ArticleFrontiers in chemistry2026
Quantitative analysis of vitamin E, 5-methyltetrahydrofolate, and glutathione in seminal plasma by liquid chromatography-tandem mass spectrometry.
Article in Frontiers in chemistry, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
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, 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.
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