Quantitation of meiosis activating sterols in human follicular fluid usingHPLC and photodiode array detection

A chromatographic assay for 4,4-dimethyl-5 alpha-cholesta-8,14,24-triene-3 beta-ol (FF-MAS), and its reduced species, 4,4-dimethyi-5 alpha-cholesta-8, 24-triene-3 beta-ol (T-MAS), has been established for analysis of human fol licular fluid (huFF). The assay also quantifies lanosterol, free cholestero l and progesterone. It was established using a pool of more than 100 indivi dual follicular fluids from women undergoing in vitro fertilization treatme nt. Both FF-MAS and T-MAS were found in huFF, and can be quantified with HP LC equipped with photodiode array (PDA) detection. The examination waveleng th for each analyte was chosen at the absorption maximum between 200 and 30 0 nm. Spike-recovery experiments revealed mean recoveries of 91 +/- 7.3% fo r lanosterol, 103 +/- 5.1% for FF-MAS, 104 +/- 5.5% for T-MAS, 103 +/- 4.5% for free cholesterol and 85 +/- 5.1% for progesterone. The lower recovery value for progesterone was due to a sub-optimal extraction procedure for th is particular analyte, as indicated by re-extraction. The minimum amounts o f FF-MAS required for quantification were 4 ng/mL and 23 ng/mL for T-MAS an d lanosterol. FF-MAS was assayed to approximately 1.6 mu M. T-MAS and lanos terol was assayed to about half of this value. No esterification of either MAS or lanosterol could be detected in huFF. Less than 10% of cholesterol w as underivatized cholesterol, as more than 10 times the amount of free chol esterol could be assayed after extended saponification. This method can be used for evaluating the accumulation of MAS in huFF and its correlation to oocyte quality and fertilization parameters in in vitro fertilization progr ammes. Copyright (C) 1999 John Wiley & Sons, Ltd.