High-performance liquid chromatographic detection of lipid peroxidation in human seminal plasma and its application to male infertility

Abstract

Background: A simple and reliable high-performance liquid chromatographic (HPLC) method has been developed and validated for the analysis of malondialdehyde (MDA) in human seminal plasma. Methods: After human seminal plasma is hydrolyzed, MDA is reacted with thiobarbituric acid (TBA) to form MDA(TBA) 2, a red-colored adduct with maximum absorbance at 532 nm. HPLC separation of the adduct in human seminal plasma was performed on a Lichrospher C 18 column. Results: A mobile phase composed of 0.025 mol/l KH 2PO 4 (pH 6.2)–methanol in the ratio 58:42 (v/v) was found to be the most suitable for this separation. Under the chromatographic conditions described, the MDA–TBA adduct had a retention time of approximately 4 min and good separation and detectability of MDA in human seminal plasma sample was obtained. The method proved to be linear calibration in the range of MDA from 0.10 to 2.50 μmol/l. The relative standard deviations of within- and between-assay for MDA analysis were 3.1% and 3.8%, respectively. The average recovery was 90.0–98.8% for the human seminal plasma samples. The method has been successfully applied to the study of the lipid peroxidation levels in the seminal plasma of male infertility. Semen samples were obtained from healthy volunteers and infertile males. Ejaculates were classified into studied subgroups and defined as: obstructive azoospermia, non-obstructive azoospermia, oligozoospermia, asthenozoospermia, oligoasthenozoospermia and oligoasthenoteratozoospermia. With the exception of obstructive azoospermic group, MDA concentrations of seminal plasma in control group had very significant difference with those in other infertile groups ( P<0.01). Conclusions: This indicated that lipid peroxidation could be harmful to male sperm and reproductive system, which may lead to male infertility.