Search Results (43)

Background: Crude oil refinery waste sludge (COWS) ranks among the most compositionally complex and ecotoxicologically hazardous industrial residues. Although bulk total petroleum hydrocarbon (TPH) and summed polycyclic aromatic hydrocarbon (PAH) removal are routinely reported, the metabolite-level biochemical fate of individual petrogenic compounds, spanning ring dihydroxylation, catechol cleavage, and entry into central carbon metabolism, remains largely unmapped under co-composting with diverse animal manures. Objectives: This study aimed to construct a metabolite-resolved, microbially anchored biochemical fate map of crude oil sludge during co-composting. Methods: Aerobic microcosms combining crude oil sludge, garden soil, and a wood-chip bulking agent were amended separately with poultry, horse, cow, or swine/pig manure alongside an unamended control and then incubated at 22 °C for 300 days. Analyses integrated untargeted gas chromatography-mass spectrometry (GC-MS) metabolomics, targeted PAH quantification (EPA Methods 3541/8270), 16S rRNA gene amplicon sequencing (Illumina MiSeq, V1–V3, paired-end 300 bp), physicochemical monitoring, and culture-dependent isolation, with National Institute of Standards and Technology (NIST) Mass Spectral library annotation. Results: GC-MS resolved 1169 metabolite features across 17 samples, comprising 538 annotated compounds within 11 chemical classes and 631 unknowns, of which 151 recurred in at least 10 samples. Petrogenic markers (n-alkanes C₁₄–C₃₆, hopanoids, steranes, and alkylated dibenzothiophenes) and ring-cleavage intermediates (2-hydroxyfluorene, 1,4-naphthoquinone, phenanthrene-methanol, benzenediols, butanedioic acid, fatty alcohols C₁₆–C₂₀) elucidated a four-stage degradation cascade consistent with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways map01220 and map00624. PAH mean-removal ranked swine/pig (88.0%) > horse (87.0%) > poultry (80.5%) > cow (79.1%) > control (68.2%). Sequencing recovered 2969 operational taxonomic units (OTUs) enriched in Pseudomonas, Achromobacter, Stutzerimonas, Dietzia, Gordonia, and Mycobacterium, with Pseudomonas dominating high-removal systems; respiration peaked at 18.7 mg CO₂-C g⁻¹ in poultry treatments. Conclusions: This work establishes a metabolite-resolved map linking hydrocarbonoclastic taxa to separate degradation steps. The co-occurrence of oxygenated PAH intermediates with decreasing parent PAH concentrations serves as an indicator of transformation processes and may assist in identifying potential residual-risk signals, thereby supporting remediation evaluation and process optimization. Full article

Background/Objectives: Mesoporous silica nanoparticles (MSNs) are widely investigated as nanocarriers for drug delivery, gene transfer, and bioimaging. However, the mechanisms underlying their size-dependent cytotoxicity at the metabolic level remain incompletely understood. This study aimed to determine whether different-sized MSNs induce distinct patterns of subcellular injury and metabolic reprogramming in lung epithelial cells. Methods: A549 cells were exposed to 80 nm and 600 nm MSNs at 50 and 200 μg/mL for 24 h. Ultrastructural changes were examined by transmission electron microscopy (TEM). Intracellular reactive oxygen species (ROS) and Ca²⁺ were measured by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and Fluo-4 AM fluorescence, respectively. Inflammatory gene expression (IL1B, IL6, TNFA, HIF1A) was quantified by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Untargeted metabolomics were performed using combined gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS) platforms, followed by principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and MetaboAnalyst-based pathway enrichment. Results: TEM revealed distinct size-dependent subcellular distributions: 80 nm MSNs were predominantly associated with mitochondrial abnormalities, including cristae disruption, swelling, and mitophagy-like features, whereas 600 nm MSNs accumulated in endocytic vesicles with membrane disruption. Metabolomic profiling showed that 80 nm MSNs were associated with TCA cycle blockade—characterized by the accumulation of early intermediates (citrate, oxaloacetate) and the depletion of distal intermediates (fumarate, malate)—with compensatory glycolytic activation (increased glyceraldehyde-3-phosphate and pyruvate) and reduced deoxynucleotide pools (dCDP, dUMP). By contrast, 600 nm MSNs triggered broad nucleotide triphosphate accumulation (ATP, CTP, dGTP, dCTP), amino acid depletion, and robust inflammatory activation, including a ~136-fold increase in IL1B expression and HIF1A transcriptional upregulation. PCA and PLS-DA confirmed distinct size-dependent metabolic phenotypes. Conclusions: MSN size strongly influences subcellular targeting—80 nm particles were predominantly associated with mitochondrial injury while 600 nm particles disrupted endocytic vesicles—driving qualitatively distinct patterns of metabolic reprogramming and inflammatory signaling. These findings establish a correlative mechanistic framework linking particle size to organelle-specific injury and provide candidate metabolic markers for nanotoxicological evaluation. Full article

Global warming exacerbates high-temperature stress, disturbing the growth, metabolic homeostasis and quality formation of tea plants (Camellia sinensis L.). Trehalose, a multifunctional osmolyte, can enhance abiotic stress tolerance, but its systematic metabolic mechanism against heat damage in tea remains unclear. Here, we applied integrated gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS) non-targeted metabolomics to compare control (CK), heat-stressed (T), and trehalose-treated heat-stressed (TT) tea leaves. We identified 163 differential volatile metabolites in GC-MS and 1619 differential non-volatile metabolites in LC-MS. Metabolite classification showed that organic oxygen compounds dominated differential volatile metabolites, while lipids and lipid-like molecules dominated differential non-volatile metabolites. The Kyoto Encyclopedia of Genes and Genomes enrichment showed that alanine, aspartate and glutamate metabolism, arginine biosynthesis, aminoacyl-tRNA biosynthesis, and flavone and flavonol biosynthesis were core shared pathways. Quantitatively, exogenous trehalose under heat stress significantly increased carbohydrate accumulation, restored lipid homeostasis, and elevated alanine, arginine, and related intermediates, thereby maintaining carbon–nitrogen balance. Trehalose also remodeled the amino acid substrate pool for aminoacyl-tRNA biosynthesis. In flavonoid metabolism, trehalose enhanced high-antioxidant flavonoid aglycones while reducing most glycosides and inhibiting excessive hydroxylation of flavonols. Although total flavonoid content decreased in TT relative to T, this reflected alleviated oxidative damage and reduced dependence on flavonoid-based defense. Combined with total amino acid and flavonoid quantifications, we conclude that exogenous trehalose enhances tea plant thermotolerance by coordinately regulating primary amino acid metabolism and secondary flavonoid metabolism. These findings provide a theoretical basis for using trehalose in heat-resistance cultivation and quality improvement of tea plants. Full article

Temperature extremes represent a major constraint for the cultivation of yellow passion fruit (Passiflora edulis Sims f. flavicarpa), a tropical crop increasingly exposed to heat waves and chilling events under climate change. Glycine betaine (GB) is a widely studied osmoprotectant in plants, yet its influence on metabolic responses of passion fruit under contrasting temperature stresses remains poorly characterized. This study investigated the effects of exogenous GB on primary metabolite profiles of passion fruit seedlings subjected to heat (25, 35, and 45 °C) and cold (25, 15, and 5 °C) conditions. Seedlings were treated with GB (100 mM) or left untreated, and leaf metabolites were quantified using GC–MS-based metabolomics. Heat exposure was associated with pronounced changes in amino acids, organic acids, sugars, polyamines, and γ-aminobutyric acid (GABA), while GB-treated plants showed altered levels of proline, GABA, polyamines, and selected tricarboxylic acid intermediates. Under cold conditions, several amino acids and organic acids decreased, whereas soluble sugars accumulated, particularly in GB-treated plants. Principal component analysis revealed distinct metabolic configurations under heat and cold treatments and indicated that GB modified metabolite profiles in a stress-dependent manner rather than restoring control-like states. These findings describe how GB is associated with shifts in central carbon and nitrogen metabolism under contrasting temperature regimes, providing a metabolomic perspective on stress-related metabolic adjustments in passion fruit. Full article

To characterize the key odorants and elucidate the flavor profiles of compound fermented beverages after fermentation, single-compound fermented beverages (GW, AW) and a compound fermented beverage (CW) were prepared using Italian Riesling grapes and SirPrize apples as raw materials. The flavor and metabolite profiles were systematically analyzed by integrating flavoromics (comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry, GC × GC–TOF MS) and metabolomics (LC–MS/MS). The results demonstrated that CW exhibited the most favorable acid/reducing sugars (2.18), imparting a drier taste and superior stability. Compounds with relative odor activity values (rOAV) greater than 1—including 3-methyl-1-butyl acetate, ethyl hexanoate, ethyl butanoate, and ethyl octanoate—collectively contributed prominent fruity, floral, and sweet aromas to all three wine types. Ethyl decanoate provided an additional distinctive traditional fruity note specifically to AW, while 1-octen-3-ol contributed a mushroom-like aroma to both GW and CW. Moreover, 3-methylbutanal, 4-ethyl-2-methoxyphenol, and ethyl 3-methylbutanoate added additional significant aroma contributions to CW, imparting floral, clove-like, and fruity notes, respectively. Notably, ethyl hexanoate (fruity aroma) exhibited a remarkably high rOAV of 27.43 in CW, significantly surpassing its levels in the single-substrate fermentations. Lipid metabolism and the phenylpropanoid pathway were significantly activated in CW, facilitating the coordinated synthesis of esters and phenolic compounds. Sensory attribute network analysis further confirmed that CW possessed more pronounced “sweet”, “fruity”, and “floral” characteristics. Correlation analysis revealed significant relationships between volatile organic compounds (VOCs) and total soluble solids (TS), titratable acidity (TA), the TA/TS ratio, and metabolite levels, underscoring the close connections among physicochemical properties, precursor/intermediate metabolites, and flavor formation. Comprehensive analysis of non-volatile metabolites and flavor-associated VOCs revealed variety-specific characteristics and compounding effects, providing valuable insights for enhancing the quality of compound fermented beverages. Full article

The ability of plants to survive oxygen deficiency is associated with significant changes in metabolism. Metabolic profiling of wheat seedlings under anoxia and subsequent reoxygenation conditions was performed using GC-MS. A total of 374 and 298 compounds were detected in root and shoot metabolomes, respectively. All intermediates of central metabolism were identified. Early anoxic responses of root and shoot metabolomes showed similarity, leading to the accumulation of amino acids (Ala, GABA and Tyr), carboxylates (lactate and succinate), nucleotides and amines, together with a decrease in sugars. The metabolic response to long-term anoxia varied significantly in the roots and shoots of wheat seedlings and was related to the redistribution of carbon flux from glycolysis predominantly to lipids in the roots, while it was directed to carboxylates and GABA in the shoots. Imposition of 24 h of reaeration after short-term anoxia (6 h) switched the metabolome toward a normoxic profile, predominantly in roots. Anaerobically down-regulated metabolites were accumulated, while anaerobic intermediates were depleted post-anoxia. The effects of more prolonged anoxia on wheat seedling metabolomes were less reversible, particularly in shoots. Interestingly, several metabolites with not fully understood roles (e.g., hydroxyl carboxylates, α,ω-dicarboxylic acids, polyols) were detected under anoxic conditions in wheat seedlings, which could potentially serve as markers of plant sensitivity to oxygen deficiency. Full article

Background: Land use change fundamentally alters soil microbial communities and biochemical processes, yet the integrated effects on rhizosphere microbiome–metabolome networks remained poorly understood. Objective: This study investigated land uses as forest, grassland and intermediary edge shape the rhizosphere biochemical networks of naturally grown Barbarea vulgaris. Methods: Rhizosphere soils of Barbarea vulgaris were analysed for microbial community structure abundance, and metabolomic profile applying phospholipid fatty acid (PLFA) profiling and mass spectrometric untargeted metabolomics (GC–MS/MS and MALDI–TOF/TOF MS). These were coupled with co–inertia analysis to assess microbiome–metabolome interactions. Results: Microbial community analysis revealed significant effects of land use on bacterial community structure (G+/G−, p < 0.001). Untargeted metabolomics identified 248 metabolites, of which 161 were mapped to KEGG pathways. Amino acids and derivatives (21.1%) followed by organic acids (16.8%) were the most representative among identified metabolites. Pathway enrichment analysis revealed coordinated reprogramming of central carbon and nitrogen metabolism across land use gradients, particularly in the amino acid metabolism, TCA cycle, and glycolysis/gluconeogenesis pathways. Microbiome–metabolome coupling analysis revealed distinct correlation patterns between microbial phenotypes and metabolite classes, with forest environments showing the strongest biochemical network integration (RV = 0.91). Edge habitats presented intermediate signatures, supporting their role as transitional zones with unique biochemical properties. Conclusions: The environmental context fundamentally shapes rhizosphere biochemical network organization through coordinated shifts in bacterial community structure and metabolic pathway activity. These habitat-specific metabolic signatures suggest that land use change triggers adaptive biochemical responses that may influence plant performance and ecosystem functioning across environmental gradients. Full article

Background/Objectives: Gestational diabetes mellitus (GDM) alters maternal metabolism during pregnancy and may impact the biochemical composition of breast milk. Given the critical role of human milk in early-life metabolic programming, identifying metabolic alterations in GDM milk and understanding the effects of insulin therapy has important implications for neonatal health. This study aims to investigate the metabolomic profile of transitional breast milk in mothers with gestational diabetes mellitus compared with healthy controls and to evaluate the impact of insulin therapy on milk metabolite composition. Methods: Breast milk samples were collected between postpartum days 10 and 15 from 80 mothers with GDM and 80 matched controls. Metabolomic profiling was performed using gas chromatography–mass spectrometry (GC–MS), and data were analyzed using multivariate and univariate statistical techniques including PCA, PLS–DA, logistic regression, and ROC analysis. Conclusions: A total of 133 metabolites were identified, and GDM mothers exhibited a distinct metabolomic signature characterized by significant alterations in carbohydrate, amino acid, and microbial-derived metabolites. In particular, galactinol, arabitol, and pyrogallol were significantly decreased, while α-ketoglutaric acid and citric acid were elevated in the GDM group. Insulin-treated mothers showed unique metabolic changes involving glycolytic intermediates (glycerone phosphoric acid), purine metabolism (xanthine), and oxidative pathways (isocitric acid, gluconic acid lactone). Multivariate models based on the top metabolites achieved moderate discriminatory performance (AUC = 0.68). GDM is associated with substantial metabolic changes in transitional breast milk, and insulin therapy appears to modulate these alterations further. These findings suggest that maternal metabolic status and its treatment can shape the neonatal nutritional environment, potentially influencing early metabolic programming. Full article

Ankylosing spondylitis (AS) displays wide inter-patient variability that is not accounted for by HLA-B27 alone, suggesting that additional immune and metabolic modifiers contribute to disease severity. Using a genetically matched design, we profiled peripheral blood mononuclear cells from two brother pairs discordant for AS severity and one healthy brother pair. Strand-specific RNA-seq was analyzed with a family-blocked DESeq2 model, while untargeted metabolites were quantified using gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS). Differential features were defined as follows: differentially expressed genes (DEGs) (|log₂FC| ≥ 1 and FDR < 0.05) and metabolites (VIP > 1, FC ≥ 1.2, and BH-adjusted p < 0.05). Pathway enrichment was performed with KEGG and Gene Ontology (GO). A total of 325 genes were differentially expressed. Type I interferon and neutrophil granule transcripts (e.g., IFI44L, ISG15, S100A8/A9) were markedly up-regulated, whereas mitochondrial β-oxidation genes (ACADM, CPT1A, ACOT12) were repressed. Metabolomics revealed 110 discriminant features, including 25 MS/MS-annotated metabolites. Primary bile acid intermediates were depleted, whereas oxidized fatty acid derivatives such as 12-Z-octadecadienal and palmitic amide accumulated. Spearman correlation identified two antagonistic modules (i) interferon/neutrophil genes linked to pro-oxidative lipids and (ii) lipid catabolism genes linked to bile acid species that persisted when severe and mild siblings were compared directly. Enrichment mapping associated these modules with viral defense, neutrophil degranulation, fatty acid β-oxidation, and bile acid biosynthesis pathways. This sibling-paired peripheral blood mononuclear cell (PBMC) dual-omics study delineates an interferon-driven lipid–bile acid axis that tracks AS severity, supporting composite PBMC-based biomarkers for future prospective validation and highlighting mitochondrial lipid clearance and bile acid homeostasis as potential therapeutic targets. Full article

Babesia divergens is a tick-borne apicomplexan parasite that causes human babesiosis, a malaria-like disease. B. divergens metabolism remains poorly characterized. Here, we employed a multiplatform mass spectrometry-based metabolomics approach (using CE-TOF/MS, GC-QTOF/MS, LC-QTOF/MS, and LC-QqQ/MS) to profile intra- and extracellular metabolic changes in B. divergens-infected and uninfected red blood cells (RBCs) and their supernatants. Our results indicate alterations in the metabolome caused by B. divergens infection and proliferation within RBCs. These findings are consistent with the major metabolic dependencies of B. divergens, including extracellular glucose, glutamine, and arginine, accompanied by the accumulation of glycolytic and TCA cycle intermediates. We identified altered nucleotide metabolism, pentose phosphate pathway activity, and redox imbalance. Depletion of lysoglycerophospholipids, glucose, arginine, and glutamine, and accumulation of free heme and sphingolipids suggested pathogenic effects. Growth experiments indicate that glucose and glutamine, but not hypoxanthine, are required for parasite growth. We additionally discovered a phosphorylated HEPES derivative (PEPES) produced upon B. divergens infection of RBCs in vitro. Collectively, these findings and their global interpretation provide insights into B. divergens metabolism and metabolic dependencies and host–parasite metabolic interactions and outline potential directions for future studies on human babesiosis diagnosis, prognosis assessment, and treatment. Full article

Breast cancer (BC) is a neoplasm characterized by high heterogeneity and is influenced by intrinsic molecular subtypes and clinical stage, aspects that remain underexplored in the Colombian population. This study aimed to characterize metabolic alterations associated with subtypes and disease progression in a group of newly diagnosed, treatment-naive Colombian women using an untargeted metabolomics approach. To improve metabolite coverage, samples were analyzed using LC-QTOF-MS and GC-QTOF-MS, along with amino acid profiling. The Luminal B subtype exhibited elevated levels of long-chain acylcarnitines and higher free fatty acid concentrations than the other subtypes. It also presented elevated levels of carbohydrates and essential glycolytic intermediates, suggesting that this subtype may adopt a hybrid metabolic phenotype characterized by increased glycolytic flux as well as enhanced fatty acid catabolism. Tumor, Node, and Metastasis (TNM) staging analysis revealed progressive metabolic reprogramming of BC. In advanced stages, a sustained increase in phosphatidylcholines and a decrease in lysophosphatidylcholines were observed, reflecting lipid alterations associated with key roles in tumor progression. In early stages (I-II), plasma metabolites with high discriminatory power were identified, such as glutamic acid, ribose, and glycerol, which are associated with dysfunctions in energy and carbohydrate metabolism. These results highlight metabolomics as a promising tool for the early diagnosis, clinical follow-up, and molecular characterization of BC. Full article

Background: Fish escape events from aquaculture facilities are increasing and pose significant ecological, economic, and traceability concerns. Accurate methods to differentiate between wild, cultured, and escaped fish are essential for fishery management and seafood authentication. Methods: This study analyzed muscle tissue from Sparus aurata, Dicentrarchus labrax, and Argyrosomus regius using a multiomics approach. Heavy metals were quantified by ICP-MS, fatty acid profiles were assessed via GC-MS, and metabolomic and lipidomic signatures were identified using 1H NMR spectroscopy. Multivariate statistical models (MDS and PLS-LDA) were applied to classify fish origins. Results: Wild seabream showed significantly higher levels of arsenic (9.5-fold), selenium (3.5-fold), and DHA and ARA fatty acids (3.2-fold), while cultured fish exhibited increased linoleic and linolenic acids (6.5-fold). TMAO concentrations were up to 5.3-fold higher in wild fish, serving as a robust metabolic biomarker. Escaped fish displayed intermediate biochemical profiles. Multivariate models achieved a 100% classification accuracy across species and analytical techniques. Conclusions: The integration of heavy metal analysis, fatty acid profiling, and NMR-based metabolomics enables the accurate differentiation of fish origin. While muscle tissue provides reliable biomarkers relevant to human exposure, future studies should explore additional tissues such as liver and gills to improve the resolution of traceability. These methods support seafood authentication, enhance aquaculture traceability, and aid in managing the ecological impacts of escape events. Full article

Background: Pulmonary arterial hypertension (PAH) is a rare and severe condition characterized by increased pulmonary arterial pressure and vascular resistance. Women are more susceptible to PAH yet have higher survival rates than men, a phenomenon called the “estrogen paradox”. This study aims to investigate the sex-based differences in PAH using plasma untargeted metabolomics. Methods: Plasma samples were collected from 43 PAH patients and 37 healthy controls. The samples were analyzed using two complementary analytical techniques: gas chromatography–mass spectrometry (GC-QqQ/MS) and liquid chromatography–mass spectrometry (LC-Q-ToF/MS). The metabolic differences between male and female PAH patients and controls were identified using multivariate statistical analyses. Results: Our results show changes in the lipid, fatty acid, and amino acid metabolism in both sexes. Women presented additional changes in the carbohydrate, bile acid, and nucleotide metabolism. The metabolites affected by PAH in women included decreased threonine, tryptophan, and lipid intermediates and elevated bile acids. Men were found to have additional changes in the heme catabolism, cholesterol synthesis, and lipoxygenase pathways. The metabolites affected by PAH in men included decreased branched-chain amino acids and increased bilirubin, phospholipids, and oxidized fatty acids. Conclusions: The gender differences observed in the development of PAH are likely multifactorial. While estrogens and potentially other sex hormones have been implicated in modulating relevant biological pathways, their exact role in disease progression and pathogenesis remains to be fully elucidated. The specific metabolic changes in women and men point to distinct disease mechanisms, potentially explaining the differences in prevalence, prognosis, and treatment response of patients with PAH. The obtained results should be validated with the use of targeted quantitative analyses and larger numbers of patients. Full article

The widespread application of pesticides in agriculture has raised increasing concerns regarding their ecological impact, particularly in aquatic environments. Among these, α-cypermethrin, a highly active isomeric form of cypermethrin, has been extensively used due to its potent insecticidal efficacy and low mammalian toxicity. However, its toxicity to non-target aquatic organisms remains insufficiently understood at the metabolic level. In this study, a targeted metabolomics approach was employed to investigate the biochemical effects of α-cypermethrin in adult zebrafish. Acute toxicity was first determined to establish sublethal exposure concentrations (0.15 µg/L and 1.5 µg/L), followed by a 48 h exposure under a controlled flow-through system. GC-MS/MS-based analysis quantified 395 metabolites, and multivariate statistical models (principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA)) revealed clear dose-dependent metabolic alterations at two time points. Pathway analysis identified disruptions in glycolysis, glycerolipid metabolism, amino acid turnover, and glutathione pathways. Notably, glutamate depletion and associated reductions in GABA (4-Aminobutanoate) and TCA (Tricarboxylic acid) cycle intermediates suggest oxidative stress-induced metabolic bottlenecks. These results provide mechanistic insights into α-cypermethrin-induced toxicity and demonstrate the utility of metabolite-level biomarkers for environmental monitoring. This study contributes to a systems-level understanding of how sublethal pesticide exposure affects vertebrate metabolism, offering a basis for improved ecological risk assessment and pesticide regulation. Full article

Hongqujiu, one of the three principal varieties of yellow wine, is a traditional fermented beverage originating from China that employs Hongqu as the fermentation agent. In this study, an untargeted metabolomics approach based on gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS) was applied to systematically analyze the volatile compounds (VOCs) and non-volatile compounds (NVCs) in Hongqujiu across different aging years for the first time. The analysis identified a total of 262 VOCs and 2564 NVCs in samples of Hongqujiu aged for six distinct years. Based on metabolic differences, the samples were categorized into two groups: the low-year group (5-year, 6-year) and the high-year group (8-year, 10-year, 15-year, 20-year). Nineteen VOCs (e.g., 4-amino-butyric acid and diethanolamine) and thirty NVCs (e.g., palmitoylethanolamide and erinacine D) were identified as key differential metabolites distinguishing the low-year group from the high-year group. The higher-year group is enriched with a variety of substances with different flavors or biological activities, such as sugar derivatives, amino acids and their complexes, organic acids and their intermediate metabolites, steroids and terpenoid compounds, lipids and their derivatives, nitrogen-containing heterocycles, and aromatic compounds. The accumulation of these substances not only shapes the unique and rich flavor characteristics of aged red rice wine (such as the caramel aroma and umami peptide flavor), but also endows red rice wine with potential health benefits due to the physiological regulatory functions of some active ingredients. This study contributes to a deeper understanding of the composition and dynamic variations in metabolites in Hongqujiu, offering a scientific foundation for identifying aged Hongqujiu and conducting further research to enhance its quality. Full article