Background: Oxidative stress contributes to male infertility, but quantifying non-enzymatic antioxidants in seminal plasma remains challenging. This study aimed to establish liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methods for measuring vitamin E (VE), glutathione (GSH), and 5-methyltetrahydrofolate (5-MTHF) in human seminal plasma. Methods: Analyte-specific pretreatments were established: liquid–liquid extraction for VE, 96-well phospholipid removal plate for 5-MTHF, and sulfosalicylic acid precipitation for GSH without derivatization, with stable isotope-labeled internal standards. LC-MS/MS analysis was performed on AB SCIEX 4500 MD and Waters Xevo TQ-S systems. The method was validated in accordance with the CLSI C62-A guidelines. Preliminary method-specific reference intervals (RIs) were established in 120 healthy reproductive-aged men. Serum-seminal plasma correlations were assessed in 22 paired samples, and exploratory clinical associations were evaluated in 47 infertile men. Results: High linearity (R > 0.99) was achieved, with lower measuring interval limits of 78.10, 1.31, and 15.37 ng/mL for VE, 5-MTHF, and GSH, respectively. The intra- and inter-assay coefficients of variation were <6.8% and <5.5%, respectively. Recovery rates ranged from 88.78% to 108.10%. No significant matrix effects, carryover, or interference were observed. No significant seminal plasma-serum correlations detected. Seminal 5-MTHF and GSH levels were significantly lower in infertile men than in healthy controls [5-MTHF: 139.50 (68.02–198.50) vs. 387.80 (152.50–623.80) ng/mL, P < 0.001; GSH: 4314.00 (3051.00–5974.00) vs. 9049.00 (6128.00–11828.00) ng/mL, P < 0.001], whereas VE levels showed no significant difference [199.00 (138.00–252.00) vs. 168.50 (129.00–237.80) ng/mL, P = 0.211]. Conclusion: The validated LC-MS/MS methods enable reliable quantification of seminal VE, 5-MTHF, and GSH and provide preliminary method-specific RIs in healthy Chinese men. Lower seminal 5-MTHF and GSH levels in infertile men suggest their potential relevance to male infertility. Further multicenter studies are needed to validate their clinical applicability.

Heat stroke is a life-threatening heat-related emergency that often leads to multiple organ dysfunction syndrome (MODS), with liver injury among its most prominent manifestations. HS-associated acute liver injury (HS-ALI) is a major determinant of prognosis, yet its pathogenesis remains incompletely understood. Current evidence suggests that HS-ALI results from the interplay of direct thermal stress, systemic inflammation, oxidative imbalance, coagulation dysfunction, and regulated cell death. Emerging experimental studies have implicated pyroptosis, ferroptosis, necroptosis, and gut–liver immune crosstalk in HS-associated tissue injury; however, the strength of evidence varies considerably among these mechanisms, and direct validation in human HS-ALI remains limited. Accordingly, these pathways should be considered potential contributors to disease progression rather than uniformly established mechanisms.