Abstract
The method of HEAP for the determination of progesterone was modified by introducing thin layer chromatography (TLC) and improving some conditions for coloration.
TLC was applied to isolation and purification of progesterone and 20 β-hydroxyprogesterone. Satisfactory results were obtained by developing a sample twice with n-hexane: ethylacetate (5:2) and once with benzene: ethyleacetate (2:1).
The most intense and stable fluorescence was obtained when the color was developed with concentrate sulfuric acid: ethanol (3:2) at 60°C for 10 minutes. When determined by the spect-rofluorometer, the maximum wavelength of this fluorescence presented peaks at 468 nm for excitation and 525 nm for emission. Under the same conditions as these the minimum amount of detection of 20 β-hydroxy progesterone was 2.5 ng.
The blood level of progesterone was determined by the modified method during the estrous cycle in four Japanese beef cows. Its minimum value was 0.2-0.8 ng/ml during the estrous period, and its maximum value 2.6, 6.2 ng/ml during the luteal phase (8 to 20 days after ovul-ation).
