Search Results (1,191)

Background: Distinguishing between osteoarthritis (OA), rheumatoid arthritis (RA), and psoriatic arthritis (PsA) remains challenging despite different underlying mechanisms. Synovial fluid reflects metabolic changes within affected joints, yet comprehensive metabolomic comparisons across these conditions are limited. We aimed to identify disease-specific metabolic signatures in synovial fluid that could improve differential diagnosis and reveal therapeutic targets. Methods: We collected synovial fluid from 39 patients (20 OA, 5 RA, and 14 PsA) during routine knee arthrocentesis between January 2023 and February 2024. Following metabolite extraction, we performed untargeted metabolomic profiling using quadrupole time-of-flight liquid chromatography–mass spectrometry (Q-TOF LC/MS). Data underwent multivariate statistical analysis, including principal component analysis (PCA) and partial least squares–discriminant analysis (PLS-DA), to identify discriminatory metabolites. Results: While unsupervised analysis showed overlap between groups, supervised PLS-DA achieved clear metabolic separation. RA samples showed elevated itaconic acid, indicating inflammatory macrophage activation, and increased O-acetylserine, suggesting altered one-carbon metabolism. Hypoxanthine was decreased, which reflected severe metabolic stress. PsA exhibited the unique elevation of 4,4-dimethylcholestane and 2-oxoarginine. These metabolites have previously been unreported in this disease. OA demonstrated increased hippuric acid and indoleacetic acid, which are both gut microbiota products, supporting the gut–joint axis hypothesis. Conclusions: Each arthritis type displayed distinct metabolic fingerprints in synovial fluid. Candidate discriminatory metabolites, including gut-derived metabolites in OA and specific lipid alterations in PsA, open new diagnostic and therapeutic avenues. Given the limited RA sample size (n = 5), RA-related results should be viewed as exploratory and requiring validation in larger independent cohorts. These metabolites may, after rigorous validation in larger and independent cohorts, contribute to multi-metabolite biomarker panels for earlier diagnosis and to the rational design of targeted therapeutics addressing disease-specific metabolic disruptions. Full article

Background: Atherosclerosis and its associated chronic inflammation of the arterial wall disrupt fatty acid metabolism, leading to changes in plasma fatty acid composition. These alterations can be used to improve disease diagnosis and risk stratification by the development and application of specific lipidomic indices. Objectives: The objectives of this study are to evaluate the performance of conventional fatty acid indices and enhance diagnostic efficiency in atherosclerosis by introducing novel index based on plasma PUFA n-6 and n-3 content (Omega-6/3 Balance Index, O6/3-BI), as well as the perspective SFA/MUFA ratio (stearic/oleic acid ratio, C18:0/C18:1n-9) and a logit function combining PUFA and SFA/MUFA biomarkers. Methods: Plasma fatty acids were quantified by LC-MS/MS in healthy controls (n = 50) and patients with carotid atherosclerosis (n = 52), stratified by atorvastatin, rosuvastatin, or no statin therapy. The conventional indices (the Omega-3 Status (EPA + DHA), AA/EPA, and the omega-6/omega-3 ratio), and pathway ratios (C18:0/C18:1n-9; and C20:4n-6/C22:4n-6), as well as the newly introduced PUFA index and combined PUFA-SFA/MUFA logit function, were calculated. Their diagnostic performance for distinguishing atherosclerosis was assessed by a receiver operating characteristic (ROC) analysis with the cross-validation and calculation of Cliff’s Δ effect size. Results: The conventional parameters demonstrated a poor to low discrimination ability of the atherosclerosis patients’ groups from healthy controls (area under the ROC curve, AUC 0.548–0.711). In statin-treated patients, these conventional markers lost significance. The newly introduced PUFA index and SFA/MUFA ratio demonstrated improved patients’ discrimination with AUC 0.734–0.780 for the former and strong predictive power with AUC 0.831–0.858 for the latter marker and maintained their diagnostic value under statin therapy. The most significant positive effect size was observed for the SFA/MUFA ratio with Cliff’s Δ = 0.67–0.71. The combined PUFA-SFA/MUFA logit function also demonstrated a strong predictive power with AUC = 0.880 (Cliff’s Δ = −0.76), outperforming any single index. Conclusions: The newly introduced lipidomic index based on the PUFA content, SFA/MUFA ratio, and a logit function combining PUFA-SFA/MUFA biomarkers demonstrated a substantially better discrimination of atherosclerosis-related fatty acid metabolic disturbances than conventional fatty acid biomarkers. Full article

Nocardia seriolae (N. seriolae) is a significant bacterial pathogen in global aquaculture, causing substantial economic losses. Live-attenuated vaccines represent a promising control strategy, but their molecular mechanisms remain poorly understood. This study employed a quantitative proteomic approach to compare the proteomic profiles of a virulent wild-type strain (F1) and an attenuated vaccine strain (F110) of N. seriolae. Using a data-independent acquisition (DIA)-based LC-MS/MS analysis, we identified 4516 proteins, with 540 showing significant differential expression (311 upregulated, 229 downregulated). Bioinformatic analysis revealed that upregulated proteins in F110 were primarily involved in metabolic processes, including phosphatidate cytidylyltransferase and various enzymes related to amino acid and nucleotide metabolism. Conversely, downregulated proteins were enriched in virulence-associated functions, including HtpX and MFS transporter permease. These findings suggest that attenuation involves a complex reprogramming of metabolic pathways coupled with a reduction in key virulence factors, providing insights into the potential molecular basis of vaccine development and potential targets for novel therapeutic strategies. Full article

Parkinson’s disease (PD) is a progressive neurodegenerative disorder primarily characterized by the degeneration of dopaminergic neurons, leading to significant motor and non-motor symptoms. This study investigates glycosylation patterns with a significant emphasis on male Parkinson’s Disease (PD) patients, revealing unique alterations distinguishing PD from healthy states, utilizing high-performance liquid chromatography coupled with mass spectrometry (HILIC-LC-MS). Findings reveal significantly altered serum N-glycosylation profiles between male and female patients, with increased levels of high-mannose glycans and reduced mono-sialylated glycans in male patients. ROC curve analysis indicates that these glycan changes are the most important features for distinguishing PD from healthy states, with AUC values of 0.71 for M5 and 0.85 for M6. This study underscores the critical role of glycosylation in the pathophysiology of Parkinson’s disease and highlights its potential in early detection and monitoring of disease progression. Full article

Background/Objectives: Numerous studies have documented cardioprotective effects of adiponectin in animal models of cardiometabolic disease (CMD). Adiponectin receptor agonist ALY688 has demonstrated functional significance against pressure overload-induced cardiac remodeling events in a mouse model of heart failure with reduced ejection fraction (HFrEF), potentially through modulation of the systemic metabolome. However, the specific metabolites and their pathophysiological contribution to cardioprotection in cardiac hypertrophy or heart failure remain unclear. This study aimed to characterize systemic metabolic alterations across five tissues in HFrEF and determine how ALY688 modifies these pathways to mediate cardioprotection in the transverse aortic constriction (TAC) model. Methods: Targeted metabolic profiling was performed on heart, liver, muscle, epididymal white adipose tissue (eWAT), and serum collected five weeks post-surgery from wild-type male C57BL/6 mice. Mice underwent either Sham or TAC-induced left ventricular pressure overload, with or without daily subcutaneous ALY688 administration. Metabolites were quantified using liquid chromatography–tandem mass spectrometry (LC–MS/MS) and statistically analyzed at the tissue level. Results: Consistent with pathological cardiac remodeling, the comprehensive metabolomic analysis revealed that TAC induced widespread disruption of systemic metabolic homeostasis. ALY688 treatment significantly modified several key metabolite classes, including triglycerides (TGs) and glycosylceramides (HexCer). Notably, ALY688 also altered multiple gut-derived metabolites, including trimethylamine N-oxide (TMAO), 5-aminovaleric acid (5-AVA), and glycodeoxycholic acid (GDCA), highlighting a potential gut–liver–heart axis mediating its cardioprotective effects. Conclusions: These findings demonstrate that ALY688 mitigates TAC-induced metabolic dysregulation across multiple tissues. The identified metabolic signatures suggest that ALY688 exerts cardioprotective effects, at least in part, through restoration of systemic metabolic homeostasis and engagement of a gut–liver–heart metabolic axis. These results provide mechanistic insight into adiponectin receptor agonism and support further exploration of ALY688 as a potential therapeutic strategy for HFrEF. Full article

The present study involves the synthesis of polyvinylpyrrolidone (PVP)-stabilized iron-based trimetallic nanoparticles with different metal addition sequences (Fe/Ag/Zn, Fe/Zn/Ag and Fe/(Zn/Ag)) using the sodium borohydride reduction method. In order to investigate the catalytic reactivity of the nanoparticles, a series of batch experiments were performed using methyl orange dye as a model pollutant. It was found that the Fe/Ag/Zn system showed the maximum catalytic activity compared to the other studied trimetallic systems. About 100% of the methyl orange dye was removed within 1 min and the second-order rate constant obtained was 0.0744 (mg/L)⁻¹ min⁻¹; the rate of reaction was higher than that of the other trimetallic systems. Furthermore, the effects of pH, initial dye concentration and nanoparticle dosage on the degradation of methyl orange were investigated. The results showed that the reactivity of the Fe/Ag/Zn trimetallic nanoparticles was highly dependent on the aforementioned parameters. Higher reactivity was obtained at lower pH, lower initial methyl orange dye concentration and higher nanoparticle dosage. Lastly, liquid chromatography–mass spectroscopy (LC-MS) was used to elucidate the reaction pathway and identify by-products from methyl orange degradation. The developed catalyst demonstrated exceptionally rapid and apparent degradation of methyl orange within one minute, outperforming previously reported bimetallic and trimetallic systems. This work reports a cost-effective nZVI-based trimetallic system containing minimal silver, which shows promising reactivity toward azo dye degradation and may be suitable for future application in textile wastewater treatment. Full article

Anti-Porphyromonas gingivalis mechanisms of Berberis hemsleyana bark extract remain to be elucidated, and the anti-inflammatory activity of its n-butanol fraction (BNB) in RAW264.7 cells—mediated through suppression of the NF-κB pathway—require further validation. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the crude extract from B. hemsleyana were determined against Candida albicans, Escherichia coli, Porphyromonas gingivalis, Staphylococcus aureus and Streptococcus mutans. Scanning electron microscopy (SEM) and bacterial protein leakage assays were used to evaluate its antibacterial activity against P. gingivalis. High-performance liquid chromatography-mass spectrometry (LC-MS) was applied to analyze the ethanol extract of B. hemsleyana bark, leading to the screening of 47 compounds. The antibacterial mechanisms of the compounds were predicted through Network Pharmacology analysis and Molecular docking. Anti-inflammatory activity mediated via the NF-κB pathway was verified using an LPS-induced RAW264.7 cell inflammatory model. Specifically, the BNB showed a significant antibacterial effect on P. gingivalis. Meanwhile, it was confirmed that this fraction damaged the bacterial cell membrane structure, leading to the leakage of intracellular proteins in bacteria and thus impairing their infectivity. Network pharmacology analysis and molecular docking results indicated that B. hemsleyana bark’s biologically active compounds (Calenduloside E, Limonin, Acanthoside B, Dihydroberberine) antibacterial activity by regulating cytokines and cell apoptosis, thereby coordinating the body’s microbial homeostasis and inflammation. Additionally, BNB significantly reduced the secretion of the inflammatory cytokines IL-1β, TNF-α and IL-6 in vitro via the NF-κB pathway. The crude extract from the bark of B. hemsleyana has antibacterial and Anti-inflammatory activity. The n-butanol fraction showed a significant antibacterial effect on P. gingivalis. Full article

Background: Lidocaine, classified as an amide-type agent, and tetracaine, designated as an ester-type agent, are frequently co-formulated for dermatologic procedures. Despite the extensive literature on the pharmacokinetics (PK) of these substances, there is a paucity of head-to-head comparisons of intravenous (IV) and topical administration in the same preclinical model. Absolute bioavailability (F%) is imperative for optimizing formulation design and safety. Methods: A single-dose, single-sequence, three-period pilot study was performed in male Göttingen mini-pigs. The first period of the study involved the intravenous bolus administration of lidocaine HCl and tetracaine HCl, with a dosage of 1 mg/kg for each agent. In Period 2, the topical application of Pliaglis (a combination of 7% lidocaine and 7% tetracaine, with a concentration of 10 g/100 cm² and a duration of 60 min) was utilized. In Period 3, the pharmacokinetic profile of Z4T4L4 (a formulation comprising 4% lidocaine HCl and 4% tetracaine HCl) was assessed under the same experimental conditions. Blood samples were collected up to 24 h after the administration of the drug; skin biopsies were obtained 90 min after the application of the test substance. Plasma and skin concentrations were measured by means of validated liquid chromatography–tandem mass spectrometry (LC–MS/MS). PK parameters were derived using a noncompartmental analysis approach, while F% was calculated through AUC comparison with IV dosing. Results: Subsequent to intravenous administration, the mean elimination half-lives of lidocaine and tetracaine were determined to be 1.62 h and 1.85 h, respectively. Pliaglis demonstrated higher skin concentrations of lidocaine (358 μg/g) and tetracaine (465 μg/g) compared to Z4T4L4 (33.6 μg/g and 46.1 μg/g, respectively). Despite lower skin levels, Z4T4L4 produced higher F% (lidocaine: 1.98% vs. 1.41%; tetracaine: 3.34% vs. 1.26%). The time to maximum plasma concentration (T_max) for lidocaine was found to be 2–4 h (Pliaglis) and 2–8 h (Z4T4L4), while for tetracaine, it was 1–8 h (Pliaglis) and 2–8 h (Z4T4L4). Conclusions: In this preliminary study, which included three subjects, Z4T4L4 exhibited a numerical tendency towards increased systemic bioavailability in comparison with Pliaglis. This observation was noted despite the fact that Z4T4L4 resulted in markedly lower skin concentrations. Due to the exploratory nature of the pilot study (n = 3), observed differences are reported as numerical trends. The data suggest that Z4T4L4 may enhance systemic absorption while reducing skin retention, highlighting a potential formulation-dependent dissociation between local concentration and systemic bioavailability. These preliminary findings provide in vivo evidence of a divergence between eutectic-based tissue retention and enhancer-driven systemic flux. This highlights that formulation design fundamentally dictates the safety profile of local anesthetics, necessitating a balance between local efficacy and systemic safety. Full article

Background/Objectives:Pereskia aculeata and Pereskia grandifolia belong to the Cactaceae family, despite their foliar and woody stem characteristics. Both species are commonly known as Ora-pro-nóbis (derived from Latin, meaning “pray for us”), a name rooted in their historical use in colonial Brazil due to their nutritional value, particularly P. aculeata, which is frequently described as a high-protein food source. The goal of the present study was to compare these species based on phytochemical composition and antioxidant capacity. Methods: Both species were investigated for their chemical antioxidant properties (DPPH and phosphomolybdenum complex) and cellular anti-ROS activity using the CACO-2 cell line. A comprehensive phytochemical analysis was performed using LC-MS and GC-MS. Results: P. aculeata exhibited a more abundant content of phenolics and flavonoids, with greater structural variability in phenolic compounds and glycosylated flavonoids than P. grandifolia. Still, P. aculeata showed a more potent chemical antioxidant effect. By contrast, in P. grandifolia, a series of novel saponins was now discovered and characterized. In addition, the compounds from this species exhibited a greater cellular antioxidant activity than those of P. aculeata. Tryptophan-derived alkaloids, such as abrine (N-methyltryptophan), were present in both species, but hypaphorine only in P. aculeata. Conclusions: Both species of Pereskia exhibit potential health benefits, including distinct antioxidant activity, among other unexplored effects, given their significant variability in phytochemicals. These differences could be investigated in greater depth using combined LC-MS and GC-MS, thereby enabling more confident structural investigations of these natural compounds. Full article

Cutaneous lupus erythematosus (CLE) can occur independently of lupus erythematosus. SLE, and its responsiveness to treatment, does not necessarily align with that of coexisting SLE. Extracellular vesicles (EVs) allow communication between cells and rapid delivery throughout the body. We hypothesized that EVs may support disease-specific inflammation in CLE and SLE patients. Plasma EVs from healthy controls (n = 5), CLE (n = 6), and dermatomyositis (n = 17) were purified by ultracentrifugation and size-exclusion chromatography, phenotyped by flow cytometry, and profiled by LC-MS/MS. Circulating EVs were mainly platelet-, endothelial-, and antigen-presenting cell-derived examples. CLE EVs harbored four proteins absent in the controls—mimecan, IFI27, fibulin-2, and snRNP B/B′ (anti-Sm an-tigens)—and their cumulative number increased with SLEDAI. Relative to the controls, 18 proteins were upregulated and 15 downregulated in CLE EVs. The number of upregulated proteins showed a trend toward a correlation with SLEDAI (r = 0.79, p = 0.06) but not with CLASI (r = 0.21). Among upregulated proteins, lysozyme C and hyaluronan-binding protein 2 tracked with cutaneous activity (CLASI r = 0.74 and r = 0.86) but not with systemic activity (SLEDAI r = 0.52 and r = 0.31). CLE plasma EVs were enriched in antigen-presenting cell markers and disease-related cargo, including anti-Sm antigens and proinflammatory proteins. Although overall protein diversity correlated primarily with systemic disease activity, a subset of proteins appeared to reflect cutaneous activity. Full article

Streptococcus suis is an important zoonotic pathogen responsible for severe infections in pigs and humans. Its capacity to survive within phagocytic cells is considered a key virulence mechanism that contributes to dissemination and persistence in host tissues. This study employed comparative proteomic profiling to investigate intracellular adaptation of S. suis serotypes 2 (SS2) and 14 (SS14) during infection of human U937 macrophages. Five isolates originating from humans and pigs were analyzed using gel electrophoresis with liquid chromatography–tandem mass spectrometry (GeLC–MS/MS), revealing 118 differentially expressed proteins grouped into 11 functional categories. Translation-related proteins represented the largest group (48%), including upregulated ribosomal subunits (30S: S2, S5, S7, S8, S12, S15; 50S: L1, L5, L18, L22, L24, L33, L35) and translation factors such as GidA/TrmFO and RimP. Enrichment of carbohydrate metabolism and DNA replication proteins, including phosphoenolpyruvate carboxylase (PEP), UDP-N-acetylglucosamine pyrophosphorylase (GlmU), and ATP-dependent DNA helicase RuvB, indicated metabolic reprogramming and stress adaptation under intracellular conditions. Stress-response proteins such as molecular chaperone DnaK were also induced, supporting their multifunctional, “moonlighting” roles in virulence and host interaction. Comparative analysis showed that SS2 expressed a broader range of adaptive proteins than SS14, consistent with its higher virulence potential. These findings reveal conserved intracellular responses centered on translation, energy metabolism, and stress tolerance, which enable S. suis to survive within human macrophages. Integration of these intracellular proteomic signatures with previous exoproteomic, peptidomic, and network-based studies highlights translational and metabolic proteins—particularly DnaK, enolase, elongation factor EF-Tu, and GlmU—as multifunctional candidates linking survival and immunogenicity. This work establishes a comparative proteomic foundation for understanding S. suis intracellular adaptation and highlights potential targets for future vaccine or therapeutic development against this zoonotic pathogen. Full article

This study aimed to establish a fast and efficient method for the determination of N-nitrosamines (NAs) in meat products by integrating two sample preparation techniques—QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) and FaPEx (Fast Pesticide Extraction)—with liquid chromatography–tandem mass spectrometry (LC–MS/MS). Chromatographic separation was performed on a Poroshell 120 Phenyl Hexyl column using a gradient elution of acetonitrile and 0.01% formic acid at a flow rate of 0.3 mL/min and a column temperature of 25 °C. Under these conditions, nine NAs and one internal standard were completely separated within 11 min with selective reaction monitoring mode (SRM) for detection. Samples were first extracted with QuEChERS powder using acetonitrile containing 0.1% formic acid, followed by purification with a FaPEx-Chl cartridge. This combined approach demonstrated superior performance compared with traditional solvent extraction or QuEChERS extraction alone. The recoveries of the developed method ranged from 76% to 111% and 52% to 103% at spiking levels of 50 ng/g and 20 ng/g, respectively. The limits of detection (LOD) and quantification (LOQ) were 0.002–0.3 ng/g and 0.006–1.00 ng/g, respectively. The inter-day and intra-day precisions (RSD%) ranged from 2.7% to 17% and 2.9% to 17%, respectively. These results indicate that the proposed method is among the most time-efficient and effective analytical approaches currently available for the determination of NAs in meat products. Full article

Parkinson’s disease (PD) is a progressive neurodegenerative disorder shaped by genetic factors such as LRRK2 and GBA1 mutations, as well as dietary and metabolic influences. Food-derived plasma metabolites—including caffeine, paraxanthine, trigonelline, piperine, and sitosteryl hexoside—have emerged as promising, accessible biomarkers for early detection, progression monitoring, and therapeutic targeting, yet their longitudinal behavior and genetic interactions remain insufficiently characterized. Using the Parkinson’s Progression Markers Initiative (PPMI) cohort (n = 455; 303 PD patients, 152 controls), we quantified plasma levels of these metabolites by quantitative LC-MS/MS with batch correction, examining sporadic PD and genetically defined subgroups (LRRK2-PD [PDL], GBA1-PD [PDG], dual-mutation PD [PDGL], and prodromal equivalents). Baseline one-way ANOVA showed significantly lower caffeine and paraxanthine in PDL (p = 0.0467, p = 0.0178) and PDG (p = 0.0408), reduced piperine in PDL (p = 0.0009), PDG (p = 0.0257), and prodromal LRRK2 (p = 0.0168), and elevated sitosteryl hexoside in PDG (p = 0.0184). Longitudinal regression analyses revealed that in sporadic PD, caffeine negatively correlated with MDS-UPDRS parts I (β = −2, p = 0.0475) and III (β = −7.2, p = 0.007), trigonelline declined over time and was inversely associated with part III (β = −1.7, p = 0.0069), and sitosteryl hexoside negatively correlated with parts II (β = −68.3, p = 0.042) and III (β = −74.1, p = 0.0425). In PDL, sitosteryl hexoside inversely correlated with part I (β = −54.2, p = 0.0049), while in PDGL, paraxanthine showed negative associations with part II (β = −18.5, p = 0.00327). These findings demonstrate subgroup-specific alterations in food-derived metabolites and consistent inverse associations with PD severity, supporting their potential as non-invasive biomarkers, particularly in LRRK2/GBA1 mutation carriers, and highlighting the need for longitudinal validation and dietary intervention trials to advance personalized PD management. Full article

Salvia rosmarinus Spenn. (rosemary) is a medicinal and aromatic plant of notable pharmacological value. This study evaluated the therapeutic properties of rosemary leaves collected from two Serbian continental (L1, L2) and one Montenegrin Mediterranean (L3) locations, harvested in November (N), March (M), and July (J). Extracts prepared with 70% methanol, 70% ethanol, and water were analyzed for chemical composition and biological activity. L3 extracts exhibited the highest polyphenolic content, with L3M methanolic extract showing the greatest total phenolic (134.60 mg GAE/g) and phenolic acid levels (211.96 mg CAE/g), and L3M ethanolic extract the highest flavonoid content (25.54 mg QE/g). LC/MS analysis identified 28 previously unreported compounds in Rosmarinus sp. extracts, revealing hydroxycinnamic acid derivatives and flavonoid O-glycosides as the main constituents in S. rosmarinus. The alcoholic extracts were rich in 1,8-cineole, camphor, borneol, terpinen-4-ol, and verbenone. L3 extracts demonstrated the strongest antioxidant and enzyme-inhibitory activities, often surpassing positive controls. L3J showed pronounced cytotoxicity against HCT-116 colorectal cancer cells (IC₅₀ = 13.08 µg/mL after 24 h incubation), while showing non-cytotoxic effects on normal human keratinocytes (IC₅₀ > 500 µg/mL). Finally, rosmarinic acid alone synergistically enhanced the cytotoxic effect of 5-fluorouracil (combination index < 0.8). This comprehensive study highlights the influence of geography, season, and solvent on phytochemical profile and bioactivity of rosemary extracts, emphasizing the therapeutic potential of distinct rosemary populations. Full article

Methenamine, a urinary antiseptic with antimicrobial properties, decomposes into toxic formaldehyde under acidic conditions. Its use is prohibited in dairy cattle in Korea to prevent harmful residues in milk. This study was designed to develop and validate a sensitive and reliable LC–MS/MS method for determining methenamine in raw milk and bovine muscle in compliance with the Positive List System (PLS) regulations. Samples were extracted with acetonitrile (ACN)–methanol (MeOH) (7:3, v/v) containing ammonia water, followed by defatting with n-hexane and purification with primary secondary amine (PSA). Chromatographic separation was performed on a hydrophilic interaction liquid chromatography (HILIC) column, and quantification was conducted using matrix-matched calibration to minimize matrix effects. The method showed excellent linearity (R² > 0.999), low limits of quantification (LOQ) (0.49 μg/kg for raw milk; 0.64 μg/kg for bovine muscle), and acceptable recoveries (78.1–102.8%) with precision (CV ≤ 8.75%), meeting Codex CAC/GL 71-2009 criteria. Stability studies demonstrated that methenamine remained stable in stock solutions, working standards and processed extracts under the storage and handling conditions used. Application to incurred samples resulted in the detection of methenamine in 2 of 32 raw milk samples (0.65 and 1.14 μg/kg) but in none of the 25 bovine muscle samples, with all detected levels below the Korean PLS limit. These findings confirm that the developed method is accurate, sensitive, and applicable for routine surveillance of methenamine residues to ensure consumer safety. Full article