Tianwei Zhang, Bin Liu, Yang Fu, Yan Jin, Fang Luan, Yong Wang
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.
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.