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

A chromatographic assay for 4,4-dimethyl-5alpha-cholesta-8,14, 24-triene-3beta-ol (FF-MAS), and its reduced species, 4, 4-dimethyl-5alpha-cholesta-8,24-triene-3beta-ol (T-MAS), has been established for analysis of human follicular fluid (huFF). The assay also quantifies lanosterol, free cholesterol and progesterone. It was established using a pool of more than 100 individual follicular fluids from women undergoing in vitro fertilization treatment. Both FF-MAS and T-MAS were found in huFF, and can be quantified with HPLC equipped with photodiode array (PDA) detection. The examination wavelength for each analyte was chosen at the absorption maximum between 200 and 300 nm. Spike-recovery experiments revealed mean recoveries of 91 +/- 7.3% for 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 this particular analyte, as indicated by re-extraction. The minimum amounts of FF-MAS required for quantification were 4 ng/mL and 23 ng/mL for T-MAS and lanosterol. FF-MAS was assayed to approximately 1.6 microM. T-MAS and lanosterol 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 was underivatized cholesterol, as more than 10 times the amount of free cholesterol 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 programmes.