Further examination weie made on the double isotope derivative dilution method by the use of thiosemicarbazide-³⁵S as the labeled reagent and ³H-labeled corticosteroid for correcting the loss during extraction and separation procedure, and fractional determination of free and protein-bound cortisol and corticosterone.Δ⁴-3-Ketosteroids with similar chemical structure present in plasma, namely cortisol, corticosterone, aldosterone, deoxycorticosterone, and cortisone can be separated by repeating thinlayer chromatography at a low temperature of 10 °C, more than four times. Cortisol and corticosterone can be determined correctly and with good precision, relative standard deviation being 1-2%. Other three corticosteroids can be determined correctly and with good precision, relative standard deviation being 3-6%.
It was found that the protein-bound cortisol and corticosterone in plasma are dissociated during solvent extraction and the total amount is determined in free form. It was also found that the protein-bound and free cortisol and corticosterone can be separated by gel filtration, and the combination of gel filtration and the present method of determination has made it possible to carry out fractional determination of bound and free forms of cortisol and corticosterone. From its results, the quantity per 100ml of plasma was found to be 1.83 and 0.18 μg of free cortisol and corticosterone, and 17.45 and 1.75 μg of protein-bound cortisol and corticosterone, respectively.