolomic profiling allows identifying important markers of the state of a water body under the influence of pollutants, which is necessary for the development of comprehensive regulations and guidelines for the effective handling of these pollutants.

Stability of the Glycated Amino Acid 6-(2-Formyl-5-hydroxymethyl-1-pyrrolyl)-l-norleucine (Pyrraline) During Oxidation.

Food proteins may be modified during processing and storage through reactions with reducing sugars (Maillard reaction, glycation) or by reactive oxygen species (protein oxidation). Little is known about particular reactions at the interface of glycation and oxidation. In the present study, the glycated amino acid pyrraline (6-(2-formyl-5-hydroxymethyl-1-pyrrolyl)-l-norleucine) and the proteinogenic amino acids tyrosine and tryptophan were subjected to different types of oxidation. The stability of the amino acids was assessed by HPLC with UV detection, whereas oxidation products were assigned by HPLC with triple quadrupole or time-of-flight mass spectrometric detection. Conditions that lead to oxidation of aromatic proteinogenic amino acids can also lead to oxidation of pyrraline. Pyrraline was particularly unstable in the presence of permanganate, hypochlorite, and under hydroxyl radical-generating conditions (iron, ethylenediaminetetraacetic acid, ascorbic acid). Evidence obtained by high-resolution mass spectrometry revealed the oxidation of pyrraline to 6-(2,5-diformyl-1-pyrrolyl)-l-norleucine, 6-(2-carboxy-5-hydroxymethyl-1-pyrrolyl)-l-norleucine, 6-(2-formyl-5-carboxy-1-pyrrolyl)-l-norleucine, and 6-(2,5-dicarboxy-1-pyrrolyl)-l-norleucine in the presence of potassium permanganate. The latter product was isolated by semipreparative HPLC and characterized by NMR. Under hydroxyl radical-generating conditions, pyrraline is hydroxylated at the ring under formation of 6-(2-formyl-4-hydroxy-5-hydroxymethyl-1-pyrrolyl)-l-norleucine or 6-(2-formyl-3-hydroxy-5-hydroxymethyl-1-pyrrolyl)-l-norleucine. This study shows that the so-called "advanced glycation end products" are no end products of the Maillard reaction, but may undergo further chemical degradation reactions.

Administration Route Differentiation of Altrenogest via the Metabolomic LC-HRMS Analysis of Equine Urine

Altrenogest, also known as allyltrenbolone, is a synthetic form of progesterone used therapeutically to suppress unwanted symptoms of estrus in female horses. Altrenogest affects the system by decreasing levels of endogenous gonadotrophin and luteinizing and follicle-stimulating hormones, which in turn decreases estrogen and mimics the increase of progesterone production. This results in more manageable mares for training and competition alongside male horses while improving the workplace safety of riders and handlers. However, when altrenogest is administered, prohibited steroid impurities such as trendione, trenbolone, and epitrenbolone can be detected. It has been assumed that greater concentrations of these steroid impurities are present in injectable preparations and, therefore, pose a greater risk of causing anabolic effects when administered. For this reason, and due to the necessity of this therapeutic substance for the safety of thoroughbred racing participants, a metabolomic approach investigating the differentiation of two main administration routes was conducted. Liquid chromatography high-resolution mass spectrometry analysis of equine urine samples found five sulfated compounds, estrone sulfate, testosterone sulfate, 2-methoxyestradiol sulfate, pregnenolone sulfate, and cortisol sulfate, with the potential to differentiate between oral and intramuscularly administered altrenogest using a random forest classification model. The best model results, comparing two horses’ administration normalized peak area datasets, gave an AUC score of 0.965 with a confidence level of 95% (between 0.931 and 0.995). Identifications of these compounds were confirmed with assistance from the Shimadzu Insight Explore Assign feature, together with MS/MS spectrum and retention time matching of purchased and synthesized reference standards. This study proposes a new potential application for metabolomic multi-tool workflows and machine learning models in a forensic toxicological context.