Microfluorometric determination of 4-pregnen-20alpha-ol-3-one separated from rat ovarian venous blood and ovary by divided thin layer chromatography.

Abstract

A highly specific fluorometirc technique was devised to estimate a minute amount of 4-pregnen-20α-ol-3-one (20α-OH-P). In this method 20α-OH-P was extracted from the aqueous phases of ovarian venous blood and ovary with CH2Cl2. After the neutral lipids and phenolic fraction were removed, the evaporated extract was applied to a newly designed thin layer chromatography (17cm in length, 1cm in width, less than 200g in thickness; x 3 strip on a glass plate; 20×5cm). Standard 20α-OH-P was applied on the first strip while the other two strip were available to sample and blank, respectively. After development in ligroin: benzene: ethylacetate at 22°C, these 3 strips were examined under UV (2537A) light and eluted. Fluorescent product was formed with the eluted 20α-OH-P and 65% ethanolic sulfuric acid. The product gave a peak at 485μE (band; 380-540) in the absorption spectrum, a peak at 490mμ(band: 380-560) in fluorescence excitation spectrum and a peak at 530mμ(band; 440-620) in fluorescence emission spectrum. Accurate measurement was possible in nano gram (λ=0.284). Recovery rate was 69.6±1.38% in the ovary and 68.7±5.30%in blood.Identification was made by comparison with an authetic standard. The melting point, absorption spectra in methanol or sulfuric acid chromogen, infrared spectra and chromatographic behavior were all identical. Acetylated or oxidized isolated substances run singly or in mixture were identical with the standards. The amonts of 20α-OH-P was 0.173±0.0145μg in one ovary, 25.408±6.645μg/100ml in the ovarian venous blood and 0.748±0.1528μg/100ml in the cervical flow blood obtained upon decapitation.