Separation and quantification of cholesterol and major phospholipid classes in human semen by high-performance liquid chromatography and light-scattering detection

A high-performance liquid chromatographic method coupled with light-scattering detection for the separate and accurate quantification of cholesterol and main phospholipid classes was applied to human spermatozoa and seminal plasma (SP). This method is based on normal-phase chromatography with silica gel as stationary phase and a ternary gradient with hexane, mixtures of chloroform–methanol and water as mobile phase. Lipids are separated with a good resolution and a high reproducibility. About 5·106 spermatozoa or 25 μl of seminal plasma are sufficient to accurate quantitative analysis of phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidycholine (PC), sphingomyelin (SM) and cholesterol. PC is the predominant phospholipid class in spermatozoa (102±8 nmol/108 spermatozoa) whereas SM is the major in the SP (163±6 nmol/ml). Both in spermatozoa and SP, PI is the minor class of the phospholipids (12±1 nmol/108 spermatozoa and 24±2 nmol/ml). In conclusion, this method offers interesting perspectives for analysis of sperm lipid composition in semen samples with low quantities of spermatozoa.