Quantitative analysis of desmosterol, cholesterol and cholesterol sulfate in semen by high-performance liquid chromatography

A simple, rapid and accurate method to separate and quantify cholesterol, d esmosterol and cholesterol sulfate in human spermatozoa and seminal plasma (SP) is described. This high-performance liquid chromatographic procedure i s based on reversed-phase chromatography on a Inertsil ODS2 5 mum silica co lumn with a binary gradient of mixtures of chloroform-methanol and chlorofo rm-methanol-water as the mobile phase at a flow-rate of 0.25 ml/min. Sterol s are separated with good resolution and high reproducibility. The eluted s terols are quantified using a light-scattering (mass) detector. As little a s 64, 64 and 68 pmol of cholesterol, desmosterol and cholesterol sulfate, r espectively, can be quantified under these conditions. Cholesterol is the p redominant sterol both in spermatozoa (107 +/-7 nmol/10(8) spermatozoa) and SP (0.83 +/-0.10 mu mol/ml) whereas the concentrations of desmosterol were 38 +/-6 nmol/10(8) in spermatozoa and 0.18 +/-0.02 mu mol/ml in SP Cholest erol sulfate represents about 6% of total cholesterol in the spermatozoa an d SP In conclusion, this method offers interesting perspectives for the qua ntitative analysis of these sterols not only in semen, but also in other bi ological samples. (C) 2001 Elsevier Science B.V. All rights reserved.