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 5.10(6) spermatozoa or 25 mu l 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/10(8) 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/10(8) 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. (C) 2000 Elsevier Science B.V. All rights reserved.